The addition of 30% FA to manufactured sand concrete causes a significant reduction in early strength under steam curing, which is not beneficial to the formwork removal and tensioning of precast members. Notably, manufactured sand concrete with 20% FA under steam curing had the highest late strength. The filling effect of FA and the additional gel produced by the pozzolanic reaction would result in the reduction in large pore content, refinement of pore size, improvement of microstructural compactness, and increase in gel system strength. Therefore, the addition of 20% FA to the manufactured sand concrete can improve the long-term strength, which is beneficial to the production of precast beams in cold regions.Steam curing, a common way of curing precast concrete, can greatly improve its 1-day strength. However, the thermal effect of prolonged high-temperature curing can negatively impact the concrete’s performance, thus compromising production of precast products in cold regions. Fly ash (FA) is used as a supplementary cementitious material to improve part of the properties of concrete. In this paper, we investigated the effect of FA (10~30%) on the compressive strength and microstructure of manufactured sand concrete at the steam curing and later stages. Specifically, we analyzed the behavior of FA in the constant temperature phase under steam curing. Results indicated that the pozzolanic reaction of FA started to occur at 24 h of constant temperature curing. Early hydration under steam curing produces a large amount of Ca(OH) Precast concrete products play an important role in infrastructural development. The sleepers and precast beams used in China‘s high-speed railway are precast prestressed concrete components ,2. To im2 and Al2O3, which can react with calcium hydroxide (CH) produced by cement hydration to produce more calcium silicate hydrate (C-S-H), calcium aluminate hydrate (C-A-H) and calcium aluminum silicate hydrate (C-A-S-H). These gels have been shown to improve the late strength and reduce the capillary content of concrete [Reducing the harmful effects of prolonged steam curing on concrete properties and microstructure is imperative to achieving the engineers’ goals. Fly ash (FA), a solid waste from coal burning, is widely used in concrete as a typical supplementary cementitious material ,20. Partconcrete ,29,30. A3+ and Si4+ into the C-S-H gel structure and connected silicon short chains, thereby increasing the content of silicate tetrahedra coordinated with aluminate tetrahedra, and increasing in the polymerization of C-S-H gels [FA‘s low reactivity, coupled with its delayed effect on setting time ,26, makeS-H gels . ChindapS-H gels  found thAs mentioned above, although much research has been conducted in this field, several limitations still exist. For example, controlling constant temperature duration in the current steam curing regime does not effectively meet the tensioning requirements of precast beams, especially for the production of precast beams in cold regions. Studies on FA concrete have mainly focused on normal temperature curing, and there is a lack of research on the strength and microstructure of FA concrete under steam curing, especially the steam curing process, because it is a critical period for the formation of concrete properties and microstructure. It is certain that high temperature curing can have harmful effects on the long-term strength and microstructure of concrete, but only a few studies have explored how to reduce the thermal damage effect.In this paper, we targeted production of precast beams in cold regions with the aim of understanding the compressive strength and microstructure of FA concrete at the constant temperature stage of steam curing. Next, we discuss the effect of FA on the long-term strength of steam-cured manufactured sand concrete. We used X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) techniques to investigate the properties, microstructure and pore structure of the hydration products and FA’s pozzolanic reaction products under steam curing.The Portland cement (P Ⅱ 52.5) produced in Lanzhou Qilianshan Cement Plant, with a specific surface of 351 m2/kg, meets the requirements of GB 175-2007 . Class FNormally, a maximum of 35% by mass of FA can be used to replace cement in concrete ,31, so tAll concrete specimens were prepared and tested at room temperature (20 ± 2 °C). Briefly, materials were first weighed, according to the proportions of the mixture, then the crushed stone, manufactured sand, cement and fly ash were poured into the forced mixer in turn and mixed for 90s. Next, water and polycarboxylate superplasticizer were added into the mixture and the mixing continued for 2 min. The slump of fresh concrete was 160–190 mm, which met the production requirements of precast beams in the plant. The fresh concrete was loaded into a cube mold, measuring 100 mm × 100 mm × 100 mm, for compressive strength and NMR testing. The concrete specimens loaded into the molds were placed on a vibrating table and compacted to reduce air bubbles. The specimens were then subjected to both steam curing and standard curing. SEM specimens were prepared by selecting the mortar portion of fresh concrete to reduce the negative effects of the concrete crushing sampling process on the microstructure of the cement matrix. It uses the same curing regime as concrete.The steam curing regime commonly used for the production of precast beams for the high-speed railway in China  was usedThe compressive strength of concrete was tested according to the China standard GB/T 50081  at a loaThe concrete microstructure was observed by Scanning electron microscopy (SEM), with a Zeiss Gemini SEM 500. Briefly, test specimens were obtained from mortar specimens using a small cutting machine, then a plane surface without any grinding treatment was chosen as the test area. To obtain clear microstructure photos, the specimens were sprayed with gold to increase their electrical conductivity.X-ray diffraction (XRD) method was used to analyze the phase composition of the specimen. The specimen was taken from the slurry portion of the crushed concrete and ground into a fine powder The test equipment was a Rigaku Miniflex 600 with an X-ray source of 60 V. The scan speed was 2°/min and the 2-theta range was 5° to 65°.1H) always spin around their own axis at a certain frequency, which is equivalent to a small constant magnetic field. In the presence of an external fixed main magnetic field, part of the hydrogen nuclei in the field is magnetized, which causes them to be distributed parallel to the direction of the main magnetic field. By applying a set of radio frequency pulses of characteristic frequencies to the main magnetic field, the low-energy hydrogen protons gain energy to enter the high-energy state. Consequently, the macroscopic magnetization is deflected and starts precession. Free induction decay (90-FID) and spin-echo pulse sequence (CPMG) are the two commonly used pulse sequences. After turning off the radio frequency pulse, the macroscopic longitudinal magnetization vector gradually returns to the equilibrium position before excitation under the effect of the main magnetic field. This process becomes longitudinal relaxation, also called spin–lattice relaxation. The macroscopic transverse magnetization vector gradually decays, a process called transverse relaxation or spin–spin relaxation. The time of lattice relaxation is T1 and the time of spin relaxation is T2 [T2 can be calculated according to [T2, bulk is the bulk relaxation time. Free water has a large bulk relaxation time, about seconds, and thus can be neglected. T2, surface is the surface relaxation time. T20 diffusion is the diffusion relaxation time, which can also be neglected when the magnetic field gradient is constant. Therefore, transverse relaxation time T2 can be approximated by T2, surface. The relationship between surface relaxation time with pore volume (V) and pore surface area (S) is calculated as follows:ρ is the material surface relaxivity and takes the value of 12.5 nm/ms [r and k denote the pore radius and geometric factor of the pore, respectively. In this study, we assumed that the pore was columnar pore, and the value of k is 2. From Equation (3), it is evident that the transverse relaxation time of pore water is positively correlated with the pore size.Nuclear magnetic resonance techniques have been widely used to test the pore water content, pore structure and pore size distribution of cement and concrete materials ,44. Baseon is T2 ,46,47. T.5 nm/ms . Based oTesting of transverse relaxation time was conducted on a Macro MR12-150H-I , with a constant magnetic field of 0.3 ± 0.05 T, a magnet temperature of 32 ± 0.02 °C and a radio frequency coil diameter of 150 mm. The equipment working frequency was 12.638 MHz, and can test the size of samples that are less than 150 mm ,49. WhenR2 above 0.85; but after 24 h, R2 decreased to below 0.7. Notably, the greater the amount of FA replacement, the higher the degree of reduction in early strength under steam curing. The strength difference between FA concrete and pure cement concrete was reduced after 24 h of constant temperature curing. Subsequent XRD, SEM and T2 spectra analysis revealed that the reduced content of hydration products and increased total pore content were the main reasons for the low strength of FA concrete, and the increase in gel content by the pozzolanic reaction of FA caused the strength difference between FA concrete and pure cement concrete to gradually become smaller.The compressive strength of manufactured sand concrete with different FA contents under the constant temperature stage of steam curing is shown in The compressive strength of manufactured sand concrete with different FA contents at 28 days and 90 days under both steam and standard curing is shown in SEM images of manufactured sand concrete under the constant temperature stage of steam curing and the late stage are presented in 2 and Al2O3 in the glassy FA, thereby accelerating pozzolanic reactions [2+ and OH− into the interior of FA at a later stage and promoted the pozzolanic reaction.Occurrence of the pozzolanic reaction of FA under the constant temperature stage during steam curing can be attributed to three reasons. Firstly, accelerated hydration of cement under high-temperature curing and early production of large amounts of calcium hydroxide provides the preconditions for pozzolanic reactions. Secondly, a large amount of calcium hydroxide produced over a short period of time increased alkalinity of the pore solution. The high pH facilitated the dissolution of active SiOeactions . Lastly,Under steam curing, pure cement concrete exhibited coarse and numerous microscopic pores, which were accompanied by uneven distribution of hydration products and the presence of microcracks e,f. Thes3S (PDF #49-0442) and C2S (PDF #49-1673) in all four groups of concrete specimens. The diffraction peak of ettringite (PDF #41-1451) can also be observed, which was a hydration product of tricalcium aluminate (C3A). Gismondine (PDF #39-1373) can be observed, but the intensity of its diffraction peak was low, which was a product of cement hydration [3S in F0 was the lowest. According to the principle of the hydration reaction of Portland cement, cement hydration degree can be expressed by the content of CH [Next, we used XRD to explore the effect of FA on the hydration of manufactured sand concrete during the constant temperature stage of steam curing. XRD patterns of F20 concrete cured at constant temperature for 6, 12, 24 and 36 h are shown in ydration . Moreovent of CH ,53. The 3S and C2S significantly decreased in all four groups of specimens, while that of the diffraction peaks of CH increased after 12 h of constant temperature curing. This indicated the intense occurrence of the hydration reaction of the cement. The intensity of the CH diffraction peak in F30 was not significantly reduced, indicating that the pozzolanic reaction of FA did not appear. The diffraction peaks of C3S and C2S were still observed at a constant temperature of 24 h, but the intensity of their diffraction peaks was significantly lower. The intensity of the diffraction peak of CH continued to increase, with the largest increase observed in F0. The low increase in CH diffraction peak intensity in FA concrete may be attributed to the following reasons: Firstly, addition of FA resulted in lower cement content in concrete and reduced the amount of CH produced by hydration. Secondly, part of the CH produced by cement hydration started to be consumed by the pozzolanic reaction of FA, as evidenced by SEM images. We found no obvious change in the intensity of diffraction peaks of CH, C2S and C3S across all four groups of samples after 36 h under constant temperature curing. This indicated that the hydration reaction became significantly slower.The Intensity of the diffraction peaks of C2S and C3S were still observed at 28 days and 90 days, but the intensity of the diffraction peak at 90 days was lower. This indicated that C2S and C3S were still slowly hydrated, and the CH produced by late hydration also provided the conditions for the persistence of the pozzolanic reaction.Profiles of XRD patterns during later stages of steam-cured manufactured sand concrete are illustrated in T2 spectral distribution curve reflected the pore volume of the concrete, and the relaxation time represented the pore size [T2 spectra of steam-cured manufactured sand concrete at 28 and 90 days with different FA contents are shown in T2 spectrum in FA concrete had significantly higher relaxation-signal intensity and spectral area than those of pure cement concrete. The peak signals of the second and third peaks shifted to the left. This indicated that the volume of both gel and capillary pores increased, while the pore size of large pores decreased in FA concrete compared to pure cement concrete. Based on SEM images, we attributed this phenomenon to the pozzolanic reaction and filling effect of FA. Increase in age caused a decrease in the peak signal intensity of T2 spectra in all four groups of specimens. Moreover, the peak signals of the second and third peaks moved to the left, with a large left shift in the FA concrete. These results indicated that both the subsequent continuous hydration and pozzolanic reaction could reduce the pore content and refine the pore size. FA concrete had a dual effect of hydration and pozzolanic reaction, which subsequently generated a significant variation in the peak signal intensity and location.The area integral of the ore size ,48. We dore size . DistribT2 spectrum distribution curves of manufactured sand concrete with different FA contents under steam curing are shown in The area integral of the T2 spectra of manufactured sand concrete with different FA contents is illustrated in The ratio of the area of the first, second and third peaks to the total integrated area in the (1)The steam curing time required to reach the demoulding and prestress tensioning strengths of F30 was 100% and 50% longer than that of F0, respectively. This phenomenon was caused by the dilution effect of FA.(2)The intensity of the diffraction peak of CH in FA concrete decreased after 24 h of constant temperature under steam curing. FA particles were dissolved and the smooth spherical surface changed to a fibrous network, which proved that the pozzolanic reaction of FA occurred, indicating that steaming could remarkably advance the pozzolanic reaction time of FA.(3)Appropriate amounts of FA need to be added in the steam-cured manufactured sand concrete. The reason was that the filling effect and pozzolanic reaction of FA would result in lower large pore content, increased microstructural compactness and higher strength of the gel system, which in turn reduced the adverse effect of steam curing on the microstructure, and therefore had a beneficial effect on the long-term strength. The amount of FA admixture in steam-cured manufactured sand concrete was suitable to be controlled at 20%.In summary, our findings indicate that the addition of appropriate amounts of FA can alleviate the negative effects of steam curing on the long-term strength of manufactured sand concrete. This is expected to facilitate production of precast concrete beams in cold regions. The following main conclusions are drawn from this study:Although it was proved that the pozzolanic reaction of FA could happen at 24 h of steam curing, the following aspects still need further study: (1) Reaction kinetics of FA concrete under steam curing . (2) Eff


Immunoglobulin G4-related disease (IgG4 RD) has a fair prognosis but its diagnosis has been difficult due to the condition's wide range of clinical manifestations, limited awareness among common practitioners, and various differentials. Here, we present a case of an elderly male who presented with recurrent dental caries, recurrent sinusitis, persistent dry mouth, and dry eyes along with bilateral parotid gland enlargement without any lymphadenopathy. The patient was evaluated further and found to have elevated levels of IgG4 and on histopathological examination of the parotid gland showed lymphocytic infiltrate with germinal centers without any granulomatous lesions and IgG4-positive plasma cells on immunohistochemistry (IHC). The patient was diagnosed with IgG4 RD and was started on corticosteroids, after which there was a symptomatic improvement. Immunoglobulin G4-related disease (IgG4 RD) is a recently recognized disease around two decades ago and is a fibro-inflammatory condition characterized by tumefactive lesions, a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, storiform fibrosis, and often elevated serum IgG4 concentrations . This diA 73-year-old man formerly employed as a glass factory manager had a 15-year history of recurrent dental caries for which he has had to replace all of his teeth with artificial dentures as well as a nine-month history of recurrent sinusitis. For five months, the patient had complained of persistent dry mouth as well as a persistent need to drink fluids with meals and associated with recurrent feelings of foreign body sensation in the eyes and dryness without reddish discoloration or blurring of vision (sicca symptoms). This was also associated with gradual onset progressive painless swelling over both sides of the face, and gradual onset persistent feeling of heaviness in the eyelids. No history was suggestive of nasal polyps, asthma, eczema, or other tumefactive lesions that resemble malignancy. He denied any rash, photosensitivity, Raynaud's phenomenon, joint pain, headache, abdominal pain, chest discomfort, or shortness of breath. On examination, a firm, fixed, non-tender bilateral symmetrical enlargement of the parotid gland was noted without any redness or pus discharging sinus, no lymphadenopathy, pallor, clubbing, icterus, and the rest of the general physical and systemic examinations were unremarkable.Routine investigations revealed azotemia and mild anemia. Initially, he was worked up along the lines of Sjogren's syndrome as the patient is having significant xerostomia, and xeropthalmia after ruling out diabetes, HIV, mumps, alcoholism, hypothyroidism, and heavy metal exposure. The ophthalmological evaluation was done for sicca symptoms and was not satisfactory with Sjogren's syndrome. His antinuclear antibodies (ANA)-immunofluorescence assay (IFA) came to be negative, Sjögren's-syndrome antigen A (SSA) antibody low titer positive, low C3, normal C4, and angiotensin-converting enzyme (ACE) levels within normal limits. With high suspicion index of IgG4 RD, he was further evaluated for IgG4 levels which were found to be elevated. As per the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) Classification and Diagnostic criteria for IgG4 RD, a workup was done. Immunohistochemistry (IHC) showed highlights of IgG4-positive plasma cells Figure . HistopaThere are many close differential diagnoses for IgG4 RD. The clinical presentation of sarcoidosis matches this disease. In our patient, after ruling out the main causes for sicca symptoms and bilateral symmetric painless parotid enlargement, Sjogren’s syndrome is of great interest in our case and SSA low titer positivity further makes Sjogren’s a very close differential here . ImmunogThe first step is entry criteria which need a demonstration of IgG4 RD manifestation in at least one of 11 possible organs in a manner consistent with this disease; our patient has characteristic ocular and major salivary glandular enlargement and on histopathology with IHC has a picture consistent with IgG4 RD. The second step is excluding the other common misdiagnosis made with this disease by using the following criteria which have been divided into clinical, serological, radiological, and pathological features that should not be present to make a diagnosis of IgG4 RD Figure .Our patient did not have any systemic inflammatory features; on serology, he had SSA (Ro) positive but in low positive titer, and on CECT abdomen, he had pyloric thickening which made gastric malignancy a possibility. Hence an upper GI endoscopy with a biopsy was done which ruled out malignancy. In histopathology, there were no features elucidated above. The third step is the application of weighted inclusion criteria based on clinical, serological, radiological, and pathological assessments with scoring.The IgG4 RD classification criteria are fulfilled if entry criteria are met, no exclusion criteria are present, and the total points add up to 34 for our patient ( ≥20) Table . The newImmunoglobulin G4-related disease is a clinically evident condition that typically manifests sub-acutely but it can occasionally progress over several months, years, or even decades. Many individuals with IgG4 RD are misdiagnosed as having other diseases, including malignancies because of their findings, which can be attributed to non-specific inflammation. This hinders early initiation of treatment and reduces the patient's quality of life. Many patients with the disease develop an indolent form of the disease that remains asymptomatic for years without progressing to other disease manifestations; therefore, not all disease manifestations require prompt medical attention; in some cases, watchful waiting is recommended, but vital organ involvement must be treated aggressively. However, IgG4 RD can cause serious organ dysfunction and failure. Steroids are still the first line of treatment for IgG4 RD patients because they are efficacious, particularly when started early in the disease's course. In some cases, a combination of immunosuppressive agents is required.


Since glass fiber-reinforced polymer (GFRP) bars have a lower modulus than steel bars, the design of GFRP-reinforced concrete (GFRP-RC) is often governed by the serviceability limit state (deflection and cracking) rather than the ultimate state. A new design method has been proposed in this paper for GFRP-RC beams based on the flexure crack width. The state when the maximum flexure crack width in the tensile zone reaches the limit of 0.5 mm specified by ACI 440.1R-15 was used as the design limit state. The concrete compressive strain at the extreme compression fiber of concrete under the design limit state was obtained by four-point bending tests of eight full-scale GFRP-RC beams and finite element analysis. Based on the concrete compressive strain under the design limit state and cross-sectional analysis, a design method for calculating the longitudinal reinforcement ratio of GFRP-RC beams under the design limit state is proposed. This design method is proven to be feasible by the experimental and the finite element results. In addition, the flexural capacity coefficient was discussed to investigate the safety reserve of the design method. Fiber-reinforced polymer (FRP) bars are characterized by strong corrosion resistance, high tensile strength, light weight, and low modulus of elasticity. In the marine environment with higher humidity and saline mist, FRP bars can be adopted to replace corrosion-prone steel rebars to overcome the corrosion issue. According to fiber types, FRP bars can be classified into glass fiber-reinforced polymer (GFRP) bars, basalt fiber-reinforced polymer (BFRP) bars, carbon fiber-reinforced polymer (CFRP) bars, aramid fiber-reinforced polymer (AFRP) bars, etc. ,2,3,4. CCurrent design methodology for GFRP-RC flexural members is based on the design method for conventional steel-RC flexural members only with some modifications and adjustments to incorporate GFRP bar-related parameters ,15,16,17Moreover, the elastic modulus of GFRP bars is smaller than that of steel bars, which makes the actual mechanical performance of GFRP-RC beams very different from steel-RC beams. When the flexure crack width of GFRP-RC beams reaches the limiting value, the stress level of GFRP bars is still far lower than its design strength; in the same loading condition, the ratio between the midspan deflection of GFRP-RC beams and that of steel-RC beams with the same rebar arrangement is about 3.0; the flexure crack width of GFRP-RC beams is usually more than three times as much as that of steel-RC beams with the same rebar arrangement ,21,22,23For the above-mentioned reasons, the design limit state of GFRP-RC beams should be controlled by the flexure crack width rather than the ultimate load. Therefore, the paper proposes a design method in which the flexure crack width determines the design limit state of GFRP-RC beams. The design codes ACI 440.1R-15  and CAN/Because the elastic modulus of GFRP reinforcement is small, the flexure crack width of GFRP-RC beams under loading easily exceeds the limit of the specification. Consequently, the design limit state of GFRP-RC beams should be the state when the maximum flexure crack width reaches the limit of the specification.Wlim, the concrete compressive stress of GFRP bars at the extreme compression fiber of concrete (abbreviation: concrete compressive strain) is εcd, and the corresponding tensile strain of GFRP bars is εfd, as shown in εcd under the design limit state is an important topic of research for GFRP-RC beam design. Experimental research and finite element analysis were conducted on eight full-scale GFRP-RC beams, considering the influence on the concrete compressive strain exerted by those factors, such as longitudinal reinforcement ratio, concrete strength grade, and longitudinal reinforcement arrangement.It is shown that when the flexure crack width reaches the limit value ρfb, where strains in concrete and GFRP bars simultaneously reach their maximum values. This is the common design concept for FRP-RC sections [Eight full-scale GFRP-RC beams were tested under four-point bending until failure. All the specimens were 3000 mm long having a rectangular cross-section, which was 200 mm wide and 300 mm high. Both the longitudinal bars and stirrups were of GFRP bars. The entire beams were reinforced in compression (top) with two 12 mm diameter GFRP bars. The assembled GFRP cages are shown in sections . In thisAll beams were tested under four-point bending over a simply supported clear span of 3000 mm. The distributive girders were used to apply symmetric and simultaneous load at two loading points, forming a pure bending section in the midspan of the test beam. Five linear variable differential transducers (LVDTs) were installed on each beam to measure deflection at different locations during testing. The crack width gauge was used to measure the flexure crack width. The reinforcing bars and the compression concrete zone of the beams were instrumented with electrical resistance strain gauges to capture the strains at the desired locations. The load was applied in loading control at 2 kN/min until the specimens were about to crack. After the initiation of cracking, the loading rate was decreased to 1 kN/min and kept for 2 min to record the experimental data in the stable condition of load and deformation.Due to the small number of specimens, the test results have a certain degree of discreteness. In this paper, accurate finite element modeling was used to reproduce the crack development process of the specimens. This can ensure the effectiveness of the design method of GFRP-RC beams proposed in this paper.Ec and Poisson’s ratio (0.2). In the plastic stage, the concrete damage plastic model (CDP) was used to simulate the inelastic behavior of concrete. The nonlinear concrete behavior was calculated by using uniaxial compression/tensile stress–strain data and the following parameters. According to Refs. [σc)–compressive strain (εc) relationship of concrete is expressed as:In the linear elastic stage, the behavior of concrete was determined using the elastic modulus to Refs. ,26, the to Refs. , the comσt)–tensile strain (εt) relationship of concrete is expressed as:fc is the concrete compressive strength; ε0 is the concrete compressive strain corresponding to the concrete compressive strength; εcu is the concrete compressive strain under the ultimate limit state, εtr is the tensile cracking strain.The tensile stress  has developed to become a standard tool in fracture mechanics with a sharp crack representation and can well describe the propagation of macrocracks ,29. In tA three-dimensional FE model for FRP-RC beams was developed in ABAQUS. The geometry, loading mode, and boundary conditions were similar to those used in the experiments. The FE model is shown in At each load stage, the flexure crack width and the corresponding concrete compressive strain can be measured. The flexure crack width measured is at the height of the reinforcement. When the flexure crack width reaches the limit of 0.5 mm, this state is determined to be the design limit state of GFRP-RC beams. By averaging the concrete compressive strain measured by the two strain gauges, the concrete compressive strain is obtained under the design limit state, and its finite element calculation value is obtained as well. Mn , as shown in From the above results, under the design limit state, the concrete compressive strain of each beam obtained by test and finite element analyses is close to 0.001, and it is virtually not influenced by the factors, such as reinforcement ratio, concrete strength, reinforcement arrangement, etc. Thus, the concrete compressive strain under the design limit state can be naturally set to be 0.001, which provides a reliable basis for the further design of GFRP-RC beams.h is the height of beam section; h0f is the effective height of the beam section; xcd is the height of the concrete compression zone under the design limit state; εcd and fcd are the concrete compressive strain and stress under the design limit state, respectively; εfd and ffd are the tensile strain and stress of the GFRP bar under the design limit state, respectively; αd and βd are the strain-dependent parameter and stress-dependent parameter under the design limit state, respectively. As can be seen from εcd, does not reach the ultimate concrete compressive strain εcu, under the design limit state. The stress distribution in the concrete can be approximated with an equivalent rectangular stress block using two parameters, αd and βd, as shown in αd and βd, the stress–strain relationship in concrete needs to be determined. In this study, the concrete stress–strain relationship was determined by the current Chinese Code for Design of Concrete Structures GB50010-2010 [010-2010 , as showF and resultant force moment M of the beam section in the compressive zone can be depicted as: In According to plane cross-section assumption, providingαd and βd can be calculate as:Then, the block parameters By substituting Equations (6) and (7) into Equation (8), it is obtained thatαd = 0.6 and βd = 0.7.In the second section of Md is the bending moment under the design limit state.It is shown in The depth of the compressive zone can be obtained by the plane cross-section strain relation:Substitute Equation (10) into Equation (11), obtainingCombine Equations (12) and (13), obtainingρfd of the GFRP bar in the tensile zone under the design limit stateCombine Equations (10) and (14), obtaining the longitudinal reinforcement ratio So far, the longitudinal reinforcement ratio can be determined in the GFRP-RC beam with a rectangular section.xcu is the height of the concrete compression zone under the ultimate limit state; εcu and fcu are the concrete compressive strain and stress under the ultimate limit state, respectively; εfl and ffl are the tensile strain and stress of the GFRP bar under the ultimate limit state, respectively; αu and βu are the strain-dependent parameter and stress-dependent parameter under the ultimate limit state, respectively.The ultimate limit state is that the failure of the GFRP-RC beam is initiated by the crushing of the concrete and the strain, and the stress distribution in the cross section in the concrete can be described by The concrete strain at the maximum compressive concrete strength is assumed to be 0.0033 according to GB50010-2010 . The coeαu = 0.97, βu = 0.82.Additionally, Af = ρfbh0f into Equation (10), obtainingλ = αuβufcεcu/(Efρfb).Similarly, according to the force equilibrium condition, we substituted So, the ultimate bending moment of the GFRP-RC beam is:Md,ex/Md,th are 1.06 and 0.08, respectively. The average and COV of Md,ex/Md,fe are 1.07 and 0.05, respectively. The theoretical and FEM bending moments are in good agreement with the experimental bending moment, meaning the calculation method in this paper is feasible.Mu,ex/Mu,th and Mu,ex/Mu,fe is close to 1 and the COV is smaller, which proves that the theoretical calculation method and the finite element method are effective.SJ) of GFRP-RC beams can be represented as [The flexural capacity coefficient (ented as :(20)SJ=M(1)If the longitudinal reinforcement ratio is greater than the balanced reinforcement ratio, when the flexure crack width of the GFRP-RC beam reaches the standard limit value of 0.5 mm, the concrete compressive strain at the extreme compression fiber of the concrete is about 0.001. The longitudinal reinforcement ratio, concrete strength, and longitudinal reinforcement arrangement have little influence on the concrete compressive strain of this state.(2)Great changes take place in the stress–strain relation of GFRP-RC beams under the design limit state and the ultimate limit state. By setting the concrete compressive strain at the extreme compression fiber of concrete as 0.001, the equivalent rectangular stress block coefficient, the depth of the concrete compressive zone, and the strain of the tensile bar are calculated again. Finally, a formula for the calculation of the longitudinal reinforcement ratio under this state was derived.(3)The theoretical bending moment under the design limit state was compared with its experimental value. The ratio between them ranges from 0.92 to 1.04. Their good agreement proves that the calculation in this paper is correct.(4)When the concrete of the test beams in the compressive zone is crushed under the ultimate limit state, the concrete crack width in the tensile zone reaches 1.2–2.0 mm, far beyond the standard limiting value of 0.5 mm, which explains further that it is appropriate to treat the moment when the concrete crack width in the tensile zone reaches the limiting value as the design limit state, reasonably reflecting the property of the GFRP bar and the actual working conditions of the beam. Furthermore, the ratio between the theoretical and experimental flexural bending moments under the ultimate limit state ranges from 0.85 to 0.96, and hence it is in a good agreement.(5)GFRP-RC beams based on the design method proposed in this paper have the flexural capacity coefficient of about 2.5, which means the beams have a very high factor of safety and reserve strength. Generally, the stress of the tensile bar material is no more than 30% of the ultimate strength, which makes it safe against creep failure too, thereby ensuring its long-term performance.This paper mentions the derivation of formulas for the calculation of the longitudinal reinforcement ratio while setting the moment when the flexure crack width reaches the limiting value of 0.5 mm as the design limit state of GFRP-RC beams. It also describes the experimental and finite element analyses of eight GFRP beams, drawing the conclusion as follows:


In recent years, the bottom-up approach has emerged as a powerful tool in the fabrication of functional nanomaterials through the self-assembly of nanoscale building blocks. The cues embedded at the molecular level provide a handle to control and direct the assembly of nano-objects to construct higher-order structures. Molecular recognition among the building blocks can assist their precise positioning in a predetermined manner to yield nano- and microstructures that may be difficult to obtain otherwise. A well-orchestrated combination of top-down fabrication and directed self-assembly-based bottom-up approach enables the realization of functional nanomaterial-based devices. Among the various available molecular recognition-based “host–guest” combinations, cyclodextrin-mediated interactions possess an attractive attribute that the interaction is driven in aqueous environments, such as in biological systems. Over the past decade, cyclodextrin-based specific host–guest interactions have been exploited to design and construct structural and functional nanomaterials based on cyclodextrin-coated metal nanoparticles. The focus of this review is to highlight recent advances in the self-assembly of cyclodextrin-coated metal nanoparticles driven by the specific host–guest interaction. Recent years have witnessed an increasing use of metal nanoparticles as building blocks for the fabrication of various nano- and microstructures that find applications in various areas of biomedical and material sciences ,2. Whileper-6-thio-β-cyclodextrin (β-CD-(SH)7) is synthesized from β-cyclodextrin (β-CD) by converting the primary hydroxy groups to iodo groups, followed by their conversion to thiol groups upon treatment with thio-urea  (poly-(TMSMA-r-PEG-r-NAS)) followed by branched PEI (BPEI). Free amino units on SPIONS were then modified by tosylated cyclodextrins and magnetic responsive clusters were obtained by paclitaxel-β-CDs inclusion complex after mixing with pendant paclitaxel-containing polymers. In the presence of an external magnetic field, those clusters exhibited superior antitumor effects against HeLa, MCF-7, and CT26 cancer cell lines and showed remarkable tumor inhibition efficacy in a subcutaneous CT26 tumor model . Su. Su52]. According to another report published by Harada and Takahashi, β-CD and γ-CD form inclusion complexes with ferrocene and its derivatives . Based oFang and coworkers reported the construction of an electrochemical aptasensor for thrombin detection based on a host–guest molecular recognition. In this work, using a dabcyl- and thiol-labeled thrombin-binding aptamer (TBA), a single-stranded 15-mer DNA was immobilized on an Au electrode surface via Au-S bond and used as a probe for sensing the target thrombin protein . This wa32+/β-CD-AuNPs/nanographene (NGP) composites were used to modify the glassy carbon electrode (GCE) surface, and then aptamers (TBA1 and TBA2 with a 1:1 M ratio) were labeled with Fc to act as the probed and were attached to the composites via the host–guest recognition between β-CD and Fc. In the absence of thrombin, the quenching of Fc to [Ru(bpy)3]2+ was maintained, and “signal-off” ECL was observed. However, because of the specific combination of the aptamer probes and thrombin, the configuration of aptamer probes changed and escaped from the electrode surface once thrombin appears, which results in the quenching disappearance, and the ECL signal was changed from “off” to “on”.In a more recent work, Fan and coworkers developed a functionalized nanocomposite-based electrochemiluminescence (ECL) sensor for detecting thrombin . In thisThe recent attention being paid to electrochemical sensors has let the investigation of different types of materials being used in sensors. Among these, nanomaterials have preferable advantages of stability, sensitivity, and selectivity. Especially in designing sensors for required properties, metal nanoparticles are crucial to achieving selective determination of specific compounds ,60,61,62Another important approach based on nanoparticle self-assembly is supramolecular layer-by-layer (LbL) systems. In 2005, Huskens and coworkers published a report on multivalent host–guest interactions between AD-functionalized dendrimers and CD-coated gold nanoparticles . Since t2, 350 nm), ferrocenyl-functionalized silica nanoparticles  and CD-functionalized Au , respectively. Each nanoparticle took part in specific adsorption onto CD-mobilized silicon oxide surfaces (CD SAMs) by alternating assembly through their host–guest recognition units. Additionally, generation 1-adamantyl-functionalized poly(propyleneimine) dendrimers (G1-PPI-(AD)4) were used as a molecular glue for absorption of cyclodextrin-coated nanoparticles onto CD SAMs via host–guest recognition technique. Researchers focused on the comparison of the impacts of the sequence of the nanoparticle assembly steps, from large to small and from small to large nanoparticles, which were observed by AFM imaging.In a similar way, Huskens and coworkers revealed another supramolecular LbL system using nanoimprint lithography for the confinement of the nanostructures . Nanopar3+ and MEL in milk were used as two competitive guest molecules. While β-CD provides a relatively hydrophobic cavity for Fe3+ and MEL, fluorophore CNPs provide excellent fluorescence properties to β-CD-CNPs (ON). Thus, When Fe3+ is presented to the medium, it interacts with the β-CD cavity and makes a host–guest complex which causes fluorescence quenching of the system (OFF). On the other hand, when MEL enters the medium, it competes with Fe3+ and makes a stronger host–guest complex with β-CD. Finally, Fe3+ is removed from the β-CD cavity while MEL takes its place, and the fluorescence of the system switches ON. The sensor was applied to an analysis of MEL in milk samples, and it gave a high level of sensitivity and selectivity in MEL detection in these real-life samples.In another study, Yue and coworkers demonstrated an “ON-OFF-ON” sensor that uses competitive host–guest interaction for melamine (MEL) detection in milk . β-CD-mocis form under UV irradiation at 360 nm . The valuable electrical, thermal, and optical properties of carbon nanotubes and their exceptionally high mechanical strength and stability have made them suitable for many applications ,72. Liu t 360 nm . Due to r system ,75. LeveThe field of host–guest chemistry in materials science has attracted great attention since this promising method allows the development of different scaffolds enabling multivalent interactions, one of which is colloidal microcapsules (MCs). Among the number of examples for the colloidal microcapsules ,77,78,79In a following study, Rotello, Sanyal, Crosby, and coworkers reported the fabrication of buckled microparticles via host–guest interactions of 1-adamantoyl-6-(4-phenyl)azobenzoyl-hexane (AB-Hex-AD), 1,6-di(4-(phenyldiazenyl)benzoyl)-hexane (AB-Hex-AB), and 1,6-diadamantoylhexane (AD-Hex-AD) linkers dissolved in chloroform and β-CD-coated AuNPs in water. Divalent molecular guest linkers crosslinked the interfacial nanoparticles to generate stable microcapsules with a robust nanoparticle membrane shell. The inner oil phase containing dicyclopentadiene and catalysts was polymerized to obtain stable microparticles with measurable buckled surfaces. Interestingly, the mechanical strength of the outer membrane was observed to depend on the binding affinities between the host NPs and the guest linkers .trans form can interact with CD under visible light, while after irradiating with UV azobenzene turns into the cis form and can no longer interact with CD. By using this photo-controlled molecular recognition feature, reversible jammed (solid-like droplets) and unjammed (liquid-like droplets) states of NPS were generated. In terms of easy manipulation in changing of shapes and dispersion states of droplets, the designed NPS can be utilized for delivery in fluid systems. In a subsequent report, Shi, Russell, and coworkers proposed a supramolecular redox-responsive system utilizing host–guest interaction of β-CD and ferrocene (Fc) at the toluene–water interface [l-lactic acid (Fc-PLLA), by using the assembly of reduced Fc with β-CD via NaClO and disassembly of oxidized Fc via Na2S2O4, reversible encapsulation of cargo (Rhodamine B) and its release were achieved. As an impressive demonstration, the authors were able to 3D print dye-encapsulated tubules of water in toluene using the liquid structuring ability of the supramolecular colloidal assembly at the interface and release the encapsulated cargo through disassembly of the interface by changing the redox state of Fc.More recently, Shi and coworkers designed a photo-responsive nanoparticle surfactant (NPS)  which wanterface . In the In this review, recent advances in the self-assembly of cyclodextrin-coated nanoparticles driven by the specific host–guest interactions have been summarized. In the past decade, the bottom-up approach has arisen as a powerful tool in nanofabrication for the field of materials and biomedical sciences. Such an approach for the fabrication of functional materials requires the utilization of specific molecular interactions. In this regard, the cyclodextrin-mediated interactions embody several attractive features such as high specificity, stimuli-responsive association, and dissociation, but, most importantly, the assembly takes place in an aqueous environment. The latter attribute enables engineering functional constructs appropriate for many biomedical applications spanning from disease diagnostics to drug delivery. One can see from the present review the several attractive features of cyclodextrin-coated nanoparticles that serve to excite and fire the imagination of new and established researchers to utilize these fascinating building blocks to create new functional materials for addressing global challenges in the area of biomedical sciences.


In view of the easy cracking of the high-performance concrete (HPC) of the subway underground station floor, the effects of fly ash, basalt fiber, expansive agent, and water reducer on the compressive strength, initial crack time, through-crack time, and crack area of the HPC on a subway underground station floor at different ages by orthogonal experiment are examined. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and mercury intrusion porosimetry (MIP) are used to further analyze the microstructure and product composition of the optimal ratio HPC and reference concrete. The results show that with the increase in the content of fly ash and expander, the 7 d and 28 d compressive strength of the HPC gradually decreased. However, as the content of basalt fiber increased, the 7 d and 28 d compressive strength of the HPC gradually increased. The 7 d and 28 d compressive strength of the HPC increased and then decreased with the increase in water-reducer content. When the content of fly ash, basalt fiber and expander increased, the initial crack and through-crack time of the HPC delayed gradually, and the crack area gradually decreased. When the fly-ash content reached 30%, the cracking area accounted for 65.1% of the concrete with 15% fly-ash content. When the basalt fiber content reached 0.4%, the cracking area accounted for 56.5% of the concrete with 0.1% basalt fiber content. When the expander content reached 10%, the cracking area accounted for 60.5% of the concrete with 4% expander content. With the increase in the content of water reducer, the initial crack and through-crack time of the HPC gradually advanced, and the crack area gradually increased. When the water-reducer content reached 1.3%, the cracking area accounted for 105.7% of the concrete with 1.0% water-reducer content. The addition of fly ash and expander can produce a large number of crystalline products to fill the pores, and the disordered distribution of the added basalt fibers increases the compactness of the structure; moreover, the internal micro-pores increase, and the macro-pores decrease, thus improving the crack resistance. With the rapid development of urbanization, underground space has been developed and utilized on a large scale; as one of the most effective ways to make full use of underground space to relieve the pressure of surface traffic, the subway has developed rapidly recently. At the same time, it has brought some problems: the concrete of the subway underground station faces various constraints and loads, early cracks are hard to avoid , and cra3 whisker are mixed into the cement matrix, they can cooperate with each other when damaged, inhibit the generation and expansion of micro cracks and cracks, and improve the strength and toughness of the cement matrix. The mixing effect of steel fiber and polyester fiber is better, and the total crack area is reduced by 99%. Calcium and magnesium expansion agent can expand in the early and middle stages of concrete, inhibit the concrete shrinkage deformation, and the expansion amount, is easy to control, which has a good compensation effect and an anti-cracking function [Scholars around the world have conducted a large number of experiments on the crack resistance of concrete. The shrinkage cracking performance of concrete can be improved by adding auxiliary cementitious materials . Salah Afunction ,18,19. Hfunction  simulatefunction . If the function ,23. Babafunction  studied function ,26; howefunction , so it ifunction  found thfunction  found thScholars worldwide have used different methods to conduct concrete-crack-resistance experiments. Reza Hanifeh  derived The multi-component studied in this paper is mainly mixed with different amounts of mineral admixtures, fibers, and additives. HPC for the subway underground station floor was prepared by orthogonal tests, and orthogonal experiments were designed to evaluate the influences of mineral admixture, expansion agent, basalt fiber, and water reducer on the compressive strength, initial crack time, through-crack time, and crack area of the HPC at different ages. In addition, the microstructures and product compositions of the HPC under the optimal and benchmark proportions were further studied by scanning electron microscopy (SEM), X-ray diffraction (XRD), and mercury intrusion porosimetry (MIP).According to the requirements of Chinese standards, Cement: P·II 52.5 Portland cement, class I of fly ash, silica fume, slag, natural river sand with a fineness modulus of 2.56, basalt gravel with a particle size of 5–30 mm, basalt fiber, calcium oxide expander, and water reducer are used to prepare the experimental specimens. Among them, the performance indicators of cement meet the technical requirements of GB175-2007, the performance indicators of fly ash meet the requirements of GB/T1596-2005, the performance indicators of silica fume meet the requirements of GB/T 27690-2011, the performance indicators of slag powder meet the requirements of GB/T18046-2000, and the water reduction rate of the water reducer is 30–40%. The expander and water reducer meet the requirements of GB50119-2013, the chemical composition of calcium oxide expansion agent is shown in 16 (44) is adopted. In order to study the effect of fly-ash content, basalt fiber content, expansion agent content, and water reducing agent content on the crack resistance of the HPC, combined with raw material chemical composition and performance parameters, the orthogonal test of LThe demould cubic specimens of 100 mm × 100 mm × 100 mm were placed in the curing chamber for curing for 7 and 28 days. According to the relevant test methods in GB-T50081-2011, the compressive strength of the cubic specimens cured for 7 and 28 days was measured with a 500 t micro-electro-hydraulic servo universal testing machine. The concrete crack resistance test was carried out according to CCES 01-2004, and the specimen of 60 cm × 60 cm × 6.3 cm was formed. The mold and specimen are shown in 0; when the first crack was observed, it was recorded as t1. After the crack appeard, it was observed and measured every 20 min until the crack penetrated; at this time, it was recorded as t2. It was observed and measured every hour after the crack penetrated, once every two hours after two hours, once after 10 hours, and once after 24 hours. The contents of observation and measurement include the number of cracks, the average crack width of each crack, the average crack length of each crack, the maximum crack length, and the maximum crack width.The formed specimen was cured in an environment with relative humidity of 60 ± 5% and temperature of 20 ± 2 ℃. After the concrete was poured and leveled, the time was tT1 is initial crack time of concrete (min), T2 is crack penetration time of concrete (min), A is cracking area of concrete (mm2), t0 is the initial moment after concrete leveling, t1 is the momen of the first crack in concrete, t2 is the moment of concrete crack penetration, N is the total number of concrete cracks, iW is average width of the NO.i crack in concrete (mm), and iL is average length of the NO.i crack in concrete (mm).The initial cracking time, crack penetration time, and cracking area were calculated in accordance with Equations (1)–(3):The 10 mm × 10 mm × 5 mm samples that had been cured for 28 days were soaked in anhydrous ethanol for 3 d, and the samples were taken out and dried in an oven at 60 °C for 48 h. The JSM-6510 high-resolution scanning electron microscope was used for SEM scanning analysis.The samples were taken from absolute ethanol that had been cured for 28 days, dried in the oven at 60 °C for 24 h, partially ground to powder, and sieved through 80 μm sieve. D8 advance X-ray diffractometer produced by Bruker Corporation  was used for XRD analysis under a scanning range of 10° to 80° at a scanning rate of 0.30 s/step, step size = 0.02°. Some samples were analyzed by mercury intrusion, and the test was performed by Auto Pore IV 9510 mercury injection meter produced by the Micromeritics Instrument Corporation .2 required for hydration of fly ash may be insufficient. At the same time, the moisture in the concrete migrates to the surface early, and the free water decreases, which affects the hydration of fly ash, so the compressive strength gradually decreases [The effect of each component on the compressive strength is displayed in ecreases .As illustrated in As shown in As shown in As illustrated in As illustrated in The microstructure of the HPC was analyzed by scanning electron microscope. BG is the reference concrete, and OG is the optimal mix HPC in The X-ray diffraction analysis of the high-performance concrete was performed. BG is the reference concrete, and OG is the optimal mix HPC in 2, C-S-H, Aft, Ca(OH)2, and C2S. The main components of sand was SiO2; C-S-H, AFt, and Ca(OH)2 were hydration products; and C2S was the clinker of cement. The positions of the characteristic peaks of each phase of the two groups of concrete basically coincided, and no new phase was found. The diffraction peak of C2S in OG concrete became lower than BG concrete, which indicated that the C2S in the optimal mix concrete was more completely hydrated and the strength was more developed than in BG concrete; the diffraction peaks of AFt and Ca(OH)2 in the optimal mix concrete OG became higher, which showed that the addition of expander produced more hydration products.In order to analyze the internal pore characteristics of the HPC, mercury injection analysis was carried out. BG is the reference concrete, and OG is the optimal mix HPC. The pore size distribution diagram is shown in Through the experimental research on the crack resistance of the HPC for the subway underground station floor with different material components, the following conclusions are obtained:(1) Considering the effect of the fly ash, the basalt fiber, the expander, and the water reducer, the 7 d and 28 d compressive strength of the HPC will gradually decrease with the increase in fly ash and expander content. On the contrary, as the content of basalt fiber increases, the 7 d and 28 d compressive strength of the HPC will gradually increase, but when the content is more than 0.3%, the 28 d compressive strength does not increase significantly. With the increase in the content of the water reducer, the 7 d and 28 d compressive strength of the HPC increases first and then decreases, but the effect on the compressive strength of the HPC is not obvious.(2) As the content of the fly ash, the basalt fiber, and the expansion agent increases, the initial crack time and through-crack time of the HPC are gradually delayed. On the contrary, with the increase in the content of water reducer, the initial crack time and through-crack time of the concrete will gradually increase.(3) When the content of the fly ash, the basalt fiber, and the expansive agent increase, the cracking area of the concrete gradually decreases. However, with the increase in the amount of water reducer, the cracking area of the concrete gradually increases. When the water-reducer content reached 1.3%, the cracking area accounted for 105.7% of the concrete with 1.0% water-reducer content.(4) The addition of the fly ash and the expansive agent can cause a large number of crystalline products to fill the pores, and the cement hydration is more sufficient. At the same time, the disorderly distribution of the basalt fibers can also increase the compactness of the structure; as a result, the proportion of small pores in the concrete increases, and the proportion of large pores decreases significantly, which effectively improves the crack resistance of concrete.The conclusions obtained can provide guidance for the construction of subway concrete engineering in China and can also be used as a reference for the cracking research of concrete engineering abroad.


Hexahydrocurcumin-encapsulated chitosan nanoparticles (HHC-CS-NPs) were formulated by oil-in-water emulsification and ionotropic gelation and optimized using the Box–Behnken design. The particle size, zeta potential, and encapsulation efficiency of the optimized HHC-CS-NPs were 256 ± 14 nm, 27.3 ± 0.7 mV, and 90.6 ± 1.7%, respectively. The TEM analysis showed a spherical shape and a dense structure with a narrow size distribution. The FT-IR analysis indicated no chemical interaction between the excipients and the drugs in the nanoparticles, but the existence of the drugs was molecularly dispersed in the nanoparticle matrices. The drug release profile showed a preliminary burst release followed by a sustained release under simulated gastrointestinal (GI) and physiological conditions. A stability study suggested that the HHC-CS-NPs were stable under UV light, simulated GI, and body fluids. The in vitro bioaccessibility and bioavailability of the HHC-CS-NPs were 2.2 and 6.1 times higher than those of the HHC solution, respectively. The in vitro evaluation of the antioxidant, anti-inflammatory, and cytotoxic effects of the optimized HHC-CS-NPs demonstrated that the CS-NPs significantly improved the biological activities of HHC in radical scavenging, hemolysis protection activity, anti-protein denaturation, and cytotoxicity against MDA-MB-231 breast cancer cells. Western blot analysis showed that the apoptotic protein expression of Bax, cytochrome C, caspase-3, and caspase-9, were significantly up-regulated, whereas the anti-apoptotic protein Bcl-2 expression was down-regulated in the HHC-CS-NP-treated cells. Our findings suggest that the optimized HHC-CS-NPs can be further developed as an efficient oral treatment for breast cancer. Variousobsters) . Our preobsters) . HoweverIn the present study, we developed and optimized the formulation of HHC-encapsulated CS-NPs (or HHC-CS-NPs) using the Box–Behnken design (BBD). The physicochemical properties, including the drug release profile, stability, in vitro bioaccessibility, and in vitro bioavailability, of the optimized HHC-CS-NPs were characterized. In addition, the biological activities of the HHC-CS-NPs were investigated for their antioxidant and anti-inflammatory effects as well as their cytotoxicity against MDA-MB-231 breast cancer cells.® F-127mucin powder from porcine stomach Type III, α-amylase, porcine lipase Type II and bile extract powder, pepsin from hog stomach, pancreatin from hog pancreas, and albumin from human serum (HSA) and bovine serum (BSA) were purchased from Sigma-Aldrich Co., Ltd. . Fetal bovine serum (FBS) was obtained from Life Science Production . Serum-free Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen Cop. . Hexahydrocurcumin (HHC) was synthesized from curcumin (Cur) following the method described by Srimuangwong et al. [Chitosan  was provided by Marine Bio-Resources Co., Ltd. . Sodium triphosphate (TPP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3--2,5-diphenyltetrazolium bromide (MTT), Pluronicg et al. . Wright-X1), Pluronic® F-127 (X2), and HHC (X3), which were found to be critical factors in the formulation of HHC-CS-NPs based on the preliminary experiments. As shown in Y1), zeta potential (Y2), and encapsulation efficiency (EE) (Y3) were chosen as the responses in the present study.The Stat-Ease Design-Expert Software , a 15-experiment run of BBD with 3 factors and 3 levels, was chosen to optimize the HHC-CS-NP formulation. The 3 factors include the concentrations of TPP (v/v) acetic acid solution) containing different amounts of Pluronic® F-127. A 7 mL TPP solution with different concentrations was subsequently added to the suspension using an NE-1000 automatic syringe pump  at a rate of 20 mL/h and continuously stirred for 90 min at 25 °C to form the HHC-CS-NPs. The resulting NP suspension was equilibrated overnight at 25 °C in the dark to ensure its complete formation and uniform particle size.The HHC-CS-NPs were fabricated by o/w emulsification and ionotropic gelation, as described by Calvo et al. , with mo® F-127, and HHC were selected as factors at three levels of study, respectively. The particle size, zeta potential, and EE were chosen as the responses, as shown in p-value, three regression coefficients , and the lack of fit F-value, were employed to adapt the responses to the suitable mathematical model , and quadratic models, respectively) generated by the BBD. In addition to the best-fit mathematical model, quadratic polynomial response equations with key and interaction factors were generated. Analysis of variance (ANOVA) was used to evaluate the suitability and validity of the model. The 3D response surface (3D-RSM) plots were generated by design, and the optimal formulation was identified using a numerical optimization technique with the desirability function approach. The optimal formulation was chosen based on the highest desirability value (close to 1), and it was quantitatively compared with the results of the experimental values of the responses. Finally, a checkpoint analysis was performed by calculating the percentage error between the model-predicted and the experimental values to confirm the roles of the obtained polynomial response equation and the 3D response surface plots in predicting the responses.BBD was used to optimize the HHC-CS-NP formulations. The concentrations of TPP, Pluronic−1.The particle size, zeta potential, and polydispersity index (PDI) of the NPs were analyzed by the dynamic light scattering (DLS) technique using the Zetasizer . The NP morphology was evaluated by transmission electron microscope . The NP suspension was diluted with ultrapure water at 30× before being dropped onto a copper grid coated with formvar film and allowed to dry at room temperature before analysis. The particle size of the optimized NPs was measured using the ImageJ software version 1.53t , Bethesda, MD, USA) . The cheg rpm at 4 °C for 45 min using Hitachi Ultracentrifugation . The supernatant containing unencapsulated HHC was collected and further analyzed at 280 nm using a UV-Vis spectrophotometer . The EE of the HHC-CS-NPs was calculated according to Equation (1).iW is the initial amount of HHC added to the formulation, and sW is the amount of HHC found in the supernatant after centrifugation.The HHC content in the HHC-CS-NPs was determined by UV-Vis spectrophotometry, as described in our previous work . The HHCv/v) to serve as the release medium. Ethanol was added to the simulated fluids to increase the solubility of HHC because the solubility of HHC in the simulated fluids is minimal, thus ensuring the sink condition and avoiding the aggregation of HHC [® T1, Membrane Filtration Products, Seguin, Texas, USA). The end-sealed dialysis bag was then immersed in 100 mL of dissolution medium. A 20 mL HHC solution at the equivalent HHC concentration in the HHC-CS-NPs was also performed to observe the effect of the NPs on the release profile of HHC. After shaking in SGF for 2 h at 37 °C, 150 rpm, the sample was transferred to SIF and SCF for 3 h each at 150 rpm at 37 °C. Each 2 mL sample was withdrawn at different time intervals (0–8 h), and an equal volume of fresh dissolution medium was suddenly replaced to maintain the sink state. The HHC concentration in the samples was analyzed by UV-Vis spectrophotometry, as described above. The results were expressed as the cumulative release of HHC versus time.The release profile of HHC from the HHC-CS-NPs under the simulated GI conditions was determined using the dialysis membrane diffusion technique, as previously described by Omer et al. , with mon of HHC . A 20 mLR2adjusted and Akaike information criterion (AIC) values with the lowest model selection criterion (MSC) values were used as indicators for selecting the best-fitted kinetic model [The in vitro release of HHC from the HHC solution or HHC-CS-NPs in a simulated body fluid  containing 30% ethanol with or without 1% human serum albumin (HSA) as a dissolution medium  was perfic model .The storage stability of the HHC-CS-NPs was assessed using the ICH guidelines  with modThe stability against UV light of the HHC-CS-NPs and HHC with an HHC concentration equivalent to that of the HHC-CS-NPs was performed following the method described by Li et al. , with mov/v) and incubated at 37 °C for up to 24 h. The sample was then taken to measure the particle size and EE at predetermined times . The morphology of the HHC-CS-NPs after 24 h incubation with BSA was also examined using TEM, as previously described.The colloidal stability in the BSA of HHC-CS-NPs was determined using the method described by Suvarna et al. , with mo2 solution , respectively . The pH of the samples was adjusted to 7.0 before incubation at 37 °C and 100 rpm for 2 h. The digested samples derived from the intestinal phase were centrifuged at 35,000× g rpm at 25 °C for 30 min. The HHC in the supernatant was extracted by mixing 10 mL of supernatant with an equivalent volume of chloroform and centrifuged at 35,000× g rpm at 25 °C for 10 min. Then, the amount of HHC contained in the chloroform layer was analyzed by UV-Vis spectrophotometry. It was noted that the supernatant containing the HHC liberated from the solid NP matrix was considered to be the micellar fraction, and the micellarized HHC available for absorption was regarded as the bioaccessible fraction. The transformation (T), bioaccessibility (BA), and bioavailability (B) of HHC were calculated using Equations (3)–(5), respectively [DigestaC and InitialC are the concentrations of HHC in the raw digesta and sample, respectively, before being subjected to digestion in the simulated GI model. BA is bioaccessibility, which refers to the HHC fraction available for absorption in the mixed micelles arriving at the small intestine phase. MicelleC is the concentration of HHC in the micellar phase. B is the bioavailability obtained by the multiplication of the T value and the BA value, which is the ratio between the MicelleC and the InitialC.The in vitro bioaccessibility and bioavailability of the HHC-CS-NPs or HHC were determined under simulated GI conditions using the protocol previously reported by Shah et al. . In thisectively .T (%) =Notably, bioavailability can only be measured by an in vivo assay of the metabolites existing in blood and/or urine after compound consumption as it has metabolic or physiological endpoints that are almost impossible to factor in an in vitro assay . In addicontrolA and sampleA are the absorbance of the control and the tested samples, respectively.The DPPH scavenging activity of HHC-CS-NPs or unencapsulated HHC was determined following the method previously described by Navarro-Hoyos et al. , with mo50). The EC50 is the sample concentration required to reduce the initial DPPH concentration by 50% and is expressed in μg/mL [The inhibition curve between the DPPH scavenging activity and the sample concentration was plotted to estimate the efficient concentration at which 50% oxidation occurs (ECin μg/mL .sA) solution , and 2 mL of 0.75 mM of ferrous sulfate solution (0.114 g FeSO4·2H2O dissolved in 1 L of ultrapure water)) was mixed with 1 mL of 0.01% (v/v) H2O2 solution and incubated at 37 °C for 1 h. A microplate reader spectrophotometer  was used to measure the sample’s absorbance at 536 nm. The damage (dA) and non-damage (nA) groups were carried out under the same conditions as the sample group, except that for the damage group 1 mL of ultrapure water was used instead of the sample, and for the non-damage group, 1 mL of ultrapure water was used instead of H2O2. The OH• radical scavenging activity from the sample was calculated using the following Equation (7).sA, dA, and nA referred to the absorbance of the sample, damaged group, and non-damaged group, respectively.The OH• radical scavenging activity of the HHC-CS-NPs or the unencapsulated HHC was performed according to the protocol reported by Lei et al. , with mog rpm for 5 min to separate the packed erythrocytes (RBCs) from the plasma. To prepare the RBC suspension, 100 μL of packed RBCs were diluted with 0.9 mL PBS (pH 7.4) to prepare the RBC suspensions. The RBC suspensions were then individually mixed with 0.8 mL samples of the HHC-CS-NPs or HHC at an equivalent HHC concentration of 20–100 μg/mL. Then, 0.8 mL ultrapure water or PBS (pH 7.4) was added to 0.2 mL diluted RBC suspensions as the positive and negative control groups, respectively. All the samples were vortexed and incubated at 37 °C for 30 min before being centrifuged at 3500× g rpm for 5 min. Furthermore, 100 μL supernatant was collected and transferred to a 96-well plate, and the absorbance of hemoglobin was measured by a microplate reader at 577 nm. The hemolysis percentage was calculated according to Equation (8).sA, nA, and pA represented the absorbance of the sample, the negative control, and the positive control group, respectively. The in vitro anti-inflammatory activity is the hemolysis protection percentage, calculated by the percentage of hemolysis protection = 100—hemolysis percentage [The in vitro anti-inflammatory activity of the HHC-CS-NPs and unencapsulated HHC was determined using a hemolysis assay, as previously described by Jiang et al. , with morcentage .To affirm the hemolysis protection activity of the NPs, the morphology of the RBCs was determined according to the method previously described by Jiang et al. , with mow/v), 4.78 mL of PBS (pH 6.4), and 0.02 mL of HHC-CS-NPs or HHC solution at an equivalent HHC concentration of 20–100 μg/mL were incubated in a water bath at 37 °C for 15 min. After incubation, denaturation was induced by heating; the samples were heated at 70 °C for 5 min and cooled at room temperature. Then, the sample was measured using a microplate reader spectrophotometer at 660 nm. PBS (pH 6.4) was used as the control throughout the experiment. The inhibition of protein denaturation percentage was calculated by using Equation (9).cA is the absorbance of the control sample, and sA is the absorbance of the tested sample.The effect of the HHC-CS-NPs or the unencapsulated HHC on the protein denaturation was determined by the protein denaturation assay according to the protocol described by Gunathilake et al. , with mov/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. The cultured cells were maintained in a humidified atmosphere of 95:5 (v/v) air:CO2 at 37 °C. The cells were seeded at a cell density of 1 × 104 cells/well/200 μL in a standard 96-well plate, grown for 24 h to obtain a confluent monolayer, and used for further experiments.The cytotoxicity of the blank NPs without HHC (CS-NPs), the unencapsulated HHC, and the HHC-CS-NPs against MDA-MB-231 breast cancer cells  was determined by the MTT assay, as previously reported by Muangnoi et al. , with mov/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. The cultured cells were maintained in a humidified atmosphere of 95:5 (v/v) air:CO2 at 37 °C. The cells were seeded at a cell density of 1 × 104 cells/well/200 μL in a standard 96-well plate, grown for 24 h to obtain a confluent monolayer, and used for further experiments.The cytotoxicity of the blank NPs without HHC (CS-NPs), the unencapsulated HHC, and the HHC-CS-NPs against the MDA-MB-231 breast cancer cells  was determined by the MTT assay, as previously reported by Muangnoi et al. , with moAfter incubation for 24 h, the culture medium was removed, and the cells were washed with serum-free DMEM. The 200 µL of DMEM containing CS-NPs at different content percentages  was then immediately added into the cells and incubated at 37 °C for 24 h. Afterward, the sample was removed, and the cells were washed with PBS and incubated with an MTT solution (0.5 mg/mL in PBS) for 4 h. The DMEM was used as a control. After incubation, the MTT solution was removed, and DMSO was added to the cells to dissolve the formazan crystals. The optical density of the solution was measured using a microplate reader spectrophotometer at 540 nm. The cell viability was calculated with Equation (10).p > 0.05) was selected for the cell viability assay of the HHC-CS-NPs or the unencapsulated HHC in further experiments.The non-toxicity dose of the CS-NPs compared with the control  and treated with the sample for 24 h. Then, the cells were re-suspended in an ice-cold hypotonic lysis buffer for 30 min at 4 °C to extract whole cellular proteins. The lysates were centrifuged at 13,500× g rpm at 4 °C for 5 min. An equivalent amount of each protein sample (40 µg) was added to 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and placed for electrophoresis. After transferring the target protein samples from the gel to a pure nitrocellulose membrane , they were blocked with 5% dry milk at 25 °C. The membranes were treated overnight at 4 °C with the specific primary antibodies: Bcl-2 (1:1000), Bax (1:1000), cytochrome C (1:1000), or β-actin (1:5000). The membranes were washed in triplicate with tris-buffered saline (TBS) and polysorbate-20 before being treated for 2 h with a species-specific horseradish peroxidase (HRP) conjugated secondary antibody reacted with Super SignalTM solution . The membrane was then subjected to an X-ray film; the attached antibody was removed, and the protein loading was confirmed using an anti-β actin antibody. ImageJ software was used to quantify the density of the protein target bands. The results were expressed as a ratio of the band intensities of the target proteins and the β-actin. Quantification of the protein target band density was performed using ImageJ software. The results were expressed as a relative ratio of the band intensities of the target proteins and the β-actin.The expression level of the apoptotic and anti-apoptosis proteins in a sample of the HHC-CS-NPs or unencapsulated HHC-treated cells was performed by a previous protocol described by Muangnoi et al. , with moThe cells were homogenized in a hypotonic buffer to obtain the part of the supernatant. The supernatant was added to a specific substrate  at the concentration of 100 µmol/L. The mixture was then incubated at 37 °C for 1 h prior to absorbance measurement at 450 nm using the microplate reader.t-test (within groups) using Microsoft Excel 365 version 2212, accepting significance at the p < 0.05 level.All the experiments were performed in triplicate, and the data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with one-way ANOVA (between groups) and a Y1), zeta potential (Y2), and EE (Y3) were separately fitted to linear, interaction (2FI), and quadratic mathematical models using linear regression to obtain the model of choice with the statistically significant (p ˂ 0.05) and R2 and R2adjusted, and R2predicted values close to one. ANOVA analysis was carried out to identify the statistically significant model terms of the selected model on the responses. The model terms with p-values < 0.05 are statistically significant. As shown in ® software and are shown in The responses of particle size (p < 0.05). The model regression equation, as shown in X1) was the major factor that had the strongest effect on the increase in particle size compared to the other factors. The 3D-RSM plot in ® F-127, or HHC increased the particle size. The highest particle size (387 ± 21 nm) was obtained when the TPP and Pluronic® F-127 were used at a high level (® F-127 (X2) or HHC (X3) concentration in the formulation. This may be due to an increase in the number of cooperating molecules in the NPs since an increase in the initial amount of formulation compositions results in an increase in the particle size [The NPs with a particle size range of approximately 40 to 400 nm are appropriate to ensure a long circulation time and an enhanced drug accumulation in the cancer cells with reduced renal clearance . As showgh level . At a hicle size 7 ± 21 nmcle size .X1) and Pluronic® F-127 (X2) (p < 0.05), while the HHC concentration (X3) had a non-significant effect (p ˃ 0.05). The model regression equation, as shown in ® F-127 were demonstrated by the 3D-RSM plot shown in +3 group of CS by the negative ions of TPP [® F-127 concentration led to a significant decrease in the zeta potential due to the neutralization of Pluronic® F-127 by the polymer network and the higher system viscosity, which causes the colloidal instability leading to unstable particle aggregation [Theoretically, the potential colloidal stability of NPs is strongly associated with the zeta potential . A high s of TPP . It was regation .p < 0.05). Based on the model regression equation, as shown in X1) and HHC (X3) concentrations had a positive effect on the EE. In contrast, the Pluronic® F-127 concentration (X2) had a negative effect, with the HHC concentration being the major factor affecting the EE. The effect of all the factors is represented by the 3D-RSM plot in ® F-127 concentration were related to a significant decrease in the EE. An increasing initial amount of HHC in the formulation led to an increase in the drug molecules encapsulated in the 3D lattice structure of the ionic gelated CS-NPs, resulting in an increase in the EE [® F-127 concentration. This might be due to the higher system viscosity, which caused particle aggregation and decreased EE.The EE of the HHC-CS-NPs ranged from 43.4 ± 1.8% to 91.2 ± 0.8%, as shown in n the EE . A highen the EE . In cont® software. The optimized formulation was selected based on the criteria shown in X1), 0.5% (w/v) Pluronic® F-127 (X2), and 3 mg/mL HHC (X3). The predicted values of the responses were a particle size (Y1) of 250 nm, a zeta potential (Y2) of 26.8 mV, and an EE (Y3) of 88.7%. After the fabrication of the HHC-CS-NPs using the optimized condition, the observed values of the characteristics were the particle size of 256 ± 14 nm, the zeta potential of 27.3 ± 0.7 mV, and the EE of 90.6 ± 1.7%. The composition check-point formulation with a percentage error between the predicted and the observed values was relatively low, with a desirability value close to 1 (0.95%), as demonstrated in p > 0.05) between the predicted and observed values was observed, indicating the good reliability of the model. Therefore, this statistically optimized condition could be used for the preparation of HHC-CS-NPs.The factors were optimized using the numerical optimization method with the desirability function provided by the Design Expert−1 correspond to O-H stretching, CH2 asymmetric stretching, and C=O stretching, respectively. The peaks at 1454, 1430, and 1364 cm−1 indicate C=C stretching, C-H bending, and CH3 bending, respectively. The peaks at 1150 and 1029 cm−1 are attributed to C-O-C stretching. The peak at 1121 cm−1 is referred to the C-O stretching [−1 indicate the bending vibrations of the -CH bond of the alkene group [−1, which are attributed to the O-NH2 and –OH group stretching, an interaction between the NH+3 groups of CS and the phosphate groups of TPP, the CONH2 group, the NH2 group, and the P=O stretching from the phosphate groups, respectively [The particle size, zeta potential, and EE of the optimized HHC-CS-NP formulation are shown in retching . The peane group . The FTIectively . The FT-It is known that the total GI transit time for drugs or active compounds may vary from one patient to another depending on their physiological conditions . In mostp ˂ 0.05), i.e., at 8 h, the HHC released in the SBF and SBF-HSA was 33% and 21%, respectively. Studies have reported that the presence of protein serum in a dissolution medium may increase the system viscosity and that the path lengths for drug diffusion cause a reduction in drug release [p ˃ 0.05) indicated the protective effect of CS-NPs against the binding and degradation of the serum proteins, leading to the sustained release of HHC.The in vitro drug release of HHC from the HHC-CS-NPs in simulated body fluid  with or without 1% HSA, which mimics the simulated physiological condition, was performed to assess the potential use of the developed NP formulation in preclinical testing prior to in vivo testing, and the results are shown in  release . The bin release . A similv/v) to investigate the release profile of hydrophobic drugs [v/v) ethanol is the most suitable for curcumin release (100% release) compared to the other release media containing various types of surfactants such as 0.5–1% w/v SLS or 30% w/v polyethylene glycol 400 (PEG 400). Nasra et al. [It is noted that the presence of ethanol in the release medium used in this release test may not accurately reflect the actual condition of human GI and the physiological environments. For hydrophobic drugs, choosing a release medium to ensure the sink condition has been challenging. Surfactants such as sodium lauryl sulfate (SLS), cetyltrimethylammonium bromide (CTAB), or polysorbate 80 and organic solvents such as ethanol, isopropyl alcohol, or acetone) are typically added to release media to enhance the drug solubility of the sink condition in in vitro drug release tests . Severalic drugs ,71,72,73ic drugs  reporteda et al.  suggesteR2adjusted and MSC values, as well as the lowest AIC values, were observed for the Peppas–Sahlin model for the in vitro release profile of HHC from the HHC-CS-NPs in both SBF and SBF + 1% HSA. This model describes the combination of Fickian (diffusion-controlled release) and non-Fickian release (the relaxation of the nanoparticle matrix). A value of the release exponent of the Peppas–Sahlin model (m) < 0.45 indicates Fickian diffusion, while a value of 0.45 < m < 0.85 indicates that the drug release is taking place through non-Fickian diffusion [k1) and relaxation (k2) constants of the Peppas–Sahlin model were compared to explore the contribution of the relaxation of the nanoparticle matrix and the drug diffusion phenomena. A value of k1 > k2 suggests that drug diffusion is more important than the relaxation of the nanoparticle matrix. Conversely, a value of k2 > k1 implies that the relaxation of the nanoparticle matrix is the dominant contributor [k1 and k2 values in According to the kinetic model fitting results, as shown in iffusion ,75. In ttributor . The k1 p < 0.05). The particle size increased to high values (˃520 nm) during the 90-day period, and particle aggregation was observed, indicating an unstable colloidal suspension (p > 0.05), but it increased significantly (p < 0.05) after 90 days (p < 0.05), whereas the EE of the HHC-CS-NPs stored at 4 °C for > 60 days showed no significant change from the initial level (p > 0.05). This might be due to the degradation of HHC being fast at high temperatures, resulting in decreases in EE. In addition, the optimized HHC-CS-NPs had better storage stability at 4 °C for up to 90 days. p > 0.05). In addition, after 150 min of exposure, about 95% of the free HHC and 10% of the HHC-CS-NPs were degraded from the initial level. This might be due to the strong photostability of CS-NPs, which prevent UV radiation from degrading encapsulated HHC. Many studies have reported that the photostability of various active compounds was significantly enhanced by encapsulation within CS-NPs due to their shielding effect and physical barrier, which can scatter and attenuate the incidence of UV light radiation [Colloidal delivery systems such as polysaccharide-based carriers must remain stable during the storage and shelf-life of drugs or active compound formulations . Thus, ispension a. Howeve 90 days a, reachiadiation ,79.On the other hand, Suvarna et al.  suggestep < 0.05) than that of the HHC solution (58.3 ± 2.3%), indicating that the CS-NPs could improve HHC stability against pH and digestive enzyme degradation in the simulated GI model. In addition, this finding was correlated with the results of the digestion stability (p < 0.05) than that of the free HHC (35.4 ± 2.4%), which indicates the higher availability for absorption (bioavailability) of the HHNPs than the free HHC. As expected, the in vitro bioavailability of the HHC-CS-NPs (64.3 ± 1.9%) was six times higher than that of the free HHC (10.7 ± 1.5%), which correlated with the bioaccessibility results. The findings might imply that encapsulating HHC within the HHC-CS-NPs greatly increased its in vitro bioavailability, owing to a large increase in bioaccessibility. In other words, the capability to preserve the structural integrity of the NPs against the degradation by the digestive enzymes present in the simulated GI fluids was evaluated by changes in the structural morphology after passing through each phase of the simulated GI model by TEM analysis, as shown in Each free HHC or HHC-CS-NPs sample was then individually passed through a three-step simulated gastrointestinal tract (GI) model, which included the oral, gastric, and small intestine stages. The in vitro bioavailability was assessed by determining the total amount of HHC remaining, including the fraction in the mixed micelle phase. The results were used to calculate the transformation, bioaccessibility, and bioavailability of HHC. As shown in tability b–d, whicp ˃ 0.05) scavenging activity of the CS-NPs (˂3% of activity), implying that the empty CS-NPs had no scavenging activity, whereas the HHC-CS-NPs or free HHC solution had significant scavenging activity (p ˂ 0.05) in a concentration-dependent manner. The highest scavenging activity was found in the HHC-CS-NPs at 80 μg/mL (~96%), which was 1.5 times higher than the free HHC solution at the equivalent HHC concentration, indicating that encapsulating HHC in the CS-NPs significantly enhanced its scavenging activity. This may be explained by the fact that the solubility, dispersibility, and stability of HHC were enhanced by encapsulation within the CS-NPs, which resulted in higher antioxidant activity. Furthermore, the half-maximal effective concentration (EC50) value of the HHC-CS-NPs (~38 μg/mL) was significantly lower (p ˂ 0.05) than that of the free HHC solution (~64 μg/mL), indicating that the encapsulation in CS-NPs enhanced the HHC antioxidant activity. To confirm the antioxidant effect of the HHC-CS-NPs, the scavenging activity of the HHC-CS-NPs toward other free radicals such as hydroxyl radicals (OH•) was needed. The OH• scavenging activity of blank CS-NPs, HHC-CS-NPs, and free HHC solution on OH• radical was found to follow the same trend as the DPPH scavenging activity results, as shown in (p ˂ 0.05) at the equivalent HHC concentration, indicating that encapsulation within CS-NPs significantly enhanced the HHC antioxidant activity.The RBC membrane is an analogous model membrane for the lysosomal membrane, which plays an important role in releasing inflammatory mediators when inflammation occurs . The antTo assess the anticancer activity of the HHC-CS-NPs, free HHC solution, and empty CS-NPs, the MDA-MB-231 cells were separately treated with each sample. The percentage of cell viability was then determined by MTT assay. Generally, toxicity-related NPs have been demonstrated to function in a dose-dependent manner. Thus, using a suitable dose of NPs in a cytotoxicity assay is key to understanding the toxicity effects of the NPs under real-world practical conditions .p ˂ 0.05) in a dose-dependent manner. The viability of the cells treated with 5% or 10% CS-NPs was shown to have a non-significant effect (p ˃ 0.05) compared to the control . The cells treated with ≥20% CS-NPs had a significant effect (p ˂ 0.05). Hu et al. [p < 0.05) in a concentration-dependent manner. Compared to the HHC solution, the HHC-CS-NPs show significantly decreased cell viability (p < 0.05) by 10% and 30% more than the HHC solution at concentrations of 5 μg/mL and 10 μg/mL, respectively. Interestingly, the HHC-CS-NPs at 5 μg/mL showed no significant difference (p > 0.05) in the cell viability with HHC at 10 μg/mL, indicating a highly enhanced cytotoxic effect of HHC by encapsulation within CS-NPs. Overall, it may be concluded that the HHC-CS-NPs were more cytotoxic than HHC.Herein, the non-toxicity dose of empty NPs (CS-NPs) was primarily determined to choose the suitable dose for the cytotoxicity assay of the HHC-CS-NPs or free HHC solution in the further step. Following the results, as shown in u et al.  suggest p ˂ 0.05), while the anti-apoptotic protein was significantly decreased (p ˂ 0.05) as compared to the control in a dose-dependent manner (p ˂ 0.05) were observed in the HHC-CS-NP- or HHC-treated cells compared to the control, as shown in p ˂ 0.05) in the HHC-CS-NP-treated cells and was 2.1 times higher than those of the HHC-treated cells at 10 μg/mL (p ˂ 0.05) in the HHC-CS-NP-treated cells, and it was 1.5 and 1.2 times lower than in the control and the HHC-treated cells at 10 μg/mL, respectively (p ˂ 0.05). It was 3.8 and 1.5 times higher than in the control and the HHC-treated cells at 10 μg/mL, respectively, suggesting that the encapsulation of HHC in CS-NPs was more effective in causing the release of cytochrome C from the mitochondrial to the cytosol. Chen et al. [p ˂ 0.05). The effect of HHC-CS-NPs on the expression of caspase-3 in treated cells was found to have the same trend as the results of the other pro-apoptotic proteins. For example, at 10 µg/mL, the caspase-3 expression was 4.4 and 1.8 times higher in the HHC-CS-NP-treated cells than in the control and the HHC-treated cells, respectively. The effect of HHC-CS-NPs on the expression of caspase-9, another member of the caspase protein family, on the treated cells followed the same trend as the caspase-3 results; at 10 µg/mL, the caspase-9 expression was 3.5 and 1.7 times higher in the HHC-CS-NP-treated cells than in the control and the HHC-treated cells, respectively. Overall, these results suggest that the HHC-CS-NPs exhibited a stronger apoptosis-inducing effect on the MDA-MB-231 cells than the free HHC. However, our present findings on the stability, the in vitro drug release profiles, and the biological activities of HHC  show that it has good stability and controlled and sustained drug release with promising biological activities. In summary, our results suggest that CS-NPs are efficient at enhancing the apoptotic mechanisms, the drug-releasing profile, the stability, and the biological activities of HHC. Therefore, it can be concluded that HHC-CS-NPs may provide mitochondrial-mediated apoptotic activities by improving multifactorial effects.To further fundamentally demonstrate the possible mechanisms of the remarkable anticancer activity of the developed nano-formulation, the four apoptotic  and one anti-apoptotic (Bcl-2) protein expression profiles of the HHC-CS-NPs or HHC solution treated MDA-MB-231 cells were determined using Western blot analysis. β-actin was used as a positive control throughout the study. Herein, the expression of all four apoptotic proteins in the HHC-CS-NP- or HHC-treated cells was significantly increased  were 0.43 ± 0.4 and 0.32 ± 0.2, respectively. The in vitro release profile of HHC from the HHC-CS-NPs under simulated GI and physiological conditions suggested a controlled and sustained release pattern. The HHC-CS-NPs performed well in physicochemical, digestive, and protein stability. The in vitro bioaccessibility, bioavailability, antioxidant activity, anti-inflammatory activity, anti-denaturation of protein activity, and cytotoxicity against MDA-MB-231 breast cancer cells of HHC were significantly enhanced by encapsulation in CS-NPs compared to free HHC. Western blot analysis showed that the HHC-CS-NP-treated cells were significantly up-regulated for the apoptotic proteins and down-regulated for the anti-apoptotic protein compared to the HHC-treated cells, suggesting that mitochondrial-mediated apoptotic activities are present. Overall, it may suggest that the CS-NPs offer promise as potential drug delivery systems for HHC as an alternative therapeutic agent for breast cancer therapy.