Aconitum carmichaelii Deb  is a potent traditional herbal medicine extensively used in treatment of cardiovascular diseases, rheumatism arthritis, and bronchitis in many Asian countries. Although Fuzi has promising therapeutic effects, its toxicities are frequently observed. Three main C19-diester-diterpenoid alkaloids (DDAs) are believed to be the principal toxins of the herb. Although toxicokinetic profiles of the toxic DDAs have already been examined in several studies, they have seldom been correlated with the toxicities of Fuzi. The current article aimed to investigate the relationship between the up-to-date toxicokinetic data of the toxic DDAs and the existing evidence of the toxic effects of Fuzi. Relationships between the cardiac toxicity and the plasma and heart concentration of DDAs in mice and rats were established. Based on our findings, clinical monitoring of the plasma concentrations of DDAs of Fuzi is recommended to prevent potential cardiac toxicities. Additionally, caution with respect to potential hepatic and renal toxicity induced by Fuzi should be exercised. In addition, further analyses focusing on the preclinical tissue distribution profile of DDAs and on the long-term toxicokinetic-toxicity correlation of DDAs are warranted for a better understanding of the toxic mechanisms and safer use of Fuzi.The processed lateral root of  Aconitum carmichaelii Deb is a famous traditional Chinese medicinal herb. Its processed lateral root  is extensively used in the treatments of cardiovascular diseases, rheumatism arthritis, bronchitis, pains, and hypothyroidism. In clinical practice in China, Fuzi is the constituent of more than 60 well-known traditional Chinese formulae frequently used [A. carmichaeli belongs to the genus of Aconitum, which consists of over 300 species distributed in the temperate regions of the north hemisphere and 211 species in China. In total, 76 species of Aconitum have been used as herbal medicine or ethnomedicine in countries such as Indian, Vietnamese, Korean, Japanese, and China [tly used . A. carmA. carmichaeli for edible plants, and contamination of A. carmichaeli in other medicinal herbs [Although Fuzi has demonstrated promising therapeutic effects, its potential cardiotoxicity and neurotoxicity are frequently observed . Thus, tal herbs ,6,7,8. Ial herbs ,11,12,1319-diterpenoid alkaloids [The principal bioactive ingredients in aconite roots are the Clkaloids . Three mlkaloids ,15,16. Tlkaloids ,14,17, wlkaloids . Based oAconitum carmichaelii Debx”, “Aconite Lateralis Radix Preparata”, and “Aconite” were used as keywords to search the herb-related articles, and keywords including “Aconitum alkaloids”, “Diester-diterpenoid alkaloids”, “Aconitine”, “Mesaconitine”, and “Hypoconitine” were used for the search of compound-related articles. Any articles contained information involving the toxicity and/or toxicokinetics of DDAs in Fuzi were considered eligible for evaluation. Studies without determining the content of any DDAs in the tested Fuzi extract were excluded from the current review. The following databases were searched to identify relevant literatures in both English and Chinese: Pubmed (from 1959 to August 2018) and China National Knowledge Infrastructure (from 1994 to Aug 2018). Both Latin and Chinese pinyin terms including “Fuzi”, “Lateral roots of A. carmichaeli comprises chemicals that range from relatively non-toxic to deadly poisonous [A. carmichaelii [20-, C19-, and C18-diterpenoid alkaloids [19-diterpenoid alkaloids, which only occur the Aconitum species [19-diterpenoid alkaloids are also believed to be responsible for some toxicities and certain pharmacological activities of A. carmichaelii [19-norditerpenoid skeleton, in which the C8 and C14 position can be occupied by an acetoxy, a benzoxy, or a hydroxyl group [oisonous . Over 12lkaloids ,19. The yl group . In the Aconitum [Aconitum poisoning [According to the Chinese Pharmacopoeia, the maximal therapeutic dose of Fuzi is 15 g/person, and the maximal total content of DDAs in Fuzi is 0.01% . The lowAconitum . Since toisoning ,3,6. Theoisoning ,3,6.Due to limited information on Fuzi toxicity mechanisms available from clinical poisoning cases, a large number of pre-clinical studies have been conducted to investigate the toxicities and their relevant mechanisms of both Fuzi and its DDAs.50) was commonly used as the toxicity assessment of Fuzi and its DDAs in the early years. The LD50 of processed Fuzi for a single oral dose was reported as 100–145 g/kg in mice. The reported LD50 for a single oral dose of AC was 1.0–1.8 mg/kg in mice [50 values were reported to be 1.9 and 5.8 mg/kg, respectively. In contrast to those of DDAs, the toxicity of the MDAs was around 1000-fold weaker, with the LD50 values  of benzoylaconine (BAC), benzoylmesaconine (BMA), and benzoylhypaconine (BHA) as 1.50, 0.81, and 0.83 g/kg, respectively [The median lethal dose  were also present in the urine [Clinical studies in the DDAs ,12,13. Athe DDAs . Toxicokhe urine . It was he urine ,43. Thesmax of AC after ingestion of pure compounds was around 25–131 min in rats and 15–35 in mice. The permeability (Papp AtoB) of AC, MA, and HA on the Caco-2 cell monolayer was 7.6 × 10−6, 8.2 × 10−6, and 21.5 × 10−6 cm/s respectively, while the permeability (Peff) of AC on a rat in-situ ileum perfusion model was around 0.5 × 10−5 cm/s. Both results from the Caco-2 cell and the in-situ intestinal perfusion model indicated medium to good absorbability of DDAs [Preclinical models provided more detailed toxicokinetic characteristics of the DDAs. After an oral dose of the pure compound and Fuzi extract, the bioavailability of AC was 8.24% and 4.72%, respectively . Absorpt of DDAs ,46,47. T of DDAs ,46,48,49 of DDAs ,46,48,49 of DDAs ,48. TakeAC has low protein binding (23.9–31.9%), leading to its rapid distribution to various organs . The livmax in comparison to that of AC. However, their plasma and tissue concentrations were much less than that of AC [O-demethylation has been found as a common metabolic pathway of the three main DDAs via Cytochrome P450 (CYP) in both liver and intestine microsome of rats and humans [O-demethylaconitine and 16-O-demethylhypaconitine were detected from rats and human plasma and urine samples after oral intake of A. carmichaeli [O-demethylated DDAs may also exert certain toxicity. Moreover, other metabolic pathways of the DDAs including hydroxylation, deoxylation, demethylation, didemethylation/deethylation, dehydrogenation, and ester exchange were discovered in an in vitro model using liver and intestine microsome or intestinal bacteria [It was previously hypothesized that DDAs may mainly be metabolized via hydrolysis. However, recent animal studies have revealed that the hydrolysis of DDAs to MDAs and NDAs may not be predominant in vivo ,52,53,54at of AC . At 120 at of AC , and theat of AC . Only a at of AC . It had at of AC , but supd humans ,52,56,57michaeli ,57. Sincbacteria ,52,56,57bacteria ,56.Urine is one of the major excretion routes for DDAs and their metabolites. After oral administration of AC, the urine concentration of AC peaked at 8 h post-dosing, and its major metabolite 16-O-demethylaconitine peaked at 6 h and remained detectable for up to 24 h post-dosing [Since DDAs have been reported to regulate the expressions and activities of some efflux transporters and drug-metabolizing enzymes, they may be able to affect the toxicokinetic profiles of themselves or other co-treated xenobiotics. DDAs and their corresponding MDAs could increase MDR1, MRP2, and breast cancer resistance protein (BCRP) expressions in cell models and mice intestine, likely via activation of the nuclear factor E2-related factor-2 (Nrf2), nuclear receptors pregnane X receptor (PXR), and constitutive androstane receptor (CAR) ,58. A loIn summary, the ADME characteristics of DDAs from animals are similar to those found in clinical observations. Additionally, more mechanisms of the ADME processes of DDAs have been derived from pre-clinical studies, including their medium to good absorbability in the intestine, limited in vivo hydrolysis, and their modulatory effect on the activities of MDR1, MRP2, BCRP transporters, and CYP enzymes.0–12 h) of AC, with no significant toxic effects observed on 0.1 mg/kg AC-treated rats [As shown in ted rats . Similarted rats , and 1 mted rats . The carmax of HA was found to be 1.53 ng/mL at such a dose [max, lower Cmax, and comparable T1/2 [A few studies also investigated the toxicity and toxicokinetic profile of HA after bolus oral administration. After oral administration of 0.05, 0.15, and 0.45 mg/kg HA to beagle dogs, linearity of the peak plasma concentrations of HA was observed, and the cardiac toxicity (manifested as QT prolongation) was dose-dependently aggravated. Abnormality on ECG can be observed even at the lowest toxic dose of 0.05 mg/kg on beagle dogs, and the Ch a dose . The toxble T1/2 .Sub-chronic toxicity was induced after seven-day oral administration of 0.146 mg/kg AC to mice. Significant ventricular tachycardia, ventricular premature, and pathological changes in the myocardial tissues (including hyperchromatic nuclei and condensation of cytoplasm) were clearly observed . It was max or AUC) at the corresponding lowest toxic bolus dose were determined. The plasma concentration of DDAs at the lowest observable cardiac toxicity dose was found as low as 1.5 ng/mL. Therefore, risk of cardiac toxicity should be revealed when the plasma concentration of DDAs reaches such a threshold. On the other hand, toxicity and toxicokinetic correlations after long-term treatment, which are critical for the study on chronic toxicity of these compounds, have never been reported.In summary, toxicity and toxicokinetic correlations on AC and HA after bolus doses have been established in animal models, while no such information regarding MA has ever been reported. The lowest toxic dose was found at 0.2 mg/kg for AC in mice and rats, and 0.05 mg/kg for HA in dogs, both of which are very close to the recommended clinical upper limit of the dose of DDAs in Fuzi . The liIn comparison to those from the oral administration of DDAs, the toxicokinetic profiles of DDAs after ingestion of Fuzi extract are more complicated. The toxicokinetic studies of the three major DDAs after oral administration of various Fuzi extracts are summarized and correlated with their toxicities as shown in Besides the cardiac abnormity demonstrated by ECG, damage in organs including the heart, liver, and kidney were also found in animal models after a single oral dose of Fuzi extract. Only one study reported the dose-dependent toxicity of Fuzi extract with its DDA contents determined. In this study, the lowest toxic dose in rats was 2 g/kg Fuzi extract, which equals to 0.116, 0.367, and 0.586 mg/kg of AC, MA, and HA, or 1.1 mg/kg total DDAs. At such a dose, no elevated biomarker indicating heart, liver, or kidney dysfunction was observed, while mild edam was noticed in the liver and heart . At a memax and AUC0-last of AC and HA after administration of Fuzi extracts were significantly lower, while the T1/2 remained similar. The Tmax of AC in pure-AC-treated rats was also comparable to that in Fuzi extract-treated rats. The toxicokinetic characteristics of each DDA after ingestion of mild to severe toxic doses of Fuzi extracts are discussed individually in the following paragraphs.On the other hand, toxicokinetic profiles of the three main DDAs after oral ingestion of Fuzi extracts have been studied in rats ,69,70,710-last and Cmax of AC was proportionally elevated as the dosage increased, and the T1/2 of AC was not significantly changed between different dosage groups [0-last (24.3 ± 8.9 min·kg/L) was significantly larger than those from the mild toxic dose (around 4.4 ± 0.5 to 8.3 ± 1.4 min kg/L) [1/2 of AC, which may be due to the low and variable plasma concentrations [Comparing with HA and MA, the doses of AC in the Fuzi extracts were relatively low (around 0.037–0.117 mg/kg AC). In a dose range of Fuzi  that refers to mild toxicity, the AUCe groups ,69,70, din kg/L) . In additrations .max and AUC0-last of HA were proportionally increased within the dose range from non-toxic  to severely toxic . The dose-normalized AUC0-last values of HA were around 4.9 ± 0.9 to 8.4 ± 0.3 min·kg/L [1/2 of HA (ranged from 104 ± 2 to 559 ± 62 min) varied among different studies using Fuzi at an equivalent dose of 0.46–4.9 mg/kg total DDAs [The HA content in the Fuzi extracts was usually much higher than that of AC . The dosmin·kg/L ,69,70. Ttal DDAs ,69,70,710-last and Cmax of MA, with dose-normalized AUC0-last of MA at around 2.8 ± 0.7 to 4.0 ± 0.8 min·kg/L, and the T1/2 (ranged from 251 ± 98 to 636 ± 210 min) was not significantly changed among different doses [0-last (6.1 ± 1.2 min·kg/L) was slightly larger than those from the low toxic dose [0-last (22.3 ± 1.8 min·kg/L), which may be due to the low and variable plasma concentrations [When Fuzi extracts were given to the rats at low toxic doses , linearity was observed in the AUCnt doses ,69,71. Htrations .0-last and Cmax of the three main DDAs were proportional to their doses with similar dose-normalized AUC0-last values at low toxic doses  of the Fuzi extract. At a high toxic dose of Fuzi extract , the dose-normalized AUC0-last values of AC and MA were significantly higher than those from the low dose. It is also noticed that, even under the same dose of AC and MA, their toxicokinetic parameters including AUC0-last and Tmax varied between the two groups of rats treated with different Fuzi extracts, suggesting that other co-existing components in different Fuzi extracts may affect the toxicokinetic profiles of AC and MA [Overall, the AUCC and MA .1/2 and Cmax remained similar [In clinical practice, intake of Fuzi can be at lower dose level for sub-chronic treatment that usually lasts for 1–2 weeks.  similar . Such ev0-last of AC and MA after a single oral dose of Fuzi extract. Correlation between the toxicokinetics of DDAs and the toxicity of Fuzi have been preliminarily studied but are still ambiguous. To further illustrate the quantitative relationship between the toxicokinetic profile and the toxicity of Fuzi extract, two issues need to be tackled: (1) Quality variability was observed among the Fuzi extracts used in different toxicity and toxicokinetic studies. The toxicokinetics of DDAs may be affected by other co-existing components in Fuzi [In summary, a high dose of Fuzi extract usually leads to severe heart, liver, and kidney damage , which i in Fuzi , and add in Fuzi . Such quThis review is the first to summarize evidence of the relationships between DDA plasma kinetics and the toxicities of Fuzi. Such relationships have been well-identified in animals receiving pure DDA compounds and have been preliminarily investigated in animals receiving different Fuzi extracts, whereas the clinical evidence of such relationships are still lacking. Based on the pre-clinical evidence, it has been demonstrated that the narrow therapeutic window and large quality variability of Fuzi extracts may significantly affect its safe use in clinical practice. Based on our findings, standardized products are essential for the safe use of Fuzi. Although correlations between the exposure of DDAs and Fuzi-induced hepatic and renal toxicities are still ambiguous, dose-dependent hepatic and renal toxicities of Fuzi have been clearly observed in pre-clinical studies. Therefore, caution with respect to potential hepatic and renal toxicity induced by Fuzi should be exercised. Since the cardiac toxicity has been well-correlated with the plasma and heart concentrations of DDAs in mice and rats, with the plasma concentration of DDAs at the lowest observable cardiac toxicity being determined, clinical monitoring of the plasma concentrations of DDAs of Fuzi is recommended to prevent potential cardiac toxicities. Further analyses focusing on the tissue concentration profile of DDAs and on the long-term toxicokinetic-toxicity correlation of DDAs are warranted to better understand the toxic mechanisms and safer use of Fuzi.


The aim of this cross-sectional study was to evaluate the knowledge and attitudes concerning antibiotic use and resistance among members of the local community in Pulau Pinang, Malaysia. The study was conducted among 326 residents of the Jelutong district, Pulau Pinang state, from August to October 2013. A self-administered five-part questionnaire was used for the data collection. The respondents exhibited inadequate knowledge of antibiotics in general. Approximately 80% of them did not know the indications for antibiotic use, while 76% believed that antibiotics were useful in terms of resolving viral fever. Additionally, 52.6% believed that antibiotics could be used to treat all types of infections. Inadequate knowledge of antibiotic resistance was also evident among the respondents, since 72.9% of them did not agree that resistant bacteria can spread from human or animal to human, while 32% were unaware that bacteria can develop resistance to antibiotics. More than 60% of respondents admitted that they took antibiotics in order to accelerate their recovery from illness, while 34.8% claimed that they only stopped taking antibiotics when they felt better. The findings hence indicate that most respondents had poor knowledge and attitudes concerning antibiotic use and resistance, which suggests the need for more community-based educational campaigns designed to improve the public’s knowledge and attitudes regarding antibiotics. To date, a wide range of viral and commonly self-resolving diseases, including the common cold, sinusitis, sore throat and bronchitis, have been treated with antibiotics worldwide despite the lack of efficacy of such a treatment modality. The use of antibiotics has also often been shown to not be supported by culture and sensitivity testing, nor is it always in line with the recommendations governing clinical practice , 2. AccoIn Malaysia, a poor level of knowledge regarding antibiotic use has previously been observed. Indeed, more than 80% of respondents in a study conducted in Putrajaya believed that antibiotics could cure viral infections , 8. The Staphylococcus aureus, other types of microorganisms, including Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia, have been reported to exhibit resistance to commonly used antibiotics  for Windows version 20.0  was used to analyse the data. The respondents’ demographic characteristics, along with their knowledge and attitudes concerning antibiotics use and resistance, were descriptively reported. The level of statistical significance was set at Of the 350 questionnaires distributed, 326 were completed (92.9% response rate). The majority of respondents were female (63.8%), above 40 years of age (72.1%), Malay (91.1%) and had received at least a secondary education (69.7%). One hundred and nine (33.4%) of them worked in the private sector, while 20% of respondents were civil servants and housewives, respectively. The majority of them had a monthly household income of below RM2,000 (62.6%) . The vasIn terms of their attitudes towards antibiotic use, 207 (63.7%) respondents admitted that they had taken antibiotics in order to accelerate their recovery from illness. Further, nearly half of all respondents reported either keeping antibiotics for future use or expecting doctors to prescribe antibiotics when they were sick. Their inadequate knowledge of antibiotic resistance was also evident, since 40.6% and 45.8% of respondents disagreed that bacteria and viruses, respectively, could develop resistance to antibiotics .To the best of our knowledge, this is the first study to assess the knowledge and attitudes of the public with regards to antibiotic use in Malaysia. The findings could, therefore, prove helpful for health-care professionals and policymakers alike in terms of better understanding the level of knowledge of the local community and their antibiotic-taking behaviours, which could subsequently help with the development of strategies for tackling the issue of inappropriate antibiotic use.Overall, the respondents demonstrated insufficient knowledge and poor attitudes concerning antibiotic use. The findings suggest that the majority of respondents in this study did not clearly understand the purpose of taking an antibiotic. The majority of them also believed that antibiotics could be used to treat all types of infections, including viral infections. Such findings are consistent with those of hospital-based studies conducted in Malaysia , 8, althIt is also noteworthy that more than half of all respondents in this study expected antibiotics to be prescribed by doctors if they were sick, even though the proportion of respondents who reported such expectations was relatively low when compared with proportions seen in previous studies , 8. It hIn is important to note that there were several limitations to this study. First, the convenience sampling method might have unavoidably caused selection bias during the recruitment of respondents. Furthermore, the generalisability of the findings is likely to be limited, since the study was conducted in only one state in Malaysia. Thus, a nationwide survey adopting a random sampling method is required in the future to provide further insight into the public’s awareness of antibiotic-related issues in Malaysia.Most respondents in this study were found to have inadequate knowledge and attitudes with respect to both antibiotic use and antibiotic resistance, which suggests that more educational campaigns are warranted in order to improve the public’s awareness and promote the rational use of antibiotics in Malaysia.


This paper assesses the mechanical and structural behavior of self-consolidating lightweight concrete (SCLWC) incorporating bloated shale aggregate (BSA). BSA was manufactured by expanding shale pellets of varying sizes by heating them up to a temperature of 1200 °C using natural gas as fuel in the rotary kiln. Fly ash (FA) and limestone powder (LSP) were used as supplementary cementing materials  for improved properties of the resulting concrete. The main parameters studied in this experimental study were compressive strength, elastic modulus, and microstructure. The fresh-state properties  showed adequate rheological behavior of SCLWC in comparison with self-consolidating normal weight concrete (SCNWC). There was meager (2–4%) compressive strength reduction of SCLWC. Lightweight aggregate tended to shift concrete behavior from ductile to brittle, causing reduced strain capacity and flexural toughness. FA and LSP addition significantly improved the strength and microstructure at all ages. The study is encouraging for the structural use of lightweight concrete, which could reduce the overall construction cost. Although the use of lightweight concrete (LWC) dates back to 3000 B.C , the mor3, and a minimum compressive strength of 17 MPa [3, as the aggregate occupies major volume in the concrete. Because of the porous structure of the lightweight aggregate (LWA), its absorption is very high, which leads to lower compressive strength. LWC has been produced by utilizing different sorts of lightweight aggregates (LWAs), such as expanded perlite [As per ACI 213R, structurally lightweight concrete has a unit weight ranging from 1350 to 1900 kg/mf 17 MPa ,16,17,18 perlite ,23,24, h perlite ,21,24,25 perlite , and exp perlite ,30,31,32However, the lightweight concrete mix design is different from conventional concrete. Using the conventional mix design will give rise to material segregation, as well as lower the strength by reducing the weight of aggregate. To avoid this problem, the mix design of self-consolidating concrete (SCC) was used for lightweight concrete . The SCCSelf-consolidating lightweight concrete (SCLWC) is a special type of concrete that has advantages of both LWC and self-consolidating concrete (SCC). Therefore, for a successful project, the use of lightweight concrete will provide an economical solution for various engineering applications. Using the mix design methodology based on SCC, it can be possible to design a SCLWC mixture with superior fresh and hardened properties ,38,39,40The fresh concrete behavior depends essentially on the workability of the concrete. Thus, it is imperative for concrete to have satisfactory fresh state properties that will affect the behavior of hardened concrete, including strength and durability ,41. The Research has been done on the self-consolidating lightweight concrete durability aspect, and it is concluded that SCLWC could achieve adequate flowability, better strength, and high durability ,44. SomeDue to the lack of research in the field of SCLWC, especially using artificial aggregate such as bloated shale, further investigations are needed. This study was carried out to develop a lightweight self-compacting shale aggregate concrete from locally manufactured material . The effect of fly ash (FA) and limestone powder (LSP) on the fresh state properties, compressive strength, homogeneity, porosity, the microstructure of concrete and density of SCLWC, were also investigated to explore the possibility of using LWA as a full replacement of NWA  for improving the various fresh and hardened characteristics of SCLWC. The cement was replaced by the SCMs, i.e., 10% of FA and 10% of LSP. Four formulations were selected. The first formulation was self-compacting normal weight concrete (SCNWC) using natural aggregate; the second formulation consisted of SCNWC and 10% of LSP and 10% FA as replacement of cement. The third formulation consisted of SCLWC, using bloated shale as coarse aggregate (SCLWC); the fourth formulation consisted of SCLWC plus 10% LSP and 10% FA as replacement of cement.3. The LSP used in this research was grayish in color and rich in CaCO3 (97.64%). It was made sure that both SCMs were free from lumps prior to mixing. The fine aggregate used was natural sand obtained from the Lawrencepur region, with a fine modulus of 2.24. Normal weight aggregate, comprising of crushed angular stone, was obtained from Margalla crush for the current research work. Coarse aggregates were used in two sizes—particle size of 2–8 mm and 8–16 mm. The maximum size for the coarse aggregate was 16 mm, conforming to ASTM C33, with a specific gravity of 2.47 (2–8 mm) and 2.44 (8–16 mm). Some of the physical properties obtained through lab tests are listed in The Ordinary Portland Cement (OPC) Type-I, conforming to ASTM C-150/C-150M-15, was selected as a binder for both SCNWC and SCLWC specimens. In this study, Glenium51, liquid superplasticizer (SP) conforming to ASTMC494, was used, while the fly ash was Class F with an apparent specific density of 2.68 g/cmThe objective of the study was to produce self-compacting yet lightweight concrete while using FA and LSP as SCM; therefore, corresponding attributes such as rheological behavior, unit weight, compressive strength, and microstructure of the resulting concrete was evaluated. Further, in order to ascertain the suitability of FA and LSP as SCM, corresponding material characterization techniques were employed. The SCC mix composition was designed by following the guidelines of EFNARC 2005 and ACI 237R-07. In this research, several trial concrete mixes using normal weight coarse aggregate and lightweight coarse aggregate with different superplasticizer dosages were prepared. From the numerous trial concrete mixes, suitable mixes satisfying the workability requirements for SCC  were selected. Four formulations for SCNWC and SCLWC were produced. For all the formulations, the water-cement ratio (w/c) was kept constant as 0.45. The mix proportion of various SCNWC and SCLWC mixes are shown in 3 were cast for each formulation of SCNWC and SCLWC as per guidelines of (BS EFNARC 12390-1). A total of 48 cylinders and 12 beams were cast for all four formulations. The casted samples were demolded after 24 hours and placed in the curing tank, which contained water at the controlled room temperature. The samples were cured in water for a specified period of time before testing. All of the materials were mixed in a pan mixer. First, coarse aggregates were put in the mixer, followed by sand and cement. One minute of dry mixing of the constituents at 180 rpm (slow rate) was done. Then, 50% of the water was added while mixing was continued at the same speed for another two minutes. The SP/viscosity enhancing agent (VEA) was then added along with the remaining water, and the mixing was further continued for three minutes at 360 rpm. The casting, curing, and testing was carried out as per EN 196-1. Concrete was cast into steel molds (three samples for each formulation for each testing age were cast). Cylinders of 150 mm diameter × 300 mm height and beams of 150 × 150 × 750 mmThe density/unit weight of fresh concrete was determined by measuring the weight of the container of known volume, fully filled with fresh concrete. The weight divided by the volume gives the density of fresh concrete.The air content in freshly mixed self-compacting concrete was measured by following the standard ASTM C231 and EFNARC 12350-7 guidelines. The pressure method was used to determine the air content in concrete.Slump flowV-FunnelL-BoxJ-RingSieve Stability Test (Segregation resistance)After the first mixing of SCC, the test was carried out in the following sequence:The density and water absorption of the concrete samples at the age of 28 days were determined as per the standard set forth by ASTM C642. The water absorption of the concrete specimen was measured as a percentage difference in the weight of concrete samples before and after the immersion in water at the age of 28 days.To assess the compressive strength of both SCNWC and SCLWC with the replacement of limestone powder and fly ash, a cylindrical specimen with a dimension of 150 mm × 300 mm cured at 3, 7, 14, and 28 days were tested in compression using a SHIMADZU universal testing machine (UTM) at a loading rate of 0.2 MPa per second as per ASTM C39.The microstructure was studied by scanning electron microscopy (SEM). For SEM, samples of both SCNWC and SCLWC after compression testing were selected to study the microstructure, morphology, and ITZ . The sample preparation for both the tests were done in the laboratory by placing the sample in acetone (to stop hydration) for 24 hours, after testing of the cylinders after 28 days.The results of the fresh properties of SCNWC and SCLWC are tabulated in All the values of slump flow and T50 are satisfactory, and within an acceptable range of SCNWC and SCLWC for a slump flow i.e., (650–800 mm) and the flow time (2–5 sec), respectively. Although it can be observed that slump flow increases when the lightweight aggregate is used, this may be due to its round shape and smooth surface. In the flow spread, SCNWC + FA + LSP has 3% more flow than SCNWC. This is because of the addition of FA and LSP, as well as higher SP content which enhanced the flow.Similarly, all the values of V-Funnel flow test are within the acceptable limit, i.e., 6–12 sec. The V-Funnel flow time indicates viscosity, which depends upon the type of aggregate used. The V-Funnel flow time for SCNWC + LSP + FA is greater than the others, and is 7% higher than the control mix, due to the SCMs, while the V-Funnel flow for SCLWC is 6% less than control mix, due to the lightweight and porous nature of the aggregate. 2/H1 shows the lesser ability of passing concrete through the L-Box. Also, the aggregate size effects the passing ability, as aggregate stuck between the steel rods reduces the passing ability. The replacement of normal aggregate with lightweight aggregate increases the passing ability and decreases the blocking tendency of the mix due to its round and regular shape and texture. Due to no interlocking of lightweight particles, the L-Box flow time also decreases with the use of lightweight aggregate. The control mix has a great height difference, which is probably due to a higher SP demand. SCLWC has 7% less value than SCNWC, which may be due to its porous nature and better flow.The L-Box test results are also within the permissible limits of SCNWC and SCLWC, i.e., 0.8–1.0. The lower value of HThe J-Ring flow and blocking index (Bj) for all four formulations are satisfactory and within the acceptable range of SCNWC and SCLWC. The J-Ring test measured the passing ability of concrete through steel rods. Literature suggests that the difference between slump flow and J-Ring values should be less than or equal to 50 mm for SCNWC and SCLWC for the better passing ability. It has been observed that J-Ring flow values increase with the use of lightweight aggregate, which may be due to better bonding and using a constant w/c ratio, i.e., 0.45. The J-Ring flow for SCLWC + LSP + FA is the highest and is 4.10% more than the control mix due to the round shape of the aggregate and the lightweight nature of the aggregate.The segregation resistance/sieve stability test values are also reasonable and within the tolerable limit of SCNWC and SCLWC, i.e., (5–15%). All the SCNWC and SCLWC show good segregation resistance. The sieve stability for SCLWC is 10.5% less than the control mix. This is because of the round shape of aggregate, which increases the workability of concrete.The compressive strength of SCLWC at 28 days was approximately the same as that of SCLWC. For typical lightweight concrete, the compressive strength decreases with a decrease in density. The compressive strength of SCNWC and SCLWC increased from 25 to 41 MPa and 23 to 40 MPa at 3-day and 28-day age, respectively. The mix design for SCLWC and SCNWC targets the same compressive strength. One can get SCLWC of the same quality as normal SCC if the appropriate amount of SP, VMA, and lower water binder (w/b) is used. Due to the replacement of cement with FA and LSP, the compressive strength increases. This is due to the pozzolanic and filler effect of SCMs. The w/c ratio played a significant role in enhancing the 28-day compressive strength. The homogeneity and unit weight of SCLWC also depends upon the w/c ratio. Strength improvement with SCMs is more pronounced in concretes with lightweight aggregate.Proper pelletizing of clay was found to be essential for the efficient production of high-quality lightweight bloated shale aggregate.3, which is about 35–40% less than SCNWC.The most efficient part was the density of SCLWC, which was about 40% less than SCNWC. This is due to the lightweight nature of the aggregate. The bulk density of SCLWC was about 1700–1750 kg/mThe 28-day compressive strength of SCLWC was 40 MPa, which was similar to SCNWC. The LWAC with fly ash as admixture increases the compressive strength of the concrete. Higher strength was achieved for a 10% cement replacement by fly ash. The optimum cement replacement by fly ash was around 10–20%. The 28-day compressive strength of SCLWC was quite comparable to SCNWC.The results show that modulus of elasticity of SCLWC was in an acceptable range. The E-value (modulus of elasticity) for the SCLWC was about 37% of that of SCNWC at the age of 28 days.Due to the acceptable workability with a medium water-to-cement ratio in SCLWC, there was no segregation or floating.Both SCMs (FA and LSP) enhanced the compressive strength up to a certain limit. The optimum replacement of FA and LSP would enhance the fresh and hardened response of the system.Based on the results and observations of an experimental investigation of the fresh and hardened properties of SCNWC and SCLWC, the following conclusions are drawn from this study:A comprehensive and thorough study was carried out to examine the behavior of lightweight bloated shale aggregate on the performance of self-consolidating concrete. Based on the rheological properties, strength, and microstructural attributes, it was concluded that bloated shale could be used in concrete for producing self-compacting as well as lightweight concrete, while the subsequent properties can be further enhanced by the addition of mineral admixtures like FA and LSP. There was no significant (2–4%) reduction in the compressive strength of SCLWC. Lightweight aggregates tended to shift concrete behavior from ductile to a brittle.Incorporating the blend of FA and LSP enhanced the compressive strength of both SCNWC and SCLWC. This is because of pozzolanic and filler effects.The addition of lightweight aggregate significantly improved the workability of SCLWC. This is due to the round shape of LWA. The slump flow of SCLWC increased (1.39%) compared with SCNWC, while the flow time of SCLWC decreased (10%) compared with SCNWC.The addition of LWA reduced the density of SCLWC up to 35%. This reduction in density could reduce the overall cost of the structure because of deadload reduction.The suitability of bloated shale aggregate for producing structurally lightweight self-compacting concrete and the positive effect of FA and LSP in improving the strength and microstructure are corroborated. This has significant applications in producing structures with greatly reduced deadloads, which would eventually reduce the overall cost of buildings due to reduced cross-sectional members and smaller foundations. However, due to the inherent brittleness of lightweight concrete, adequate techniques, like using short discontinuous fibers, should be adopted.


The anti-tumor activity was investigated using a clinically relevant in vitro tumor simulation model, which confirmed enhanced anti-tumorigenic property of liposomal Met over Met itself. To the authors’ knowledge, this is the first report of Met-loaded liposomes for improving the efficacy and therapeutic effect of Met against breast cancer. With the results obtained, it can be speculated that liposomal encapsulation of metformin offers a potentially promising and convenient approach for enhanced efficacy and bioavailability in breast cancer treatment.This study aimed at developing metformin hydrochloride (Met) encapsulated liposomal vesicles for enhanced therapeutic outcomes at reduced doses against breast cancer. Liposomal Met was prepared using thin-film hydration through various loading methods; passive loading, active loading, and drug-loaded lipid film. The drug-loaded film method exhibited maximum entrapment efficiency (~65%) as compared to active loading (~25%) and passive loading (~5%) prepared Met-loaded liposomes. The therapeutic efficacy of these optimized liposomes was evaluated for cellular uptake, cytotoxicity, inhibition of metastatic activity, and apoptosis-inducing activity. Results demonstrated significantly superior activity of positively charged liposomes resulting in reduced IC Breast cancer is the most common type of cancer found worldwide with high incidence rates making it a huge burden on global mortality . SpecifiMetformin hydrochloride (Met), an FDA-approved anti-diabetic drug, has been commonly prescribed as a first-line treatment of Type-II diabetes mellitus. Met was first discovered in 1922 by Werner and Bell . Met hasMet has been investigated clinically in breast cancer treatment as a combination therapy with other chemo-therapeutic drugs or adjuvants ,20,21. ILiposomes have been considered an effective carrier system for drugs requiring dose intensification due to their inherent ability to encapsulate drugs irrespective of their aqueous solubility . These kIn this study, we aim to develop lipid-based nanocarriers for the delivery of Met to enhance breast cancer treatment. To the authors’ knowledge, this is one of the first studies reported to explore the potential of liposomal encapsulated Met for breast cancer therapy. We hypothesize that entrapment of Met in liposomes will address the issues of a high dose requirement, along with poor tumor accumulation, which will result in the development of an effective therapy with improved therapeutic outcomes.sn-glycero-3-phosphocholine (DPPC) and cholesterol were purchased from Echelon Biosciences . 1,2-dioleoyl-3-trimethylammonium-propane  (DOTAP) was obtained from Avanti Polar Lipids . Metformin hydrochloride (Met) was purchased from Cayman Chemical Company . Phosphate Buffer saline (PBS) pH 7.5 and HPLC grade solvents—acetonitrile and water—were procured from Fisher Bioreagents . Various assay kits and other molecular biology grade reagents were obtained from various sources, which have been listed along with their respective methods.1,2-dipalmitoyl-2. 3--2,5-diphenyltetrazolium bromide (MTT) and crystal violet were procured from Fisher Bioreagents . Paraformaldehyde (PFA) was procured from Alfa Aesar . Vectashield Hardset mounting medium with DAPI was obtained from Vector Laboratories Inc. .Human breast cancer cell lines MDA-MB-231 (triple negative breast cancer (TNBC) cells) and MCF-7 (ER positive and PR positive breast cancer cells) were obtained from American Type Culture Collection (ATCC) . The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin , incubated at 37 °C under 5% COw/w). The active and passive loading method was followed as reported previously [® Extruder device  at 65 °C following 21 cycles to obtain small unilamellar vesicles (SUVs). Non-encapsulated drug was separated from Met-loaded SUVs by passing it through a Sephadex-G-25 PD-10 column (equilibrated with PBS pH 7.4). In case of active loading  of DPPC concentration was added to make the lipid constituents . The polydispersity index was evaluated to determine the homogeneity of particle size. The zeta potential was measured by determining the electrophoretic mobility of the liposomes under an electrical field using the Zetasizer nano ZS .TEM imaging of liposomes was performed in cryo mode to evaluate the morphology of vesicles. Approximately 10 μL of the sample solution was placed carefully on a lacey carbon-coated copper grids . The sample was blotted with a filter paper leaving a very thin film of sample on the grid. The sample was immediately shock-frozen and loaded into a 626-cryo-specimen holder . The samples were imaged using FEI Titan Halo 80-300 TEM operated at voltage of 100 kV. The images were recorded using 4000 × 4000 Ceta 16M CMOS camera  operated under low dose of electron voltage.v/v ratio at a flow rate of 0.7 mL/min and λmax of 237 nm on UPLC . The data were collected and analyzed using Empower 3.0 software .The UPLC analysis of Met was achieved using the ACQUITY UPLC™ HSS T3 C18 column . The mobile phase constituted of an isocratic elution of 0.1% orthophosphoric acid and acetonitrile in 50:50 g for 45 min to separate the drug from the lysed formulation. The obtained supernatant was subjected to UPLC analysis to determine the amount of drug entrapped.The entrapment efficiency was determined by comparing the amount of drug entrapped in the liposomes to the amount of drug added initially. Briefly, 20 µL of formulation was lysed using a mixture of methanol and acetonitrile and was centrifuged at 20,000 The % drug loading was evaluated by comparing the amount of drug entrapped in the liposomes to total amount of formulation (amount of lipids + amount of drug).In Vitro drug release studies from the liposomal formulations were carried out using dialysis cassettes . Briefly, the cassettes were hydrated previously in PBS (pH 7.4) and 300 µL of formulation was loaded in the cassette using a syringe. The cassettes were immersed in 150 mL of release media (1% tween-80 dissolved in PBS (pH 7.4) and were incubated at 37 ± 1 °C with stirring at 100 rpm. Samples were withdrawn at specific time intervals and release media was replenished with equal volume of fresh media maintained at 37 ± 1 °C. The samples were analyzed using UPLC method as described previously in The differential scanning calorimetry was performed using DSC 6000  connected with intra-cooler accessory. Approximately 3 mg of sample was sealed in aluminum pan and analyzed over a temperature range of 30 °C to 300 °C against an empty pan maintained as reference using the closed pan technique. The heating rate was 10 °C/min under a nitrogen purge maintained at a flow rate of 50 mL/min.λ of 1.5418 Å) obtained at a voltage of 40 kV and a current of 30 mA. The samples were analyzed after spreading as a thin film on a micro glass slide to obtain uniform spreading of the sample. Analysis was performed over 10° to 80° 2θ range at a scanning rate of 2° 2θ/min.The diffractometry was performed on an XRD-6000  using a graphite monochromator and copper source with Kα radiation , and 37 °C; and samples were withdrawn at specific time intervals. The samples were analyzed for size, zeta potential, and amount of drug entrapped.The formulations were also exposed to 40 ± 1 °C and 75 ± 2% relative humidity (RH) and were analyzed using DSC and PXRD to study effect of accelerated stability conditions.4 cells/chamber and were incubated overnight at 37 °C with 5% CO2. The cells were incubated with either plain coumarin or coumarin-loaded formulations at a concentration of 1 µg/mL for 3 h. After incubation, the cells were washed thrice with ice-cold PBS (pH 7.4) and fixed using 4% paraformaldehyde. The cells were again washed twice with PBS; the coverslip was removed from the chamber and mounted with VECTASHEILD hardset mountant containing DAPI . The mounting media was allowed to harden overnight at 4 °C, and slides were then imaged using a fluorescence microscope. .Cellular uptake of the liposomes was assessed using coumarin-loaded liposomes prepared using the same method by replacing Met with coumarin. For intracellular uptake, MCF-7 and MDA-MB-231 cells were seeded in 8-well chamber slides  at a density of 1.0 × 1050 of the formulations were evaluated in the cell lines mentioned above and were compared to Met. Briefly, human breast cancer cell lines MDA-MB-231 and MCF-7 were seeded in 96-well plates with a density of 2.5 × 103 cells/well and incubated overnight at 37 °C with 5% CO2 to allow surface attachment. Various concentrations of Met-loaded liposomal formulations were prepared by diluting liposomal stock with media. The cells were treated with different concentrations of plain Met and Met-loaded liposomes, and then incubated for 72 h. Subsequent to 72 h treatment, media was removed and MTT (1 mg/mL) was added, followed by 2 h of incubation. The formazan crystals formed during incubation were dissolved in DMSO after aspirating the MTT solution. Plates were shaken for 30 min on plate shaker and the optical absorption was measured at 570 nm using a TECAN plate reader . The cytotoxic potential of treatment groups  were determined by comparing cell viability against the non-treated cells used as control groups. The cytotoxicity data were transformed and normalized for calculation of IC50. IC50 values were calculated graphically by non-linear curve fitting module in Graph Pad prism 6.01 software .Cytotoxicity of the formulations were evaluated using an MTT assay as described previously . The IC5Cytotoxicity of blank DOTAP liposomes was evaluated on LLC-PK1 cells, porcine kidney tubular epithelial cells; HEK-293, human embryonic kidney cells; and MCF-7, breast cancer cells by following the same protocol.5 cells/well and were incubated overnight to adhere. After incubation, a scratch was made in the cell monolayer using a sterile 200 µL micropipette tip. The scratched cells were removed by washing twice with sterile PBS (pH 7.4) and then incubated with different concentrations of Met and Met-loaded liposomes for 24 h. (n = 3) time period. The scratch was imaged using an inverted microscope  and every image was examined and captured at the same location. The first image of the scratch captured before treatment was considered as time zero. The migration areas were observed as the gap created by the scratch, and the migration was calculated by measuring the distance (gap) between the two edges of scratch at each time interval using ImageJ software (Version 1.44).The cell migration ability of cells was assessed using the wound healing assay as described previously . Briefly2 cells per well and incubated overnight at 37 °C (5% CO2). The cells were then treated with varying concentrations of Met and Met-loaded liposomes for 48 h, followed by its removal and addition of fresh media. The cells were further cultured for 7 days by replacing the media every 2 days. After the incubation period, colonies were washed twice with ice-cold PBS (pH 7.4) and fixed using 4% paraformaldehyde for 10 min. The colonies were incubated with 0.2% crystal violet, followed by three rinses with tap water to remove excess dye. After air drying, the colony numbers were counted using OpenCFU colony counter software (Version 3.8) and the images were captured using a digital camera.To determine in vitro cell survival after treatment, clonogenic assay was performed as reported previously ,33. Both® caspase-3 assay kit  as previously reported [6 cells/plate were seeded in petri dishes followed by treatment for 6 h with Met, blank DO lipo, and Met DO lipo along with a control group maintained. After treatment, cells were washed twice with ice-cold PBS (pH 7.4) and were scraped to obtain a cell suspension. The obtained cell suspensions were centrifuged to get cell pellets, which were then lysed using lysis buffer. The lysed pellets were centrifuged to separate the cell debris and the supernatant obtained was transferred to 96-well plate. Equal volume of substrate was added to the supernatant and the fluorescence was measured at excitation/emission 342/441 nm. Control without enzyme was used to estimate the background florescence of substrate in each assay.Caspase enzymatic activity assay was performed to determine the apoptotic effect of Met and Met-loaded liposomes. The assay was performed using an EnzChekreported ,35. This6 cells/plate and incubated overnight. The following day, cells were treated with Met and Met DO lipo formulations and incubated for 72 h. After treatment, the cells were scraped to collect the pellets, washed 3 times with PBS, and subjected to lysis using 1% Triton® X-100  and 1% Halt™ Protease and Phosphatase Inhibitor Cocktail  in PBS followed by sonication for 90 min at 4 °C. Samples were centrifuged for 15 min at 4 °C at 13,000 g and the supernatants were collected. Cell lysate protein was quantified by the DC™ Protein Assay Kit . The samples were mixed with 2× Laemmli buffer  and 2-mercaptoethanol and denatured at 110 °C for 10 min.MDA-MB-231 cells were seeded in petri dish at a density of 1.0 × 10® TGX™ Precast Protein Gels (Bio-Rad) using a Bio-Rad PowerPacTM Basic Power Supply and transferred to Trans-Blot® Turbo™ Midi PVDF membranes (Bio-Rad) using a Bio-Rad Trans-BlotTM Turbo Transfer System.For Western blot analysis, 10 µg of protein was loaded and separated on 4–20% Mini-PROTEANThe membranes were blocked with 5% bovine serum albumin in PBS and probed with corresponding primary antibodies (1:1000 dilution) overnight at 4 °C. The following antibodies were used: β-actin ; p-mTOR ; and p-AMPK . Membranes were then incubated with the corresponding secondary HRP-conjugated antibodies ab6789  and ab6721   for 1 h at room temperature and were subjected to Western Bright chemiluminescence . Protein bands on the membranes were visualized using the Chemi mode by the Omega Lum™ G Imaging System . The band intensity was quantified using ImageJ software (Version 1.44).2 cells/well and were incubated overnight at 37 °C with 5% CO2. The cells were then subjected to two individual treatment regimens; (i) single dose and (ii) multiple dose. Briefly, single-dose treatment involved treating the cells only once and the media was replaced with treatment free-media every 3 days until 15 days. In the case of multiple doses, repeated drug exposure was performed every 3 days for 15 days. In both the doses, the replacement was done only for half of the media volume to avoid any disturbance to the developed spheroid or evade any chance of aspirating spheroid during complete media removal. The spheroids  were imaged at specified time intervals using inverted microscope . The volume of spheroids was calculated using ImageJ software (Version 1.44).It is well established that the tumor microenvironment plays an important role in its progression and development . This cat-test, one-way ANOVA and Tukey’s post-hoc multiple comparison. Statistical significance was considered at p < 0.05.The results are presented as mean ± SD (unless otherwise stated). Statistical analyses were performed with GraphPad Prism 6.01  using Student’s The liposomes were formulated using various techniques as shown in Tm) temperature of DPPC (~39–41 °C) [In Vitro release of drug from optimized liposomal formulations  was evaluated in PBS, pH 7.4, along with 1% Tween-80 at 37 °C. The data in 9–41 °C) , which ir2). As can be seen from results in 2. Thus, the in vitro release of Met from liposomal vesicles can be described by first-order release kinetics indicating a concentration dependent release. While the in vivo environment cannot be completely mimicked by in vitro studies, the in vitro drug release only provides an insight. Further studies are required to evaluate the in vivo release of Met from liposomal delivery system.The release data were further subjected to mathematical modeling using zero-order, first-order, Higuchi, and Korsmeyer-Peppas models to understand the release kinetics . In ordeThe calorimetric data obtained from DSC reveal a sharp endothermic peak representing the melting point of Met at 234 °C A. This cθ range as can be seen in θ values. The analysis of blank ND lipo and blank DO lipo also shows a few characteristic peaks of the liposomal formulation at a range of 25°–32° 2θ values, specifically at 27.25° and 31.7° 2θ values, which coincides with peaks of Met. In addition, other peaks for both blank formulations are found to be at 45.32°, 56.5°, 66.2°, and 75.2° 2θ values. The diffractogram of physical mixtures comprising of blank ND lipo/blank DO lipo and Met shows presence of Met characteristic peaks along over range of 12°–40° 2θ values and blank liposomal peaks in 30°–80° 2θ values, thus confirming the presence of both liposomal formulation as well as Met molecules. Upon analyzing Met-loaded liposomal formulations , Met characteristic peaks were absent over the entire range of 10°–40° 2θ values, however, the liposomal characteristic peaks were consistently found in both diffractograms at their respective 2θ values . Additionally, there is loss of liposomal peaks as reported by diminishing of peaks at 56.5°, 66.2°, and 75.2° 2θ values while these peaks are present in both the formulations exposed to 4 °C and 25 °C for 45 days. Similarly, upon analyzing DSC data of accelerated study (Tm) of DPPC (~41 °C) resulting in distorted integrity of liposomes and subsequent leaching out of drug from the vesicles. The in vitro stability studies performed on optimized liposomal formulations indicate that both formulations were found to be stable at 4 °C (storage temperature) as evaluated for a period of 45 days while stable at room temperature (~24 °C) for two weeks.Accelerated stability studies were conducted on Met ND lipo and Met DO lipo formulations stored at 40 °C/75% RH for period of 45 days. As shown in ed study D, it canLiposomal-mediated drug delivery has been shown to enhance intracellular delivery, and this method has been widely exploited in many anti-cancer treatments. The inclusion of cationic lipids (DOTAP) in liposomes is known to escalate the cellular uptake of liposomal vesicles compared to neutral or negatively charged liposomes . This enThe results show an increase in florescence due to enhanced cellular uptake of liposomal formulations as compared to coumarin. Amongst both liposomal formulations, DO lipo displayed a greater increase in florescence intensity in both cell lines as compared to ND lipo. Thus, it was shown that DO lipo possess a greater internalization potential in breast cancer cells.50 value of 5.3 ± 1.1 mM in MDA-MB-231 cells, which was reduced to 4.3 ± 0.8 mM by Met ND lipo. However, Met DO lipo produced a 4.5-fold reduction in IC50 value as compared to Met or Met ND lipo. The IC50 value for Met DO lipo was found to be 1.2 ± 0.5 mM (p < 0.01 against Met and Met ND lipo) in MDA-MB-231 cells, demonstrating enhanced anti-proliferative efficacy of Met upon encapsulation in DO lipo as compared to Met or Met ND lipo  as compared to 5.1 ± 0.5 mM for plain Met (p < 0.01) increased by ~3.9-fold via treatment of Met DO lipo, as the IC50 value decreased to 1.3 ± 1.0 mM. The reduction in IC50 value of Met DO lipo in both cell lines can be supported by the hypothesis that enhanced intracellular localization in cells, in agreement with cellular uptake studies, leads to increased cytotoxic potential. The obtained cytotoxicity results signify achievement of the study’s primary objective, which was to reduce the dose required for Met to exhibit anti-tumor activity in breast cancer cells.Investigation of the in vitro cytotoxic effect against MCF-7 cells, as shown in lain Met C. This r50 concentrations.Furthermore, evaluation of blank DO liposomes on normal kidney cells, LLC-PK1, as shown in 50 value (>20 mM) of Met in MDA-MB-231 after 24 h of treatment [p < 0.01)  B. Based The clonogenic assay is an in vitro tool used to evaluate the ability of a single cell to develop into a colony of cells (≥ 50 cells) by proliferating indefinitely. This is also known as clonal expansion ,49. It ip < 0.01) reduction in the number of colonies (53.5 ± 9.1% of control) as compared to Met (82.8 ± 13.0% of control); with control treated cells being 100% colonies . The increase in amount of caspase-3 activity has a linear relationship with apoptotic activity. p < 0.05) increase in caspase-3 enzymatic activity with Met DO lipo treatment as compared to Met or the control. With Met DO lipo (2.5 mM), a 1.5 ± 0.2-fold difference was observed as compared to the control, which is significantly higher than both plain Met and blank DO lipo (no observed difference). All comparisons were made as relative to the media control, which was normalized to 1.0  increase in spheroid volume. Therefore, Met DO lipo was shown to effectively display cytotoxic benefits against solid spheroids.The single-dose study involves drug treatment only once throughout the 15-day experimental period. This experimental design aids in evaluating the treatment effectiveness by simulating in vitro conditions. p < 0.01) difference of 18.5 ± 5.4-fold in spheroid volume as compared to control, whereas plain Met treatment (10 mM) did not show any significant difference from the control groups. Similar to MDA-MB-231 triple negative breast cancer (TNBC) cells, Met DO lipo promoted a significant difference in spheroid volumes as compared to Met and the control treated groups. Tumor growth suppression was continually observed until the end of the treatment period   B. These ®, a liposomal delivery of doxorubicin HCl, has extended the applicability of nanocarriers to a clinical setting [Met, an oral anti-hyperglycemic agent, is the most prescribed drug for type-2 diabetes treatment worldwide . Met has setting ,61. In tThe increased anticancer effect shown by liposomal carriers is usually attributed to a nanometer range size of liposomal vesicles (~100 nm), which is vital for increased tumoral accumulation and distribution of liposomal carriers due to an enhanced permeation and retention (EPR) effect . Resultsp < 0.05) superior upregulation of p-AMPK and inhibition of p-mTOR as compared to Met.Breast cancer is known to be aggressively invasive and metastatic, accounting for ~30% of early-stage cancers to invade locally in adjacent tissues and metastasize to organs such as the liver and lungs . TherefoThe in vitro assays performed may or may not necessarily translate well to in vivo experiments due to the complexity of in vivo tumor models and their uncontrolled cell growth, migration, and invasion causing angiogenesis and varied tumor microenvironment ,66. Ther50 value for both Met and nanoparticles loaded with Met was more than 5 mM. This increased dose requirement for killing 50% cells will directly impact the translational value of formulation to clinical setting.The delivery of Met for breast cancer treatment has been previously reported in only a few studies. Farajzadeh et al. co-encapsulated Met and curcumin in PLGA/PEG nanoparticles demonstrating the growth of breast cancer. While the study was successful in demonstrating the synergistic effect of Met and curcumin, it was reported that Met itself did not show any significant anti-tumor activity against breast cancer . In anot50 dose for breast cancer cells as compared to Met resulting in a dose reduction. In addition, supporting evidence from various in vitro studies demonstrate the superior anti-tumor activity of Met loaded liposomes for the treatment of breast cancer.To our knowledge, this is the first study of encapsulating Met in liposomal delivery carrier for the treatment of breast cancer. Our findings have proven that liposomal Met resulted in a 5-fold reduction in the ICThe present study acknowledges the idea of repositioning metformin for the treatment of breast cancer and delivering it by liposomal formulations. This idea is supported by extensive in vitro studies, where both Met and drug-loaded liposomes were evaluated for their therapeutic and mechanistic effect in breast cancer cells MDA-MB-231 and MCF-7. Furthermore, liposomes were modified to have a surface positive charge, as they provide improved internalization into the cells as compared to anionic liposomes. The improved internalization in turn provides enhanced therapeutic benefits as more drug is available to exert its chemotherapeutic action. This study lays solid groundwork to build on and pursue the development of Met loaded liposomes in pre-clinical and eventually a clinical setting. Our findings also suggest that encapsulation of Met in cationic lipid carriers addresses and overcomes certain limitations of Met like significant reduction of dose, along with establishing solid chemotherapeutic activity of the drug. In the future, we are planning to perform surface modification of liposomes, which can be exploited for development of targeted liposomes with enhanced specificity and thus better therapeutic efficacy. Furthermore, we will evaluate the efficacy of metformin encapsulated liposomes in A549 xenograft mouse model. To the best of our knowledge, this study is one of a kind and we can say with confidence that liposomal delivery of Met has tremendous potential to evolve as a standalone therapy for breast cancer treatment.


IgG4-related disease (IgG4-RD) is a newly recognized fibroinflammatory condition. The kidney is one of the organs commonly affected by IgG4-RD. Tubulointerstitial nephritis (TIN) is the main feature, and membranous nephropathy (MN) has also been described frequently. In MN, polyclonal immunoglobulins and complements are deposited in granular form along the glomerular basement membranes (GBMs). Unusual cases of monoclonal immunoglobulin deposition disease (MIDD) associated with membranous features have been reported. MIDD is morphologically similar to MN but contains immunoglobulins considered to be derived from single B-cell clone.We describe a 65-year-old man who was referred to our hospital because of hyperproteinaemia, eosinophilia, anaemia, and proteinuria. A renal biopsy demonstrated infiltration of plasma cells and eosinophils in the interstitium, and the ratio of IgG4-positive plasma cells to IgG-positive plasma cells was 55%. The patient was diagnosed as having IgG4-related TIN. Periodic acid methenamine silver staining under light microscopy revealed a bubbling appearance and spike formation in the GBM. On immunofluorescence, the expression of IgG and complements was negative; however, IgA was positively expressed in a granular pattern along the GBM. An IgA subclass analysis revealed a significant deposition of IgA1-lambda (IgA1-λ). Electron microscopy revealed irregular and small non-organized and non-Randall-type granular electron-dense deposits in the GBM that were shaped like snow leopard spots.After corticosteroid therapy was initiated, the patient’s eosinophilia remarkably improved and his serum creatinine, IgG, and IgG4 levels decreased to within the normal ranges. However, massive proteinuria persisted. To our knowledge, this is the first reported case of IgG4-related TIN associated with IgA1-λ-type MIDD with membranous features. Membranous nephropathy (MN) is defined as glomerulonephritis with a bubbling appearance and formation of spikes in the glomerular basement membrane (GBM) on light microscopy. On immunofluorescence, polyclonal immunoglobulins and complements are deposited in granular form along the glomerular basement membranes (GBMs). Only a few cases of non-organized and non-Randall-type monoclonal immunoglobulin deposition disease (MIDD) associated with membranous features have been reported in the literature , 2. MIDDOn the other hand, IgG4-related disease (IgG4-RD) is recognized as a new chronic inflammatory disease characterized by elevated serum IgG4 levels, mass or tissue infiltration rich in IgG4-positive plasma cells, and storiform fibrosis .The kidney is one of the organs commonly affected by IgG4-RD, and tubulointerstitial nephritis (TIN) with infiltration of numerous IgG4-positive plasma cells is the most common type of kidney lesion, and MN has been occasionally accompanied .Here, we describe a patient with IgG4-related kidney disease who developed massive proteinuria due to membranous features associated with the deposition of IgA1-lambda (IgA1-λ) along the glomerular capillary walls.A 65-year-old man was referred to our hospital because of hyperproteinaemia, eosinophilia, anaemia, and proteinuria after a 2-week history of slight fever, fatigue, and malaise.On admission, his mental status was normal, body temperature was 36.5 °C, pulse was 73 bpm and regular, and blood pressure was 118/75 mmHg. A physical examination revealed eruption and oedema in his lower extremities; however, no abnormal signs were observed in the lungs, heart, or abdomen. His lymph node and thyroid gland were not swollen. The laboratory findings on admission are summarized in Table A renal biopsy was performed. Light microscopy revealed 3 global scleroses and no crescent within the 9 glomeruli. In the interstitium, severe infiltration of plasma cells and eosinophils, with storiform fibrosis and infiltration of numerous IgG4-positive plasma cells (IgG4-/IgG-positive plasma cell ratio > 50%) were observed Fig. a, b. In On immunofluorescence, the expression of IgG and complements was negative; however, IgA was positively expressed in a granular pattern along the GBM. An IgA subclass analysis revealed significant monoclonal deposition of IgA1-λ Fig. . We cut Electron microscopy revealed GBM thickening, widespread podocyte effacement, and irregular and small non-organized and non-Randall-type granular electron-dense deposits in the GBM Fig.  that werIgG4-RD is a recently recognized systemic immune-mediated disease, typically characterized by mass-forming fibroinflammatory lesions. IgG4-RD is well recognized in the form of TIN (IgG4-related TIN), which may present marked infiltration of lymphocytes and IgG4-positive plasma cells into the renal interstitium with interstitial fibrosis and minor glomerular lesions , 8.However, several case reports have described IgG4-RD showing glomerular changes, of which MN is the most common. MN is present in approximately 7% of patients with IgG4-related TIN .In MN associated with IgG4-RD, positive deposition of IgG4 without light-chain monoclonality in the GBM has been observed, either predominantly or together with other IgG subclasses , 9–12. MAlexander et al. reported that the glomerular staining tests for anti-PLA2R in 8 biopsies of MN in the setting of IgG4-RD were all negative. Thus, they argued that IgG4-related MN is a secondary MN . In our In the present case, although serum IgG and IgG4 levels were elevated beyond the physiological level, routine techniques failed to detect an underlying B-cell disorder, with negative serum and urine protein immunoelectrophoresis and a normal free light chain (FLC) ratio. In Brief, the present case was compatible with IgG4-related kidney disease, however bone marrow examinations were not performed, so the possibility of hematological disorders could not be completely denied.According to the systematic review of 37 cases of IgG4-related kidney disease , the serWe did not examine viral antibodies such as Epstein-Barr virus and human herpesvirus-8; however, liver function was normal and atypical lymphocytes were not observed. Splenomegaly was not observed and the lymph nodes were not swollen; thus, it is unlikely that the patient had those viral infections.CT scan did not reveal splenomegaly and lymphadenopathy. Moreover, there were no abnormal findings in the pancreas, salivary gland, and parotid.IgG4-RD can be complicated with Sjogren syndrome or multicentric Castleman disease (MCD), and some cases are difficult to differentially diagnose.The underlying premise is that the histological features  associated with IgG4-RD are highly specific when viewed in conjunction with IgG4 staining .The presence of an atopic history is characteristic of IgG4-RD, whereas intense inflammation and polyclonal hyperimmunoglobulinaemia are characteristics of MCD. Moreover, IgA and C-reactive protein (CRP) levels are significantly higher in MCD than in IgG4-RD .In the present case, the IgA level was not elevated and the CRP level was 1.6 mg/dL. Moreover, also consistent with IgG4-RD, the IgG, IgG4, and complement levels promptly returned to the normal ranges after corticosteroid use.In 2004, Nasr et al. first described proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID), a newly characterized form of glomerulonephritis related to monoclonal IgG deposition that could not be assigned to any of the established categories of glomerular diseases. The glomerular deposits appeared primarily granular, resembling ordinary immune complex deposits but contained immunoglobulins considered to be derived from single B cell clone. They classified the disease, according to the pattern of proliferative changes on light microscopy, as membranous proliferative glomerulonephritis, endocapillary proliferative glomerulonephritis, mesangial proliferative glomerulonephritis, and membranous glomerulonephritis .Recently, Komatsuda et al. reported 3 patients with PGNMID with membranous features (2 IgG3-κ and 1 IgG1-κ) [Eighteen cases of non-Randall MIDD with membranous features have been reported Table , and IgGOnly one case of IgA1-λ-type monoclonal deposition with membranous features, which was associated with hepatitis C virus (HCV) infection and rectal cancer, has been reported . The eleAnother relevant interesting case was IgG4-RD associated with non-Randall MIDD with membranous features . It was In IgG4-RD, the markedly high IgG4 levels may simply be a reflection of the response to some primary inflammatory stimuli . T cellsIn PGNMID, the pathogenesis remains elusive. Because up to two-thirds of patients with PGNMID have no detectable M protein even after a long follow-up, Nasr et al. proposed that this unique glomerulonephritis may arise in the course of normal immune responses in these patients. During an immune response, 1 or more clones of B cells may proliferate and produce monoclonal IgG molecules with the ability to self-aggregate and rapidly deposit in the glomeruli .In the present case, paraprotein in blood and urine was not detected by immunoelectrophoresis, and even in serum FLC assay, no deviation of the FLC ratio could be found. Therefore, 1 or more clones of B cells may have proliferated and produced monoclonal IgA molecules similarly to PGNMID.Indeed, it is difficult to find a common mechanism between the 2 rare diseases, and they would not have a specific relationship.IgG4-RD cause disorders of the basis of the immune response such as regulatory T-cell, and tend to cause other autoimmune diseases such as membranous nephropathy. However, it is unknown whether monoclonal immunoglobulin deposition increases as in the present case.In summary, non-Randall MIDD with membranous features is rather rare, and only 1 case of monoclonal deposition from the IgA variant has been reported. To our knowledge, this is the first reported case of IgA1-lambda-type non-Randall monoclonal immunoglobulin deposition disease associated with membranous features in a patient with IgG4-related TIN.


Mesenchymal epithelial transition (MET) amplification has been regarded as a potential treatment target, the knowledge of its prevalence and prognostic importance is crucial. However, its clinical pathologic characteristics are not well known in esophageal squamous cell carcinoma (ESCC).Since MET gene status with fluorescence in situ hybridization (FISH) assay in 495 ESCC cases using tissue microarrays. Prognostic significance as well as correlations with various clinicopathological parameters was evaluated.We investigated MET FISH positive, including 5 cases (1%) with true gene amplification. There were no statistically significant associations between MET FISH-positivity and clinicopathologic characteristics. A significantly poorer prognosis was observed in 28 patients with MET FISH-positivity . Multivariate analysis revealed MET FISH-positivity was an independent prognostic factor for DFS  and OS . MET FISH-positivity was associated with DFS (P = 0.022 and 0.020) and OS (P = 0.046 and 0.024) both in stage I-II ESCC and in stage III-IVa ESCC. No statistical significance  was detected between stage I-II ESCC with MET FISH-positivity and stage III-IVa ESCC with FISH-negativity.Among 495 patients, 28 (5.7%) cases were MET gene copy number is an independent prognostic factor in ESCC, and ESCC might have potentially been up-staged by increased MET gene copy number. The results indicate that increased MET gene copy number is a very promising parameter, in clinical therapy and follow-up plans.Increased  Esophageal cancer (EC) is the ninth most common cancer and the sixth leading causes of cancer death globally . In ChinMesenchymal epithelial transition (MET) gene was first identified in 1984 in an osteosarcoma immortalized cell line [MET gene amplification, Met and/or HGF protein overexpression, or, rarely, domain-specific sequence mutations [utations , 11.MET amplification were extraordinarily susceptible to the selective MET tyrosine kinase inhibitor (TKI) [MET amplification was responsible for approximately 20% of the acquired resistance to epidermal growth factor receptor (EGFR) TKI treatment in lung adenocarcinomas [MET gene amplification in different tumors. MET gene amplification is identified in 2–5% of gastric cancers [MET amplification is thought to be associated with metastasis and poorer outcome in gastric [MET amplification, only few small studies evaluated its gene status in ESCC [Recent studies found different tumors with or (TKI) –14, and rcinomas , 16. The cancers , 18, 2–4 cancers , 1–8% of cancers , 13, 19, gastric , lung [2 gastric  and colo gastric . Despite in ESCC .MET gene copy status in a large cohort of ESCC. In addition, we sought to analyze its clinicopathological features and prognostic value.Therefore, in this study, we aimed to evaluate This retrospective study was conducted in a cohort of 495 treatment-naive ESCC patients who underwent esophagectomy at Zhongshan Hospital between January 2007 and December 2010. Patients were included in the study if the following criterias were met: (1) underwent primary resection, (2) with no prior treatment, and (3) with available complete medical records. Patients were excluded from the study if they had disease progression within three months after surgery. Clinical and histopathological data, including sex, age, smoking status, tumor size, tumor location, differentiation, vessel or nerve invasion, pT stage, and pN stage, was obtained from the patients’ medical and pathological records. The pathologic tumor-node-metastasis (pTNM) stage was performed according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. All patients were followed up every 3–6 months after tumor resection, and patients underwent follow-up examinations to identify possible tumor recurrence. Exam methods included endoscopy, computed tomography, magnetic resonance imaging, abdominal ultrasonography, and measurement of serum tumor marker levels.Written informed consent was obtained from all patients, and the study was approved by the ethical committee of the Zhongshan Hospital, in accordance with the ethical standards of the World Medical Association Declaration of Helsinki.TMA construction was performed as previously described . BrieflyMET gene status was evaluated using a commercially available FISH assay [MET Spectrum Red FISH Probe  and control Vysis CEP7 Centromere Spectrum Green Probe (Abbott Molecular) on 4 μm-thick TMA sections. The signals of each sample were counted in at least 50 well-defined nuclei using a fluorescence microscope  equipped with a Microscope Digital Camera . An average MET gene copy number ≥ 5 and a MET/CEP7 ratio ≥ 2 (true MET amplification) were regarded as MET FISH positive [SH assay , with Vypositive .MET status and clinicopathological characteristics. The primary and secondary endpoints were cancer-related death and recurrence/metastasis. Disease free survival (DFS) and overall survival (OS) were defined as periods from the date of surgical treatment until the date of disease progression  and the date of cancer-specific survival (event: cancer-related death), respectively. The Kaplan–Meier analysis with the log-rank test was performed to determine the prognostic significance for DFS and OS. The univariate and multivariate Cox proportional hazard regression analysis was used to identify the independent prognostic factors. The hazard ratio (HR) and its 95% confidence interval (CI) were assessed for each factor.The Chi square and Fisher’s exact tests were used to evaluate the association between P-values < 0.05 were considered to be statistically significant.Statistical analysis was carried out using SPSS 21.0 statistical software . All tests were two sided, and The patients’ clinicopathological characteristics are summarized in Table MET FISH positive , including 5 cases (1%) with true gene amplification (5 cases with MET: CEP7 ratio of ≥2.0)  cases were  5 cases % with tr 5 cases % with trMET FISH-positivity and clinical pathologic characteristics are listed in Table MET FISH-positivity was significantly associated with DFS  and OS . However, there were no statistically significant difference in sex (P = 0.638), age (P = 0.932), smoking (P = 0.919), tumor size (P = 0.434), tumor location (P = 0.941), differentiation (P = 0.957), vessel invasion (P = 0.194) and nerve invasion (P = 0.706), pT stage (P = 0.883), pN stage (P = 0.088), and clinical stage (P = 0.351).The correlations between The median follow-up time was 35.0 months (range 3–102 months). Two hundred sixty-nine patients (54.3%) had disease progression and two hundred sixty-one patients (52.7%) had died from esophageal cancer during the follow-up. The 5-year DFS and disease-specific OS rates for all patients were 44.1 and 44.4%, respectively.MET FISH-positivity, showing a median DFS or OS of 17.0 or 26.0 months, respectively, compared with 36.0 or 42.0 months in the group with MET FISH-negativity (P < 0.001 or P = 0.001). The 5-year DFS (17.9%) and OS (17.8%) rates for patients with MET FISH-positivity were significantly lower than the corresponding rates (45.7 and 46.0%) for patients with MET FISH-negativity. Univariate analysis indicated that MET FISH-positivity, differentiation, vessel invasion, nerve invasion and clinical stage had significant impacts on DFS, and MET FISH positive, vessel invasion, nerve invasion and clinical stage had significant impacts on OS (both P < 0.05). Multivariate analysis revealed MET FISH-positivity was an independent prognostic factor for DFS  and OS . Clinical stage was also found to be an independent prognostic factor for DFS and OS  had disease progression and one hundred one patients (37.1%) had died from esophageal cancer during the follow-up. In stage III-IVa patients, one hundred sixty-five patients (74.0%) had disease progression and one hundred sixty patients (71.7%) had died from esophageal cancer during the follow-up.P < 0.001). The 5-year DFS (23.7%) and OS (24.7%) rates for stage III-IVa patients, were significantly lower than the corresponding rates (60.8 and 60.4%) for stage I-II patients.Figure MET FISH-positivity was associated with DFS (P = 0.022) and OS (P = 0.046) in patients with stage I-II ESCC  did not reach the median survival. MET FISH-positivity was also associated with DFS (P = 0.020) and OS (P = 0.024) in patients with stage III-IVa ESCC (n = 223) , with a median DFS or OS of 20.0 or 25.0 months, respectively. What’s more, no statistical significance  was detected between stage I-II ESCC with MET FISH-positivity and stage III-IVa ESCC with FISH-negativity.SCC Fig. a and b. MET gene status was detected in 495 ESCC patients by FISH method. FISH analysis is a semiquantitative method that can be performed with two probes for determination of the number of signals for a target gene and for the centromere of the corresponding chromosome [In our study, romosome . Compariromosome –30.MET FISH positive rate was 5.7% and gene amplification rate was 1% using Cappuzzo criteria, which was consistent with the somatic copy number alteration data generated by The Cancer Genome Atlas Research Network [MET amplification is relatively low. MET genetic alterations were detected using increasing gene copy number. The increasing gene copy number can result from mainly two genetic mechanisms [MET gene copy number per cell higher than 5 and higher than 6 was similar, and worse than the other four groups with a mean copy number lower than 5 in NSCLC [MET /CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0 copies) has been widely accepted and used in other tumors, such as NSCLC [Our findings showed  Network –33, the chanisms : 1) polyin NSCLC . Gradualas NSCLC , 35, gasas NSCLC , 36, gasas NSCLC , tonsillas NSCLC , and mesas NSCLC .MET amplification and were treated with a MET inhibitor, experienced tumor shrinkage in 2011 [MET gene status has gained considerable interest in solid tumors [MET gene copy number has an established prognostic role in NSCLC, gastric cancer and gastroesophageal adenocarcinoma patients [MET FISH-positivity had a significantly worse DFS and OS than 467 individuals with FISH-negativity. Moreover, MET FISH-positivity was an independent prognostic factor for both DFS and OS, further indicating increased MET gene copy number is a negative prognostic factor in ESCC.Since Lennerz etal has demonstrated that 2% of patients (10/489) with esophagogastric adenocarcinoma, who harbored  in 2011 , MET gend tumors , 14. Incpatients , 39, 40.patients , 41, andMET amplification did not have an impact on prognosis in early TNM stage (stage I or II), unlike in advanced TNM stage (stage III or IV) [MET FISH-positivity has an impact on prognosis both in early TNM stage (stage I-II) and in advanced TNM stage (stage III-IVa). And there was no prognostic difference between stage I-II ESCC with MET FISH-positivity and stage III-IVa ESCC with MET-negativity. The findings indicate that MET gene alteration could be acquired during the early phase of ESCC development, and exaggerated the cancer progression [Subgroup analyses according to the disease stage were also conducted in our study. Lee et al. reported in gastric cancer, I or IV) . Our resgression .MET gene copy status using FISH, in a large series of ESCC. Our data show that increased MET gene copy number is an independent prognostic factor in surgically ESCC, and we firstly find that ESCC might have potentially been up-staged by increased MET gene copy number, which indicates increased MET gene copy number is a very promising parameter, in clinical therapy and follow-up plans.We investigated


Staphylococcus epidermidis strain showing minimal sensitivity to methicillin. Doxycycline to which the strain did not show resistance in complex with SWCNTs provides only minor increase in efficacy, whereas the SWCNTs/methicillin complex yields up to 40-fold efficacy enhancement over antibiotics alone, suggesting that SWCNT-assisted delivery may circumvent antibiotic resistance in that bacterial strain. At the same time SWCNT/antibiotic formulations appear to be less toxic to mammalian cells than antibiotics alone suggesting that nanomaterial platforms may not restrict potential biomedical applications. The improvement in antibacterial performance with SWCNT delivery is tested via 3 independent assays—colony count, MIC  turbidity and disk diffusion, with the statistical significance of the latter verified by ANOVA and Dunnett’s method. The potential mechanism of action is attributed to SWCNT interactions with bacterial cell wall and adherence to the membrane, as substantial association of SWCNT with bacteria is observed—the near-infrared fluorescence microscopy of treated bacteria shows localization of SWCNT fluorescence in bacterial clusters, scanning electron microscopy verifies SWCNT association with bacterial surface, whereas transmission electron microscopy shows individual SWCNT penetration into bacterial cell wall. This work characterizes SWCNTs as novel advantageous antibiotic delivery/imaging agents having the potential to address antibiotic resistance.Although conventional antibiotics have evolved as a staple of modern medicine, increasing antibiotic resistance and the lack of antibiotic efficacy against new bacterial threats is becoming a major medical threat. In this work, we employ single-walled carbon nanotubes (SWCNTs) known to deliver and track therapeutics in mammalian cells via intrinsic near-infrared fluorescence as carriers enhancing antibacterial delivery of doxycycline and methicillin. SWCNTs dispersed in water by antibiotics without the use of toxic bile salt surfactants facilitate efficacy enhancement for both antibiotics against  Staphylococcus aureus (MRSA) ) suspensions.−1 for SWCNTs/doxycycline and 0.0134 (µg/mL)−1 for SWCNTs/methicillin). We further used those to assess the concentration of SWCNTs in centrifuged suspensions.The concentration of all SWCNT suspensions was characterized via absorption spectroscopy. Using standard calibration curve constructed from absorptions of unfiltered SWCNT/antibiotic fractions with known SWCNT amounts we have experimentally derived extinction coefficients at 632 nm for SWCNT dispersed with both drugs (0.015 (µg/mL)The concentration of antibiotics in the suspensions of antibiotic/SWCNT hybrids was assessed via deconvoluting absorption spectra of those into components for SWCNTs and antibiotics. SWCNTs/DSPE-PEG 5000 spectra were used as an assessment for SWCNT component and antibiotic standards at known concentrations were used as reference component for antibiotics. This calculation showed w/w ratios of 1:4 for SWCNT/methicillin and 1:5 for SWCNT/doxycycline in stock SWCNT suspensions that were further used throughout this work.Near-infrared fluorescence of antibiotic/SWCNT suspensions was collected via Nanofluorescence NS2 Nanospecralyzer spectrometer with 637 nm laser excitation for SWCNT antibiotic suspensions after preparation and after a 24 h treatment period. Minimal agitation is applied to ensure no loose aggregation.S. epidermidis (VWR 470176-542) broth of McFarland 0.5 standard (absorption of 0.08 to 0.1) was created with stationary phase culture using Mueller Hilton Broth. This standard stabilized the cell count at an approximate 1 × 108 CFU/mL [8 CFU/mL . DilutioWe tested two different dosages of the antibacterial solutions to increase the breadth and reliability of data. Blank sensitivity discs were loaded with 10 µL and 20 µL (based on respective dosage) of stock suspensions and placed onto the surface of the agar using sterile forceps. Discs were impregnated with the test solution dropwise. Five discs were evenly placed equidistant from one another. Before tilting over the Petri dishes, discs were left to dry and gently pressed down to ensure attachment to agar. Once all Petri dishes are prepared, they were turned upside down to prevent surface condensation. Petri dishes are incubated for 24 h at 37 °C, then the zones of inhibition were measured with the inclusion of disk diameter in the measurements. The 24 h time point is chosen based on previous work  suggestiS. epidermidis broth (McFarland 0.5 standard), 100 µL was placed in the center of the agar plate. 100 µL of the respective antibacterial stock solution is added to the center. Bacteria were spread through the Petri dish and the plates were further incubated for 24 h at 37 °C. Pictures of the plates were uploaded onto the OpenCFU software to count the number of colonies grown on the plate. Two plates were prepared for each antibacterial treatment with corresponding controls.Using A serial dilution (using the factor of 2) of antibacterial solutions was conducted in 12-well plates starting with 200 µL of antibacterial solution placed in first well. 850 µL of broth and 50 µL of bacteria in broth were added to each well plate. The solubility of doxycycline and methicillin was 50 and 0.31 mg/mL, respectively according to the manufacturer  information. Thus, the antibiotic stock complexed with SWCNTs for all aqueous experiments was diluted to lower concentrations: for doxycycline: 1, 0.5, 0.25, 0.125 and 0 mg/mL and for methicillin are 1.25, 0.625, 0.313, 0.106 and 0 mg/mL. Plates were then incubated at 37 °C for 24 h. The solutions were transferred to cuvettes and their MIC turbidity was measured using a Cary 500 spectrophotometer with the broth used as a baseline. Two wells were prepared for each concentration.An MTT -2,5-diphenyltetrazolium bromide)-based cytotoxicity assay was conducted for 4 samples—doxycycline, SWCNTs/doxycycline, methicillin and SWCNTs/methicillin. Each sample was prepared via serial dilutions at the testing concentrations ranging from 0 to 3.5 µg/mL for doxycycline and SWCNTs dispersed with doxycycline and 0 to 0.25 µg/mL for methicillin and SWCNTs dispersed with methicillin. The absorbance was measured using the FLUOstar Omega microplate reader and was analyzed using Omega software to yield cell viability levels.We utilized InGaAs near-IR (NIR) camera coupled to hyperspectral fluorescence filter (Photon etc.) to perform fluorescence microscopy of SWCNTs imaged in bacterial cells 24 h after introducing them to bacterial culture. The sample was excited with 637 nm diode laser excitation at 130 mW output power. SWCNTS showed up in the NIR broadband (900–1450 nm) images as bright fluorescent objects. Non-treatment control images were taken for each antibiotic target ensuring no emission in the near-IR. Scanning Electron Microscope  was used at 5 kV to image bacterial cells. Samples were prepared by depositing bacteria from the culture onto conductive carbon tape via drop-casting of ~100 µL of SWCNT/antibiotic-treated bacteria in the media. SEM allowed imaging of the outer surface of bacterial cells and extracellular SWCNTs. Transmission Electron Microscopy  was further utilized to assess the incorporation of SWCNTs into bacteria and the coating of SWCNTs with antibiotic only. Samples for TEM were prepared by drying ~10 µL of either SWCNT/antibiotic suspensions or SWCNT/antibiotic-treated bacterial culture on the carbon-coated 200-mesh copper grid under ambient conditions.S. epidermis strain used in this work shows no/low response to methicillin suggesting some resistance and thus is intended as a control for the studies.Noncovalently complexed SWCNT/antibiotic hybrids were prepared in this work for the first time. Antibiotics are utilized both as a payload and as a surfactant for SWCNTs providing aqueous dispersions stable prior to treatment and over a treatment period showing no substantial SWCNT fluorescence intensity decrease or spectral broadening . Some lo−1 and 0.0134 (µg/mL)−1 respectively. Antibiotic concentration in the final dispersion is assessed by deconvoluting spectra of antibiotic/SWCNT hybrids via presenting those as a superposition of those of SWCNT and antibiotic alone at known concentrations, which resulted in the w/w ratios of ~1:40 for SWCNT/methicillin and ~1:50 and SWCNT/doxycycline aqueous suspensions.As SWCNTs alone are insoluble in water, their successful dispersion helps verify their complexation with antibiotics. Characteristic absorption spectra of SWCNTs and antibiotics were used to assess concentrations of those upon complexation. As absorption of both antibiotics is negligible in the visible, the value of absorption at 632 nm is used to determine the amount of SWCNTs . A calibWe use three different antibacterial sensitivity assays to verify the efficacy of SWCNT/antibiotic dispersions. The disk diffusion assay, colony formation assay, and MIC turbidity assay confirm our findings through three different procedures.The antibacterial effects of pure antibiotic solutions are first compared to non-treatment control and vehicle control provided by SWCNT/PEG formulations . Here DSS. epidermis are known to show antibiotic resistance and several are specifically resistant to methicillin [In this formulation, SWCNTs show little to no antibacterial effect, which is supportive of their sole role as drug delivery/imaging agents providing no interference with the antibacterial efficacy of the payload. Among the antibiotics, doxycycline is significantly more effective at both 0.2 and 0.4 mg doses, whereas methicillin shows no antibiotic activity with inhibition levels same as non-treatment control . Since shicillin . The comS. epidermidis in this test initially shows no response to methicillin, the appearance of significant antibacterial response suggests that SWCNT delivery may bypass such antibiotic resistance. While there are a number of theories regarding the mechanisms of internalization of carbon nanotubes into prokaryotic cells, [Concentrations of antibiotics in complexes with SWCNTs were further chosen to mimic those of unformulated antibiotics. SWCNT/antibiotic dispersions show substantial improvement in the inhibition response for both antibiotics. SWCNT/Doxycycline complexes become slightly more effective than Doxycycline alone (8% improvement), whereas SWCNT/Methicillin hybrids exhibits a drastic improvement—an increase in efficacy from no observable bacterial inhibition to the 50% of the inhibition response of doxycycline. Because c cells, ,55 many c cells, ,57,58 anc cells, . These mc cells, ,60. ThusThe statistical analysis performed in this work confirms the significance of the observed results for diffusion discs performed at different amounts of SWCNT/antibiotic complex loaded onto discs. Data analysis is performed using JMP software utilizing ANOVA  to provide statistical information to understand the predominant effects and significance of differences in data. In performing an ANOVA on a dataset, we make a null hypothesis, stating that the average means of the various treatments are the same. When the p-value of the analysis is lower than the confidence level chosen, the hypothesis is proven false and a significant difference and variation is detected .The dataset is analyzed using a comparison of means function by assigning a mean of the control group and comparing it to the means of each of the treatment groups. The degree of overlap of the circles in the graphic represents their significant similarity or difference, whereas the size is proportional to the corresponding variance .For comparison of antibiotic effects of SWCNT/Doxycycline to the free antibiotic a,b, the S. epidermis. The colony count for methicillin alone is similar to the control, as expected, whereas that for SWCNTs/methicillin is suppressed 40-fold (4000% improvement) showing significant observable  improvement in bacterial colony inhibition for the SWCNT complex with methicillin, to which S. epidermidis initially shows resistant behavior in our assays. These results confirmed by statistically significant findings from disc diffusion and a general trend provided by the MIC turbidity assays suggest that whereas for doxycycline positive variations in efficacy can be explained by potentially increased uptake facilitated by SWCNT delivery; SWCNT/methicillin complexes likely circumvent the antibiotic resistance of S. epidermidis. SWCNTs are expected to penetrate bacterial cell walls delivering methicillin and protecting it against degradation. Based on the reported interaction of SWCNTs with the cell wall and cellular membrane we consider that direct transport of the antibiotic by the SWCNTs may introduce cell wall disruption and/or facilitate enhanced delivery and increasing susceptibility of bacteria to methicillin.This work, for the first time, explores the joint delivery and imaging of antibiotics by single-walled carbon nanotubes. SWCNTs dispersed in water with doxycycline and methicillin non-covalently attached to their surface act as drug delivery vehicles facilitating the improved antibacterial effect in These internalization-based hypotheses are supported by SWCNT fluorescence imaging within bacterial cell culture subject to SWCNT/antibiotic treatment indicating substantial SWCNT fluorescence signal originating from bacteria rather than extracellular environment. SEM images confirm the association of SWCNTs with the cell wall of bacteria, whereas TEM verifies successful cell wall internalization by SWCNTs. In this work, SWCNT acts as effective multifunctional antibiotic delivery/imaging agents with the potential to circumvent antibiotic resistance. As methicillin is one of the more widely known antibiotics for developing resistance, its activation through noncovalent hybridization with SWCNTs offers an alternative potential approach to the antibiotic resistance issue. It may further provide a chance to reduce the dose, reuse and recycle the existing antibiotics for the treatment of the new resistant bacterial epidemics.