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Large pore mesoporous silica induced weight loss in obese mice
Background: There is a need for medical treatments to curb the rising rate of obesity. Weight reduction is correlated with a decrease in associated risk factors and cholesterol levels in humans. Amorphous silica particles have been found to exert a hypocholesterolemic effect in humans, making them popular dietary additives. Aim: To investigate the effect of mesoporous silica, which possess sharp pore size distributions, on: weight loss, cholesterol, triglycerides and glucose blood levels in obese mice. Materials & methods: Mesoporous silicas with differing pore size were mixed in the high-fat diet of obese mice. Results: Animals receiving large pore mesoporous silica with a high-fat diet show a significant reduction in body weight and fat composition, with no observable negative effects. Conclusion: Pore size is an important parameter for reduction of body weight and body fat composition by mesoporous silica, demonstrating promising signs for the treatment of obesity. Original submitted 30 January 2013; Revised submitted 20 June 2013 Keywords: body fat • body weight • cholesterol • high-fat diet • mesoporous silica • obesity
Obesity is associated with increased mortality and morbidity. The WHO estimates that more than 1.4 billion adults are obese worldwide, defining obesity as a BMI >30 kg/m2 (BMI: weight in kg divided by height in m2) [101]. Life expectancy is reduced significantly for patients with a BMI >40 kg/m2, as much as 20 years for men and 5 years for women [102]. Abdominal obesity increases the risk of developing a number of chronic diseases including: insulin resistance, Type 2 diabetes, high blood pressure, stroke, heart attacks, high cholesterol, sleep apnea, congestive heart failure, osteoarthritis and cancer. The pharmaceutical drug orlistat (tetrahydrolipstatin) is, at the moment, the only available drug for long-term weight reduction treatment [1]. Its effect is based on pancreatic lipase inhibition. Lipases are lipolytic enzymes that have a central role in fat metabolism and digestion. Lipases cleave triglycerides, but cannot process phospholipids or sterols. Undesirable side effects such
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as diarrhea, fecal incontinence, oily spotting, flatulence and bloating are commonly associated with orlistat treatment [2]. Hence, new strategies to decrease weight and lower cholesterol blood levels avoiding/reducing the use of drugs are highly needed, especially without compliance of dietary habit change. Silicon occurs in nature as silicon dioxide or the corresponding silicic acids that result from the hydration of the oxide. Human serum contains 11–25 µg silicon/dl [103] and remains relatively constant, implicating that it is rapidly distributed in the body and/or excreted. Absorbed silicon is mainly excreted via the urine without evidence of toxic accumulation in the body [3,103]. Jugdaohsingh et al. demonstrated that food-based silica is digested and absorbed from the GI tract in humans [4]. A mean (± standard deviation) of 40.9 ± 36.3% of the ingested silicon was excreted within 6 h after intake, with some variations depending on the silicon source, corresponding to 20 mg excreted silicon/
Nanomedicine (2014) 9(9), 1353–1362
Natalia Kupferschmidt‡,1,2, Robert I Csikasz‡,3, Lluís Ballell4, Tore Bengtsson3 & Alfonso E Garcia-Bennett*,5 Nanotechnology & Functional Materials, Department of Engineering Sciences, Uppsala University, Box 534, 751 21, Uppsala, Sweden 2 Nanologica AB, Drottning Kristinasväg 62, SE 11428, Stockholm, Sweden 3 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Svante Arrheniusväg 20B, 106 91, Stockholm, Sweden 4 Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28769 Tres Cantos, Madrid, Spain 5 Department of Materials & Environmental Chemistry, MMK, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden *Author for correspondence: [email protected] ‡ Authors contributed equally 1
part of
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Research Article Kupferschmidt, Csikasz, Ballell, Bengtsson & Garcia-Bennett day for a normal man [4]. Studies performed in rats by Peluso and Schneeman have shown that intake of silica in the form of silicon dioxide had a clear hypocholesterolemic effect on cholesterol-fed rats by reducing total levels of plasma cholesterol, with a decrease in both very-low density lipoprotein and low-density lipoprotein (LDL) cholesterol [5]. A pilot study performed on humans has demonstrated a clear lowering effect on blood cholesterol and triglycerides levels after oral intake of diatomaceous earth [6]. Diatomaceous earth is composed of amorphous silicates from sedimentary rock and is used as a dietary food additive for improving, for example, the shape of nails, hairs and skin. Diatomaceous silicates are approved by the US FDA as a food additive. Over the last decade, mesoporous silica materials have been the focus of numerous research due to their potential applications in the pharmaceutical industry and in nanomedicine. These include their use as: drug delivery vehicles to improve bioavailability; control drug release; improve drug solubility and stability; and as in vitro and in vivo diagnostic devices [7]. They have been shown to be effective and biocompatible vehicles for controlled and targeted release of a variety of drug compounds, both in vitro [8] and in vivo, as demonstrated by serological, hematological and histopathological examinations of blood samples and tissues after intravenous injection to mice at doses of approximately 100 mg/kg [9]. Mesoporous silicas have also been shown to be highly tolerated when administrated orally to rats during 7 consecutive days at daily doses of up to 1200 and 2000 mg/kg for AMS‑6- and NFM‑1-type particles, respectively [10]. Mesoporous silica particles consist of amorphous silica and can be engineered to have different pore structures, with controlled pore sizes ranging from 2 and 50 nm and with different pore connectivity [11]. It is this capacity, together with their large pore volumes and surface areas, that can be exploited in order to adsorb bile acids and lipids (e.g., cholesterol), as well as enzymes (e.g., lipases), in the passage of the particles through the GI tract. Due to their narrow pore size distributions, mesoporous silica is expected to lead to a more selective adsorption compared with other porous silica materials such as diatomaceous earths. Lipases have demonstrated selective adsorption into SBA‑15-type mesoporous silica materials for use in heterogenous enzymatic catalysis [12,13]. The aim of this study was to investigate the effect of mesoporous silica particle intake, administered in the diet on: body weight and fat composition, cholesterol (total cholesterol and high-density lipoprotein [HDL]), triglycerides and glucose blood levels. Two different types of mesoporous particles were tested,
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namely SBA‑15 [14] and NFM‑1 [15]. Both possess cylindrical 2D hexagonal pore structures, with pore sizes of 110 and 20 Å, respectively. These particles were chosen in order to investigate the effects of pore size upon weight reduction and hypocholesterolemic properties. Materials & methods Preparation of silica mesoporous particles
All chemicals were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden), unless stated, and used as received. The syntheses of SBA‑15 [14] and NFM‑1 [15] particles were performed as previously described. The synthesis of SBA‑15 is based on the self-assembly of the surfactant PEG-block-poly(propylene glycol)block-PEG (P123) in aqueous acidic conditions, while the synthesis of NFM‑1 is based on the self-assembly of folates, forming tetramer stacks stabilized by intrinsic hydrogen bonding and p–p interactions. Tetraethyl orthosilicate was used as a silica source in both syntheses. The pore templates of both materials were removed by calcination at 550°C to form the mesoporous particles. Further details of the synthesis can be found in the Supplementary Material (see online at www.futuremedicine.com/doi/suppl/nmm.13.138). Scanning electron microscopy images were obtained using a Gemini LEO 1550 (Zeiss AG, Oberkochen, Germany) operating at 2–3 kV with no gold coating. Transmission electron microscopy (TEM) of calcined and extracted samples was conducted with a JEOL-3010 microscope (JEOL Ltd, Tokyo, Japan), operating at 300 kV (spherical aberration: 0.6 mm; resolution 1.7 Å). Images were recorded using a charge-coupled device camera model Keen View, SIS Analysis Specialized Imaging (Olympus Soft Imaging Solutions, Olympus Corporation, Münster, Germany; size: 1024 × 1024; pixel size: 23.5 × 23.5 µm) at 30,000–100,000-times magnification using low-dose conditions on as-synthesized and calcined samples. Nitrogen adsorption/desorption isotherms
Nitrogen isotherms were measured at liquid nitrogen temperature (-196°C) using a Micromeritics TriStar II volumetric adsorption analyzer (Micromeritics Instrument Corporation, GA, USA). Before the measurements, the samples were outgassed for 3 h at 200°C. The Brunauer–Emmett–Teller equation was used to calculate the surface area from the adsorption data obtained in the relative pressure (P/Po) range of 0.05 and 0.3 [16]. The total pore volume was calculated from the amount of gas adsorbed at P/Po = 0.91. Pore size distribution curves were derived using the density functional theory method assuming a cylindrical pore model [16].
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Large pore mesoporous silica induced weight loss in obese mice
Animals & in vivo studies
Results & discussion
C57BL/6J female mice were bred and routinely genotyped at the institute. Prior to the start of the experiment, all mice were housed at 24°C with a 12:12‑h light–dark cycle, with free access to food (R70, Lactamin AB, Stockholm, Sweden) and water during the whole study. The study was approved by the Animal Ethics Committee of the North Stockholm Region. Mice were single-caged for 1 week at 24°C and then transferred to a thermal neutral zone at 30°C. After a 3-week acclimatization period, the diet was changed to a high-fat (HF) diet (45% kcal energy by fat; Research Diets D12451, NJ, USA) for 7.5 weeks. Glucose, cholesterol and triglyceride values were measured from mouse blood with a multi Care In© instrument (Biochemical Systems International Srl, Florence, Italy). Mice were then divided into three groups: control; NFM‑1 and SBA‑15 groups receiving a HF diet; and a HF diet containing 1.4% NFM‑1 and SBA‑15 particles. Food intake per mouse was measured during this period by weighing supplied and remaining food. From week 12 to sacrifice at week 20, mice received standard food (R70) with an additional 3-g HF diet twice a week to the control group and a HF fat diet mixed with NFM‑1 and SBA‑15 to the treated groups (5% NFM‑1 and SBA‑15 particles, respectively). Body weight and body composition were monitored every week during the whole study. Body composition was measured with a MRI technique (EchoMRI-700/100 Body Composition Analyzer; Echo Medical Systems, TX, USA). The animal’s general condition was controlled by checking for the following clinical signs: fur and/or skin changes, diarrhea, kyphosis, cleaning/ grooming affected activity and paleness. After sacrifice, blood was taken by cardiac puncture for analysis of lipids, glucose and silica content.
Mesoporous particles
Blood analyses
Plasma concentration of cholesterol, HDL, LDL and triglycerides were measured using a timed end point method, while glucose serum concentration was determined by a glucose oxidase method in a BeckmanCoulter Synchron DXC/LX at the Karolinska University laboratory (Stockholm, Sweden). Blood silica content was measured by inductively coupled plasma at Eurofins MicroKemi AB (Uppsala, Sweden). Food preparation
The HF diet was mixed into a fine powder and mixed homogenously together with NFM‑1 and SBA‑15 particles, respectively. One tablespoon of water was added to the dry mixture of NFM‑1 and SBA‑15 particles and baked into similar proportions of the original HF diet food. The diet was frozen to maintain quality.
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Two different mesoporous materials were chosen for this study: NFM‑1 and SBA‑15 particles were chosen due to their similar agglomerated particle size, hexagonal pore structure and different pore size (Figure 1 & Table 1). Both synthesis procedures have been reported previously [14,15] and synthetic details are included in the Supplementary Material. The organic pore templates of both materials were removed by calcination in order to reveal the open mesoporous structures. The powder x-ray diffraction patterns of both particles showed mesoscale peaks; unit cell parameters were consistent with previously published data [14,15], with diffraction peaks that may be indexed on the basis of a 2D hexagonal unit cell with unit cell parameter of a = 37.4 and 108.5 Å for NFM‑1 and SBA‑15, respectively (Figure 1A). Scanning electron microscopy images depicted agglomerates of spherical-shaped particles for NFM‑1 and, agglomerates of rod-shaped particles for SBA‑15 (Figure 1A). TEM studies confirm the 2D hexagonal pore structures of both materials (Figure 1A). Nitrogen adsorption–desorption isotherms of NFM‑1 and SBA‑15 particles are typical for mesoporous materials and can be classified as type IV according to the IUPAC nomenclature (Figure 1B). Both materials had similar surface areas of 653 and 709 m2/g, but differed in pore sizes of 20 and 110 Å for NFM‑1 and SBA‑15, respectively (Figure 1B & Table 1). The morphology of both particles differs, but both materials are composed of clumps of agglomerated particles of similar size. Effect of mesoporous particle content in HF diet-fed obese mice
The effect of particle content in the diet was evaluated in obese C57BL/6J mice after feeding with a HF diet, a common obesity mouse model that presents most obese characteristics of the patients with a genetic predisposition to develop Type 2 diabetes [17]. Animals were fed with a HF diet during 7.5 weeks in order to make them obese. Obesity (Figure 2), as well as elevated cholesterol values (Supplementary Figure 1 & 2) were confirmed in all mice as a result of the HF diet feeding. After 7.5 weeks of the HF diet, the mice were divided into three groups: control, NFM‑1 and SBA‑15. A particle content of 1.4% NFM-1 and SBA‑15 particles were embedded in the HF diet of the groups named NFM‑1 and SBA‑15, respectively. The total food intake per mouse was monitored by weighing supplied and remaining food. No differences in the amount of food intake was observed between the groups (Figure 3A), indicating no discomfort due to the presence of particles in the diet. Furthermore, none of
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XRD
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Figure 1. Particle characterization. (A) From left to right: powder XRD patterns showing mesoscale peaks that may be indexed on the basis of a 2D hexagonal unit cell; SEM images show agglomerates of spherical-shaped NFM-1 particles and agglomerated rodshaped SBA-15 particles, and TEM images showing pores parallel and perpendicular to the beam direction of NFM-1 (above) and SBA-15 (below). (B) Nitrogen adsorption–desorption isotherms of NFM-1 (gray) and SBA-15 (black) and their corresponding pore size distributions derived using the density functional theory method assuming a cylindrical pore geometry. SEM: Scanning electron microscopy; TEM: Transmission electron microscopy; XRD: X-ray diffraction.
the mice showed obvious general signs of discomfort (described in the ‘Materials & methods’ section) during the whole experiment. There were no differences in the silica blood content between the groups (Figure 3B), which indicate that the particles do not become systemic
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after oral intake. Although the food intake was similar between groups, the particles were expected to have an effect in blood cholesterol values, body weight or body fat composition. It was hypothesized that the materials could
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Large pore mesoporous silica induced weight loss in obese mice
Research Article
Table 1. Particle characterization. Characteristic
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SBA-15
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2D hexagonal
2D hexagonal
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665
710
DFT pore size (Å)
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110 cylindrical pores
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0.35
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BET: Brunauer–Emmett–Teller; DFT: Density functional theory.
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Effect of mesoporous particle content in a HF diet complementary to a standard diet in obese mice
At week 12 of the experiment, the diets of the mice were changed. From week 12 to the end of the experiment and animal sacrifice (week 20), mice had free access to standard R70 lactamin diet and received an additional 3 g of HF twice a week containing 5% SBA‑15 or NFM‑1 particles in the groups named
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act in a similar way to dietary fibers by adsorbing lipids and bile acids from the food (decreasing their absorption and resorption) as observed for silicon dioxide [5] and diatomaceous earth [6]. This effect may lead to an increase of hepatic bile acid biosynthesis, which results in a lowering effect on blood cholesterol levels. However, no effect on body weight, fat composition or cholesterol blood levels was observed in this part of the study. This could be due to the ad libitum HF intake (i.e., effect of the treatment may be hidden by the constant HF/sugar intake). In order to answer this question and be able to confirm the presence or absence of effects on weight
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Figure 2. Development of weight and fat body composition over time. Female C57BL/6 mice 15–21 weeks old at the experiment start were weighed once a week and MRI was used to determine fat and lean body composition. Dotted lines shows changes in the diet (i) high-fat diet (HF) ad libitum; (ii) HF containing 1.4% NFM-1 or SBA15 (only HF for the control group) ad libitum and; (iii) standard food ad libitum together with 1.5-g HF twice a week containing 5% NFM-1 or SBA-15. (A & B) Body weight development during the whole experiment and from week 12 to 20, respectively. (C & D) Fat body composition during the whole experiment and from week 12 to 20, respectively. The asterisks represents statistical significant differences between the SBA-15 (n = 6) and the NFM-1 (n = 5) and the control (n = 5) groups using two ways analysis of variance. Error bars represent standard error of the mean. **p < 0.01; ***p < 0.001.
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Research Article Kupferschmidt, Csikasz, Ballell, Bengtsson & Garcia-Bennett
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Figure 3. Silica particle content in the diet has no effect on blood silicon levels. The amount of food intake, as well as the silicon blood concentration is similar between the groups despite silica particle content in the diet. (A) Daily food intake between experiment weeks 7.5 and 12. The number of mice per group was: n = 5, 5 and 6 for the control, NFM-1 and SBA-15 groups, respectively. (B) Silica content in blood at the end of the study measured by the inductively coupled plasma technique (n = 3). Error bars represent standard error of the mean. HF: High fat; Si: Silicon.
SBA‑15 and NFM‑1, respectively. When the HF diet treats of 1.5 g were administered to the mice, they were consumed instantly. The ad libitum consumption of standard food was not monitored during this period; however, pair-fed studies (data not shown) indicate no differences in the amount of food intake between the animal groups under similar experimental conditions. Animals in the control and NFM‑1 groups showed similar weight and body fat composition developments during experiment weeks 1–20 (weeks 12–19 for body fat composition). The weight increase from week 12 to 20 was 2.7 and 3.4%, while body fat composition decreased by 12 and 14% for the control and NFM-1 groups, respectively (Figure 2B & 2D). The SBA‑15 group showed significant decreases in body weight and fat composition compared with the control and the NFM‑1 groups from week 12 to 19 (Figure 2B & 2D, respectively). The decrease in body fat composition was 24% for the SBA‑15 group. In contrast to the control and NFM‑1 group, the SBA‑15 group demonstrated a significant reduction in body weight of approximately 6% (Figure 2B). It is important to note that the body lean composition within the SBA‑15 group did not decrease (Supplementary Figure 3) indicating that the weight reduction was not a sign of health compliance. Results indicate the potential of using mesoporous silicas for weight reduction purposes. Even modest weight loss can reduce health risks in obese patients, with medical benefits already beginning after a 5–10% decrease of initial weight [18,19]. A significant positive linear relationship between cholesterol and long-term weight reduction has been determined in populations with BMI ≥28 kg/m2. It is estimated that
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for every 10 kg weight loss, a drop of 0.23 mmol/l in cholesterol may be expected for an obese person, with similar estimations for triglycerides and LDL cholesterol [20]. It has been shown that cholesterol reduction in patients with coronary disease retards or reverses the progression of atherosclerotic disease [21]. In individuals with impaired glucose tolerance, a 7% reduction in body weight has been associated with a 58% reduction of progression to diabetes [22]. High surface area mesoporous silica was expected to adsorb lipids or bile acids within the GI tract similar to dietary fibers. There were no differences in cholesterol, HDL, triglycerides and glucose serum levels between the mice groups at the end of the experiment (week 20), indicating that the particle content in the diet has no effect on lipid and glucose blood levels (Figure 4). Silica may adsorb fatty acids and bile acids leading to their subsequent excretion, but this effect is probably not as strong as expected for mesoporous silica particles. Bile acids are synthetized in the liver and secreted into the bile. A large amount is reabsorbed from the intestine and taken up by the liver, known as enterohepatic circulation [23]. Bile acids have an important role in cholesterol homeostasis. Their biosynthesis implicates the conversion of cholesterol into soluble molecules of bile acids, which are easily secreted. This process is negatively regulated by its end product, bile acid, and accounts for approximately 50% of cholesterol elimination from the body [24]. Furthermore, bile acids are essential for the absorption of lipids, cholesterol and lipid-soluble vitamins by intestinal epithelial cells [25]. The hypocholesterolemic effect observed for amorphous silica and dietary fibers may, at least, be
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Large pore mesoporous silica induced weight loss in obese mice
rials studied. Encapsulation of lipases, which vary in sizes (e.g., 4.9 × 4.9 × 4.6 nm from Rhizopus niveus; 4.6 × 2.6 × 1.1 nm for Porcine pancreatic lipase [26]) into the pores of NFM‑1 should not occur due to its smaller pore size. These, however, have been shown to be selectively encapsulated into SBA‑15 materials with pore sizes in the 10 nm range [12,13]. We have measured the size of the human pancreatic lipase (Protein Data Bank, IPLA2G) directly from structural data and a size of 8.6 × 3.5 × 3.5 nm is estimated. Furthermore, the isoelectric point of lipase also varies between different species and different lipases; for example, 6.7 and 6.1 for human and mice (PNPLA2) lipase enzymes [27]. A close proximity to the isoelectric point of the protein should favor diffusion into the pores. Furthermore, uncharged silanol groups (note that the duodenal pH is 5–7) on the pore wall surface may interact with the enzyme via hydrogen bonding or other interactions.
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partially due to a reduction in bile acid concentration in the regulating hepatic steroid metabolism in the liver. Overall, the evidence from our data from this experimental set up indicates a strong pore size-dependent effect on weight reduction in obese mice. The absence of an expected hypocholesterolemic effect by SBA‑15 and NFM‑1 particles is indicative that bile acid–cholesterol metabolism is not affected by the particle supplement in the diet in the time frame of the study presented here. Hence, the mechanism underling the weight reduction by SBA‑15 mesoporous silicas may be different than the lipid-lowering effect induced by porous diatomaceous earths and other nonporous silicon dioxides. The mode of action of SBA‑15 mesoporous silica may be due to a lipase sequestration/adsorption effect and its subsequent excretion and/or impeding its interaction with co-lipase. These possible modes of action are consistent with the pore size differences between the two mate-
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Figure 4. Particle content in the diet has no effect on lipid and glucose blood levels. Cholesterol, high-density cholesterol, triglycerides and glucose serum levels analyzed using a timed end point method at the end of the study (experiment week 20). The number of mice per group was: n = 5, 5 and 6 for the control, NFM-1 and SBA-15 groups, respectively (for P glucose, n = 4 in the control group). Error bars represent standard error of the mean. HDL: High-density lipoprotein; P glucose: Plasma glucose.
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Research Article Kupferschmidt, Csikasz, Ballell, Bengtsson & Garcia-Bennett Hence, large pore mesoporous silica through lipase encapsulation and its further excretion (decreasing fat absorption) may allow the modulation of lipase activity. This could be achieved by the use of, for example, different particle doses together with meals. Conclusion SBA‑15 particles embedded in food with HF/sugar content lead to a decrease in weight and body fat composition without observable toxicological signs or systemic absorption of silica. Mesoporous silica materials with big pores (≥110 Å) can potentially be used as pharmaceuticals or food additives for weight loss treatment. We conclude that the inability of NFM‑1 to reduce body weight and fat is primarily determined by its small pore size based on the absence of a cholesterol-lowering effect, together with previous data on lipase-specific adsorption within large pore SBA‑15 particles [28]. This suggests a mechanism based on modulation of lipase activity. This may occur through lipase sequestration into the pores and its excretion or through other mechanisms such as interference on lipase interaction with co-lipase. It is probable that other mechanism or coexisting mechanisms such as bile acid sequestration and a faster passage through the GI tract (e.g., as for digestive fibers) also play an additional role, but further ongoing experiments are required in order to fully understand the action of large pore mesoporous silicas on body weight and fat reduction. Future perspective Obesity is a serious health problem and, with the exception of surgical treatments, it has been shown to lack long-term success. For these reasons, efficient body weight reduction alternatives are highly needed. As mentioned in a recent review by Reis et al., “Modulation of lipase activity through interfacial engineering can be a potential solution” [1]. Mesoporous silica particles may offer this possibility. This study may lead to the development of nanomaterials and mesoporous materials with positive effects on weight loss and body fat composition. This can further lead to a decrease in blood lipids including cholesterol as a result of a general decrease of fat tissue and blood as mentioned above. Long-term studies are needed to reveal the potential capacity of mesoporous silicas to reduce blood lipid levels. Orlistat has been associated with a significant decrease in total cholesterol after adjustment for weight loss after 6–12 months treatment, which is indicative of improved cardiovascular risk factor profiles in obese patients [29]. Orlistat also reduced the incidence of Type 2 diabetes from 9.0 to 6.2% in a
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longer 4‑year trial [30]. If the effect of mesoporous silica on weight reduction occurs through a similar mode of action as orlistat (lipase inhibition) as hypothesized here, the long-term effect may be similar, as well as its side effects. More work including longer treatment times and normal weight animals are needed to shed more light on the mode of action of mesoporous silica materials on body weight and fat reduction, as well as their potential effect on blood lipid levels. Drug treatment of obesity can be considered as a long-term therapy as most patients regain weight upon stopping medication. Mesoporous silicas are proposed here to potentially be used in a similar way as weight reduction drugs, hence longer studies are also needed to assess possible secondary effects related to long-term consumption. The histological examination of major organs such as the liver, intestine and kidneys should be included in these future studies to provide a better assessment of potential negative effects and the mode of action of the particles when administrated orally. No elevated silica blood levels were found in the mice receiving particles in the diet compared with the control mice during this study. However, it should be noted that higher serum or plasma silicon concentrations have been found in patients with chronic renal failure compared with healthy controls [31]. The results presented here should also be taken into consideration in the development of mesoporous silicas as drug delivery systems for oral administration. Acknowledgements The authors are grateful to R Atluri and to MN Iqbal (Nanologica AB, Stockholm, Sweden) for assistance with material characterization and material synthesis. The authors would also like to thank Stockholm University for access to their transmission electron microscopy facilities.
Financial & competing interests disclosure AE Garcia-Bennett is the co-founder of Nanologica AB (Stockholm, Sweden), a company commercializing nanoporous materials for biomedical and related applications. AE Garcia-Bennett is supported by grants by the Swedish Research Council, projects 2009–4716. T Bengtsson is supported by the VR-M from the Swedish Research Council, Novonordiskfonden, Stiftelsen Svenska Diabetesförbundets Forskningsfond, Magnus Bergvall foundation and the Carl Tryggers foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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Large pore mesoporous silica induced weight loss in obese mice
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal
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experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary • A murine obesity model was used to investigate the effect of mesoporous silica material content in the diet. • Mesoporous silica particles with different pore sizes (20 and 110 Å for NFM‑1 and SBA‑15, respectively) were mixed in the high fat (HF) diet of C57BL/6J obese mice. No particles were added to the diet of control animals. • Particle content in the HF diet did not result in differences between the animal groups when this was the only dietary source. • Obese mice receiving a standard diet with additional HF complement containing SBA‑15 twice a week significantly decreased in weight and body fat composition. • The reduction in body weight and body fat composition exerted by SBA‑15 was attributed to the 110 Å pore size and its ability to encapsulate lipases. • The pore size of mesoporous silicas can be tailored to achieve reduction in body weight and fat composition. mesoporous nanoparticles.
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Kupferschmidt N, Xia X, Labrador RH, Atluri R, Ballell L, Garcia-Bennett AE. In vivo oral toxicological evaluation of mesoporous silica particles. Nanomedicine (Lond.) 8(1), 57–64 (2013).
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Derosa G, Maffioli P. Anti-obesity drugs: a review about their effects and their safety. Expert Opin. Drug Saf. 11(3), 459–471 (2012).
Wan Y, Zhao DY. On the controllable soft-templating approach to mesoporous silicates. Chem. Rev. 107(7), 2821–2860 (2007).
12
Reffitt DM, Jugdaohsingh R, Thompson RPH, Powell JJ. Silicic acid: its gastrointestinal uptake and urinary excretion in man and effects on aluminium excretion. J. Inorg. Biochem. 76(2), 141–147 (1999).
Salis A, Meloni D, Ligas S et al. Physical and chemical adsorption of Mucor javanicus lipase on SBA-15 mesoporous silica. Synthesis, structural characterization, and activity performance. Langmuir 21(12), 5511–5516 (2005).
13
Jugdaohsingh R, Anderson SHC, Tucker KL et al. Dietary silicon intake and absorption. Am. J. Clin. Nutr. 75(5), 887–893 (2002).
Kang Y, He J, Guo XD, Guo X, Song ZH. Influence of pore diameters on the immobilization of lipase in SBA-15. Ind. Eng. Chem. Res. 46(13), 4474–4479 (2007).
14
Zhao DY, Feng JL, Huo QS et al. Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores. Science 279(5350), 548–552 (1998).
15
Atluri R, Hedin N, Garcia-Bennett AE. Nonsurfactant supramolecular synthesis of ordered mesoporous silica. J. Am. Chem. Soc. 131(9), 3189–3191 (2009).
•
First report on the synthesis of an ordered mesoporous material with a nonsurfactant template.
16
Kruk M, Jaroniec M. Gas adsorption characterization of ordered organic-inorganic nanocomposite materials. Chem. Mater. 13(10), 3169–3183 (2001).
17
Bray GA, Paeratakul S, Popkin BM. Dietary fat and obesity: a review of animal, clinical and epidemiological studies. Physiol. Behav. 83(4), 549–555 (2004).
18
Karam J, Mcfarlane S. Tackling obesity: new therapeutic agents for assisted weight loss. Diabetes Metab. Syndr. Obes. 3, 95–112 (2010).
Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
2
3
4
•
Detailed in vivo study of nanoparticles and colloidal silica clearance and its adsorption through dietary intake.
5
Peluso MR, Schneeman BO. A food-grade silicon dioxide is hypocholesterolemic. J. Nutr. Metab. 124(6), 853–860 (1994).
•
First report showing a cholesterolemic effect in rats.
6
Wachter H, Lechleitner M, Artner-Dworzak E et al. Diatomaceous earth lowers blood cholesterol concentrations. Eur. J. Med. Res. 3(4), 211–215 (1998).
7
Garcia-Bennett AE. Synthesis, toxicology and potential of ordered mesoporous materials in nanomedicine. Nanomedicine (Lond.) 6(5), 867–877 (2011).
8
Vallet-Regi M, Balas F, Arcos D. Mesoporous materials for drug delivery. Angew. Chem. Int. Ed. Engl. 46(40), 7548–7558 (2007).
•
Highlights the early development of mesoporous materials in the field of life sciences.
19
9
Lu J, Liong M, Li Z, Zink JI, Tamanoi F. Biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. Small 6(16), 1794–1805 (2010).
Rallidis LS, Fountoulaki K, Anastasiou-Nana M. Managing the underestimated risk of statin-associated myopathy. Int. J. Cardiol. 159(3), 169–176 (2012).
20
Poobalan A, Aucott L, Smith WC et al. Effects of weight loss in overweight/obese individuals and long-term lipid outcomes – a systematic review. Obes. Rev. 5(1), 43–50 (2004).
••
Seminal study focusing on the in vivo use of mesoporous particles in cancer therapy and toxicological properties of
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21
Garber AM, Browner WS, Hulley SB. Cholesterol screening in asymptomatic adults, revisited. Ann. Intern. Med. 124(5), 518–531 (1996).
29
Mannucci E, Dicembrini I, Rotella F, Rotella CM. Orlistat and sibutramine beyond weight loss. Nutr. Metab. Cardiovasc. Dis. 18(5), 342–348 (2008).
22
Knowler WC, Barrett-Connor E, Fowler SE et al. Reduction in the incidence of Type 2 diabetes with lifestyle intervention or metformin. N. Engl. J. Med. 346(6), 393–403 (2002).
30
23
Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol. Rev. 83(2), 633–671 (2003).
Torgerson JS, Hauptman J, Boldrin MN, Sjostrom L. XENical in the Prevention of Diabetes in Obese Subjects (XENDOS) study: a randomized study of orlistat as an adjunct to lifestyle changes for the prevention of Type 2 diabetes in obese patients. Diabetes Care 27(3), 856–856 (2004).
24
Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 72, 137–174 (2003).
31
25
Kullak-Ublick GA, Stieger B, Meier PJ. Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126(1), 322–342 (2004).
Gitelman HJ, Alderman F, Perry SJ. Renal handling of silicon in normals and patients with renal insufficiency. Kidney Int. 42(4), 957–959 (1992).
26
Yiu HHP, Wright PA. Enzymes supported on ordered mesoporous solids: a special case of an inorganic-organic hybrid. J. Mater. Chem. 15(35–36), 3690–3700 (2005).
27
Holmes RS, Cox LA. Comparative studies of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1: evidence for a eutherian mammalian origin for the GPIHBP1 gene from an LY6-like gene. 3 Biotech 2(1), 37–52 (2012).
•
Very thorough review of bioinformatic data for lipases from different species.
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Chen ZZ, Li YM, Peng X, Huang FR, Zhao YF. The use of crude lipase in deprotection of C-terminal protecting groups. J. Mol. Catal. B Enzym. 18(4–6), 243–249 (2002).
Nanomedicine (2014) 9(9)
Websites 101 WHO. Obesity and overweight.
www.who.int/mediacentre/factsheets/fs311/en 102 Hockley T, Gemmill M. European Cholesterol Guidelines
report 2007. School of Economics, Policy Analysis Centre, London, UK (2007). www.policy-centre.com/downloads/European-CholesterolGuidelines07.pdf 103 Scientific opinion of the Panel on Food Additives and
Nutrient Sources added to Food on calcium silicate, silicon dioxide and silicic acid gel added for nutritional purposes to food supplements following a request from the European Commission. The EFSA Journal (2009). www.efsa.europa.eu/en/scdocs/doc/1132.pdf
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Large pore mesoporous silica induced weight loss in obese mice
Background: There is a need for medical treatments to curb the rising rate of obesity. Weight reduction is correlated with a decrease in associated risk factors and cholesterol levels in humans. Amorphous silica particles have been found to exert a hypocholesterolemic effect in humans, making them popular dietary additives. Aim: To investigate the effect of mesoporous silica, which possess sharp pore size distributions, on: weight loss, cholesterol, triglycerides and glucose blood levels in obese mice. Materials & methods: Mesoporous silicas with differing pore size were mixed in the high-fat diet of obese mice. Results: Animals receiving large pore mesoporous silica with a high-fat diet show a significant reduction in body weight and fat composition, with no observable negative effects. Conclusion: Pore size is an important parameter for reduction of body weight and body fat composition by mesoporous silica, demonstrating promising signs for the treatment of obesity. Original submitted 30 January 2013; Revised submitted 20 June 2013 Keywords: body fat • body weight • cholesterol • high-fat diet • mesoporous silica • obesity
Obesity is associated with increased mortality and morbidity. The WHO estimates that more than 1.4 billion adults are obese worldwide, defining obesity as a BMI >30 kg/m2 (BMI: weight in kg divided by height in m2) [101]. Life expectancy is reduced significantly for patients with a BMI >40 kg/m2, as much as 20 years for men and 5 years for women [102]. Abdominal obesity increases the risk of developing a number of chronic diseases including: insulin resistance, Type 2 diabetes, high blood pressure, stroke, heart attacks, high cholesterol, sleep apnea, congestive heart failure, osteoarthritis and cancer. The pharmaceutical drug orlistat (tetrahydrolipstatin) is, at the moment, the only available drug for long-term weight reduction treatment [1]. Its effect is based on pancreatic lipase inhibition. Lipases are lipolytic enzymes that have a central role in fat metabolism and digestion. Lipases cleave triglycerides, but cannot process phospholipids or sterols. Undesirable side effects such
10.2217/NNM.13.138 © 2014 Future Medicine Ltd
as diarrhea, fecal incontinence, oily spotting, flatulence and bloating are commonly associated with orlistat treatment [2]. Hence, new strategies to decrease weight and lower cholesterol blood levels avoiding/reducing the use of drugs are highly needed, especially without compliance of dietary habit change. Silicon occurs in nature as silicon dioxide or the corresponding silicic acids that result from the hydration of the oxide. Human serum contains 11–25 µg silicon/dl [103] and remains relatively constant, implicating that it is rapidly distributed in the body and/or excreted. Absorbed silicon is mainly excreted via the urine without evidence of toxic accumulation in the body [3,103]. Jugdaohsingh et al. demonstrated that food-based silica is digested and absorbed from the GI tract in humans [4]. A mean (± standard deviation) of 40.9 ± 36.3% of the ingested silicon was excreted within 6 h after intake, with some variations depending on the silicon source, corresponding to 20 mg excreted silicon/
Nanomedicine (2014) 9(9), 1353–1362
Natalia Kupferschmidt‡,1,2, Robert I Csikasz‡,3, Lluís Ballell4, Tore Bengtsson3 & Alfonso E Garcia-Bennett*,5 Nanotechnology & Functional Materials, Department of Engineering Sciences, Uppsala University, Box 534, 751 21, Uppsala, Sweden 2 Nanologica AB, Drottning Kristinasväg 62, SE 11428, Stockholm, Sweden 3 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Svante Arrheniusväg 20B, 106 91, Stockholm, Sweden 4 Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28769 Tres Cantos, Madrid, Spain 5 Department of Materials & Environmental Chemistry, MMK, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden *Author for correspondence: [email protected] ‡ Authors contributed equally 1
part of
ISSN 1743-5889
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Research Article Kupferschmidt, Csikasz, Ballell, Bengtsson & Garcia-Bennett day for a normal man [4]. Studies performed in rats by Peluso and Schneeman have shown that intake of silica in the form of silicon dioxide had a clear hypocholesterolemic effect on cholesterol-fed rats by reducing total levels of plasma cholesterol, with a decrease in both very-low density lipoprotein and low-density lipoprotein (LDL) cholesterol [5]. A pilot study performed on humans has demonstrated a clear lowering effect on blood cholesterol and triglycerides levels after oral intake of diatomaceous earth [6]. Diatomaceous earth is composed of amorphous silicates from sedimentary rock and is used as a dietary food additive for improving, for example, the shape of nails, hairs and skin. Diatomaceous silicates are approved by the US FDA as a food additive. Over the last decade, mesoporous silica materials have been the focus of numerous research due to their potential applications in the pharmaceutical industry and in nanomedicine. These include their use as: drug delivery vehicles to improve bioavailability; control drug release; improve drug solubility and stability; and as in vitro and in vivo diagnostic devices [7]. They have been shown to be effective and biocompatible vehicles for controlled and targeted release of a variety of drug compounds, both in vitro [8] and in vivo, as demonstrated by serological, hematological and histopathological examinations of blood samples and tissues after intravenous injection to mice at doses of approximately 100 mg/kg [9]. Mesoporous silicas have also been shown to be highly tolerated when administrated orally to rats during 7 consecutive days at daily doses of up to 1200 and 2000 mg/kg for AMS‑6- and NFM‑1-type particles, respectively [10]. Mesoporous silica particles consist of amorphous silica and can be engineered to have different pore structures, with controlled pore sizes ranging from 2 and 50 nm and with different pore connectivity [11]. It is this capacity, together with their large pore volumes and surface areas, that can be exploited in order to adsorb bile acids and lipids (e.g., cholesterol), as well as enzymes (e.g., lipases), in the passage of the particles through the GI tract. Due to their narrow pore size distributions, mesoporous silica is expected to lead to a more selective adsorption compared with other porous silica materials such as diatomaceous earths. Lipases have demonstrated selective adsorption into SBA‑15-type mesoporous silica materials for use in heterogenous enzymatic catalysis [12,13]. The aim of this study was to investigate the effect of mesoporous silica particle intake, administered in the diet on: body weight and fat composition, cholesterol (total cholesterol and high-density lipoprotein [HDL]), triglycerides and glucose blood levels. Two different types of mesoporous particles were tested,
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namely SBA‑15 [14] and NFM‑1 [15]. Both possess cylindrical 2D hexagonal pore structures, with pore sizes of 110 and 20 Å, respectively. These particles were chosen in order to investigate the effects of pore size upon weight reduction and hypocholesterolemic properties. Materials & methods Preparation of silica mesoporous particles
All chemicals were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden), unless stated, and used as received. The syntheses of SBA‑15 [14] and NFM‑1 [15] particles were performed as previously described. The synthesis of SBA‑15 is based on the self-assembly of the surfactant PEG-block-poly(propylene glycol)block-PEG (P123) in aqueous acidic conditions, while the synthesis of NFM‑1 is based on the self-assembly of folates, forming tetramer stacks stabilized by intrinsic hydrogen bonding and p–p interactions. Tetraethyl orthosilicate was used as a silica source in both syntheses. The pore templates of both materials were removed by calcination at 550°C to form the mesoporous particles. Further details of the synthesis can be found in the Supplementary Material (see online at www.futuremedicine.com/doi/suppl/nmm.13.138). Scanning electron microscopy images were obtained using a Gemini LEO 1550 (Zeiss AG, Oberkochen, Germany) operating at 2–3 kV with no gold coating. Transmission electron microscopy (TEM) of calcined and extracted samples was conducted with a JEOL-3010 microscope (JEOL Ltd, Tokyo, Japan), operating at 300 kV (spherical aberration: 0.6 mm; resolution 1.7 Å). Images were recorded using a charge-coupled device camera model Keen View, SIS Analysis Specialized Imaging (Olympus Soft Imaging Solutions, Olympus Corporation, Münster, Germany; size: 1024 × 1024; pixel size: 23.5 × 23.5 µm) at 30,000–100,000-times magnification using low-dose conditions on as-synthesized and calcined samples. Nitrogen adsorption/desorption isotherms
Nitrogen isotherms were measured at liquid nitrogen temperature (-196°C) using a Micromeritics TriStar II volumetric adsorption analyzer (Micromeritics Instrument Corporation, GA, USA). Before the measurements, the samples were outgassed for 3 h at 200°C. The Brunauer–Emmett–Teller equation was used to calculate the surface area from the adsorption data obtained in the relative pressure (P/Po) range of 0.05 and 0.3 [16]. The total pore volume was calculated from the amount of gas adsorbed at P/Po = 0.91. Pore size distribution curves were derived using the density functional theory method assuming a cylindrical pore model [16].
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Large pore mesoporous silica induced weight loss in obese mice
Animals & in vivo studies
Results & discussion
C57BL/6J female mice were bred and routinely genotyped at the institute. Prior to the start of the experiment, all mice were housed at 24°C with a 12:12‑h light–dark cycle, with free access to food (R70, Lactamin AB, Stockholm, Sweden) and water during the whole study. The study was approved by the Animal Ethics Committee of the North Stockholm Region. Mice were single-caged for 1 week at 24°C and then transferred to a thermal neutral zone at 30°C. After a 3-week acclimatization period, the diet was changed to a high-fat (HF) diet (45% kcal energy by fat; Research Diets D12451, NJ, USA) for 7.5 weeks. Glucose, cholesterol and triglyceride values were measured from mouse blood with a multi Care In© instrument (Biochemical Systems International Srl, Florence, Italy). Mice were then divided into three groups: control; NFM‑1 and SBA‑15 groups receiving a HF diet; and a HF diet containing 1.4% NFM‑1 and SBA‑15 particles. Food intake per mouse was measured during this period by weighing supplied and remaining food. From week 12 to sacrifice at week 20, mice received standard food (R70) with an additional 3-g HF diet twice a week to the control group and a HF fat diet mixed with NFM‑1 and SBA‑15 to the treated groups (5% NFM‑1 and SBA‑15 particles, respectively). Body weight and body composition were monitored every week during the whole study. Body composition was measured with a MRI technique (EchoMRI-700/100 Body Composition Analyzer; Echo Medical Systems, TX, USA). The animal’s general condition was controlled by checking for the following clinical signs: fur and/or skin changes, diarrhea, kyphosis, cleaning/ grooming affected activity and paleness. After sacrifice, blood was taken by cardiac puncture for analysis of lipids, glucose and silica content.
Mesoporous particles
Blood analyses
Plasma concentration of cholesterol, HDL, LDL and triglycerides were measured using a timed end point method, while glucose serum concentration was determined by a glucose oxidase method in a BeckmanCoulter Synchron DXC/LX at the Karolinska University laboratory (Stockholm, Sweden). Blood silica content was measured by inductively coupled plasma at Eurofins MicroKemi AB (Uppsala, Sweden). Food preparation
The HF diet was mixed into a fine powder and mixed homogenously together with NFM‑1 and SBA‑15 particles, respectively. One tablespoon of water was added to the dry mixture of NFM‑1 and SBA‑15 particles and baked into similar proportions of the original HF diet food. The diet was frozen to maintain quality.
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Two different mesoporous materials were chosen for this study: NFM‑1 and SBA‑15 particles were chosen due to their similar agglomerated particle size, hexagonal pore structure and different pore size (Figure 1 & Table 1). Both synthesis procedures have been reported previously [14,15] and synthetic details are included in the Supplementary Material. The organic pore templates of both materials were removed by calcination in order to reveal the open mesoporous structures. The powder x-ray diffraction patterns of both particles showed mesoscale peaks; unit cell parameters were consistent with previously published data [14,15], with diffraction peaks that may be indexed on the basis of a 2D hexagonal unit cell with unit cell parameter of a = 37.4 and 108.5 Å for NFM‑1 and SBA‑15, respectively (Figure 1A). Scanning electron microscopy images depicted agglomerates of spherical-shaped particles for NFM‑1 and, agglomerates of rod-shaped particles for SBA‑15 (Figure 1A). TEM studies confirm the 2D hexagonal pore structures of both materials (Figure 1A). Nitrogen adsorption–desorption isotherms of NFM‑1 and SBA‑15 particles are typical for mesoporous materials and can be classified as type IV according to the IUPAC nomenclature (Figure 1B). Both materials had similar surface areas of 653 and 709 m2/g, but differed in pore sizes of 20 and 110 Å for NFM‑1 and SBA‑15, respectively (Figure 1B & Table 1). The morphology of both particles differs, but both materials are composed of clumps of agglomerated particles of similar size. Effect of mesoporous particle content in HF diet-fed obese mice
The effect of particle content in the diet was evaluated in obese C57BL/6J mice after feeding with a HF diet, a common obesity mouse model that presents most obese characteristics of the patients with a genetic predisposition to develop Type 2 diabetes [17]. Animals were fed with a HF diet during 7.5 weeks in order to make them obese. Obesity (Figure 2), as well as elevated cholesterol values (Supplementary Figure 1 & 2) were confirmed in all mice as a result of the HF diet feeding. After 7.5 weeks of the HF diet, the mice were divided into three groups: control, NFM‑1 and SBA‑15. A particle content of 1.4% NFM-1 and SBA‑15 particles were embedded in the HF diet of the groups named NFM‑1 and SBA‑15, respectively. The total food intake per mouse was monitored by weighing supplied and remaining food. No differences in the amount of food intake was observed between the groups (Figure 3A), indicating no discomfort due to the presence of particles in the diet. Furthermore, none of
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XRD
SEM
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0 20 40 Pore width (Å)
Figure 1. Particle characterization. (A) From left to right: powder XRD patterns showing mesoscale peaks that may be indexed on the basis of a 2D hexagonal unit cell; SEM images show agglomerates of spherical-shaped NFM-1 particles and agglomerated rodshaped SBA-15 particles, and TEM images showing pores parallel and perpendicular to the beam direction of NFM-1 (above) and SBA-15 (below). (B) Nitrogen adsorption–desorption isotherms of NFM-1 (gray) and SBA-15 (black) and their corresponding pore size distributions derived using the density functional theory method assuming a cylindrical pore geometry. SEM: Scanning electron microscopy; TEM: Transmission electron microscopy; XRD: X-ray diffraction.
the mice showed obvious general signs of discomfort (described in the ‘Materials & methods’ section) during the whole experiment. There were no differences in the silica blood content between the groups (Figure 3B), which indicate that the particles do not become systemic
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after oral intake. Although the food intake was similar between groups, the particles were expected to have an effect in blood cholesterol values, body weight or body fat composition. It was hypothesized that the materials could
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Large pore mesoporous silica induced weight loss in obese mice
Research Article
Table 1. Particle characterization. Characteristic
NFM-1
SBA-15
Pore structure
2D hexagonal
2D hexagonal
BET surface area (m /g)
665
710
DFT pore size (Å)
20 cylindrical pores
110 cylindrical pores
Pore volume (cm3 /g)
0.35
1.17
2
BET: Brunauer–Emmett–Teller; DFT: Density functional theory.
i
50
Effect of mesoporous particle content in a HF diet complementary to a standard diet in obese mice
At week 12 of the experiment, the diets of the mice were changed. From week 12 to the end of the experiment and animal sacrifice (week 20), mice had free access to standard R70 lactamin diet and received an additional 3 g of HF twice a week containing 5% SBA‑15 or NFM‑1 particles in the groups named
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and/or blood lipid levels by the particles, the amount of fat and sugar in the diet was reduced as described below.
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Body weight (g)
act in a similar way to dietary fibers by adsorbing lipids and bile acids from the food (decreasing their absorption and resorption) as observed for silicon dioxide [5] and diatomaceous earth [6]. This effect may lead to an increase of hepatic bile acid biosynthesis, which results in a lowering effect on blood cholesterol levels. However, no effect on body weight, fat composition or cholesterol blood levels was observed in this part of the study. This could be due to the ad libitum HF intake (i.e., effect of the treatment may be hidden by the constant HF/sugar intake). In order to answer this question and be able to confirm the presence or absence of effects on weight
0.8 0.4 0.0
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Figure 2. Development of weight and fat body composition over time. Female C57BL/6 mice 15–21 weeks old at the experiment start were weighed once a week and MRI was used to determine fat and lean body composition. Dotted lines shows changes in the diet (i) high-fat diet (HF) ad libitum; (ii) HF containing 1.4% NFM-1 or SBA15 (only HF for the control group) ad libitum and; (iii) standard food ad libitum together with 1.5-g HF twice a week containing 5% NFM-1 or SBA-15. (A & B) Body weight development during the whole experiment and from week 12 to 20, respectively. (C & D) Fat body composition during the whole experiment and from week 12 to 20, respectively. The asterisks represents statistical significant differences between the SBA-15 (n = 6) and the NFM-1 (n = 5) and the control (n = 5) groups using two ways analysis of variance. Error bars represent standard error of the mean. **p < 0.01; ***p < 0.001.
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Research Article Kupferschmidt, Csikasz, Ballell, Bengtsson & Garcia-Bennett
0.06 Si content in blood (mmol/l)
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Figure 3. Silica particle content in the diet has no effect on blood silicon levels. The amount of food intake, as well as the silicon blood concentration is similar between the groups despite silica particle content in the diet. (A) Daily food intake between experiment weeks 7.5 and 12. The number of mice per group was: n = 5, 5 and 6 for the control, NFM-1 and SBA-15 groups, respectively. (B) Silica content in blood at the end of the study measured by the inductively coupled plasma technique (n = 3). Error bars represent standard error of the mean. HF: High fat; Si: Silicon.
SBA‑15 and NFM‑1, respectively. When the HF diet treats of 1.5 g were administered to the mice, they were consumed instantly. The ad libitum consumption of standard food was not monitored during this period; however, pair-fed studies (data not shown) indicate no differences in the amount of food intake between the animal groups under similar experimental conditions. Animals in the control and NFM‑1 groups showed similar weight and body fat composition developments during experiment weeks 1–20 (weeks 12–19 for body fat composition). The weight increase from week 12 to 20 was 2.7 and 3.4%, while body fat composition decreased by 12 and 14% for the control and NFM-1 groups, respectively (Figure 2B & 2D). The SBA‑15 group showed significant decreases in body weight and fat composition compared with the control and the NFM‑1 groups from week 12 to 19 (Figure 2B & 2D, respectively). The decrease in body fat composition was 24% for the SBA‑15 group. In contrast to the control and NFM‑1 group, the SBA‑15 group demonstrated a significant reduction in body weight of approximately 6% (Figure 2B). It is important to note that the body lean composition within the SBA‑15 group did not decrease (Supplementary Figure 3) indicating that the weight reduction was not a sign of health compliance. Results indicate the potential of using mesoporous silicas for weight reduction purposes. Even modest weight loss can reduce health risks in obese patients, with medical benefits already beginning after a 5–10% decrease of initial weight [18,19]. A significant positive linear relationship between cholesterol and long-term weight reduction has been determined in populations with BMI ≥28 kg/m2. It is estimated that
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for every 10 kg weight loss, a drop of 0.23 mmol/l in cholesterol may be expected for an obese person, with similar estimations for triglycerides and LDL cholesterol [20]. It has been shown that cholesterol reduction in patients with coronary disease retards or reverses the progression of atherosclerotic disease [21]. In individuals with impaired glucose tolerance, a 7% reduction in body weight has been associated with a 58% reduction of progression to diabetes [22]. High surface area mesoporous silica was expected to adsorb lipids or bile acids within the GI tract similar to dietary fibers. There were no differences in cholesterol, HDL, triglycerides and glucose serum levels between the mice groups at the end of the experiment (week 20), indicating that the particle content in the diet has no effect on lipid and glucose blood levels (Figure 4). Silica may adsorb fatty acids and bile acids leading to their subsequent excretion, but this effect is probably not as strong as expected for mesoporous silica particles. Bile acids are synthetized in the liver and secreted into the bile. A large amount is reabsorbed from the intestine and taken up by the liver, known as enterohepatic circulation [23]. Bile acids have an important role in cholesterol homeostasis. Their biosynthesis implicates the conversion of cholesterol into soluble molecules of bile acids, which are easily secreted. This process is negatively regulated by its end product, bile acid, and accounts for approximately 50% of cholesterol elimination from the body [24]. Furthermore, bile acids are essential for the absorption of lipids, cholesterol and lipid-soluble vitamins by intestinal epithelial cells [25]. The hypocholesterolemic effect observed for amorphous silica and dietary fibers may, at least, be
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Large pore mesoporous silica induced weight loss in obese mice
rials studied. Encapsulation of lipases, which vary in sizes (e.g., 4.9 × 4.9 × 4.6 nm from Rhizopus niveus; 4.6 × 2.6 × 1.1 nm for Porcine pancreatic lipase [26]) into the pores of NFM‑1 should not occur due to its smaller pore size. These, however, have been shown to be selectively encapsulated into SBA‑15 materials with pore sizes in the 10 nm range [12,13]. We have measured the size of the human pancreatic lipase (Protein Data Bank, IPLA2G) directly from structural data and a size of 8.6 × 3.5 × 3.5 nm is estimated. Furthermore, the isoelectric point of lipase also varies between different species and different lipases; for example, 6.7 and 6.1 for human and mice (PNPLA2) lipase enzymes [27]. A close proximity to the isoelectric point of the protein should favor diffusion into the pores. Furthermore, uncharged silanol groups (note that the duodenal pH is 5–7) on the pore wall surface may interact with the enzyme via hydrogen bonding or other interactions.
2.5
2.5
2.0
2.0 HDL (mmol/l)
Cholesterol (mmol/l)
partially due to a reduction in bile acid concentration in the regulating hepatic steroid metabolism in the liver. Overall, the evidence from our data from this experimental set up indicates a strong pore size-dependent effect on weight reduction in obese mice. The absence of an expected hypocholesterolemic effect by SBA‑15 and NFM‑1 particles is indicative that bile acid–cholesterol metabolism is not affected by the particle supplement in the diet in the time frame of the study presented here. Hence, the mechanism underling the weight reduction by SBA‑15 mesoporous silicas may be different than the lipid-lowering effect induced by porous diatomaceous earths and other nonporous silicon dioxides. The mode of action of SBA‑15 mesoporous silica may be due to a lipase sequestration/adsorption effect and its subsequent excretion and/or impeding its interaction with co-lipase. These possible modes of action are consistent with the pore size differences between the two mate-
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Figure 4. Particle content in the diet has no effect on lipid and glucose blood levels. Cholesterol, high-density cholesterol, triglycerides and glucose serum levels analyzed using a timed end point method at the end of the study (experiment week 20). The number of mice per group was: n = 5, 5 and 6 for the control, NFM-1 and SBA-15 groups, respectively (for P glucose, n = 4 in the control group). Error bars represent standard error of the mean. HDL: High-density lipoprotein; P glucose: Plasma glucose.
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Research Article Kupferschmidt, Csikasz, Ballell, Bengtsson & Garcia-Bennett Hence, large pore mesoporous silica through lipase encapsulation and its further excretion (decreasing fat absorption) may allow the modulation of lipase activity. This could be achieved by the use of, for example, different particle doses together with meals. Conclusion SBA‑15 particles embedded in food with HF/sugar content lead to a decrease in weight and body fat composition without observable toxicological signs or systemic absorption of silica. Mesoporous silica materials with big pores (≥110 Å) can potentially be used as pharmaceuticals or food additives for weight loss treatment. We conclude that the inability of NFM‑1 to reduce body weight and fat is primarily determined by its small pore size based on the absence of a cholesterol-lowering effect, together with previous data on lipase-specific adsorption within large pore SBA‑15 particles [28]. This suggests a mechanism based on modulation of lipase activity. This may occur through lipase sequestration into the pores and its excretion or through other mechanisms such as interference on lipase interaction with co-lipase. It is probable that other mechanism or coexisting mechanisms such as bile acid sequestration and a faster passage through the GI tract (e.g., as for digestive fibers) also play an additional role, but further ongoing experiments are required in order to fully understand the action of large pore mesoporous silicas on body weight and fat reduction. Future perspective Obesity is a serious health problem and, with the exception of surgical treatments, it has been shown to lack long-term success. For these reasons, efficient body weight reduction alternatives are highly needed. As mentioned in a recent review by Reis et al., “Modulation of lipase activity through interfacial engineering can be a potential solution” [1]. Mesoporous silica particles may offer this possibility. This study may lead to the development of nanomaterials and mesoporous materials with positive effects on weight loss and body fat composition. This can further lead to a decrease in blood lipids including cholesterol as a result of a general decrease of fat tissue and blood as mentioned above. Long-term studies are needed to reveal the potential capacity of mesoporous silicas to reduce blood lipid levels. Orlistat has been associated with a significant decrease in total cholesterol after adjustment for weight loss after 6–12 months treatment, which is indicative of improved cardiovascular risk factor profiles in obese patients [29]. Orlistat also reduced the incidence of Type 2 diabetes from 9.0 to 6.2% in a
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longer 4‑year trial [30]. If the effect of mesoporous silica on weight reduction occurs through a similar mode of action as orlistat (lipase inhibition) as hypothesized here, the long-term effect may be similar, as well as its side effects. More work including longer treatment times and normal weight animals are needed to shed more light on the mode of action of mesoporous silica materials on body weight and fat reduction, as well as their potential effect on blood lipid levels. Drug treatment of obesity can be considered as a long-term therapy as most patients regain weight upon stopping medication. Mesoporous silicas are proposed here to potentially be used in a similar way as weight reduction drugs, hence longer studies are also needed to assess possible secondary effects related to long-term consumption. The histological examination of major organs such as the liver, intestine and kidneys should be included in these future studies to provide a better assessment of potential negative effects and the mode of action of the particles when administrated orally. No elevated silica blood levels were found in the mice receiving particles in the diet compared with the control mice during this study. However, it should be noted that higher serum or plasma silicon concentrations have been found in patients with chronic renal failure compared with healthy controls [31]. The results presented here should also be taken into consideration in the development of mesoporous silicas as drug delivery systems for oral administration. Acknowledgements The authors are grateful to R Atluri and to MN Iqbal (Nanologica AB, Stockholm, Sweden) for assistance with material characterization and material synthesis. The authors would also like to thank Stockholm University for access to their transmission electron microscopy facilities.
Financial & competing interests disclosure AE Garcia-Bennett is the co-founder of Nanologica AB (Stockholm, Sweden), a company commercializing nanoporous materials for biomedical and related applications. AE Garcia-Bennett is supported by grants by the Swedish Research Council, projects 2009–4716. T Bengtsson is supported by the VR-M from the Swedish Research Council, Novonordiskfonden, Stiftelsen Svenska Diabetesförbundets Forskningsfond, Magnus Bergvall foundation and the Carl Tryggers foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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Large pore mesoporous silica induced weight loss in obese mice
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal
Research Article
experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary • A murine obesity model was used to investigate the effect of mesoporous silica material content in the diet. • Mesoporous silica particles with different pore sizes (20 and 110 Å for NFM‑1 and SBA‑15, respectively) were mixed in the high fat (HF) diet of C57BL/6J obese mice. No particles were added to the diet of control animals. • Particle content in the HF diet did not result in differences between the animal groups when this was the only dietary source. • Obese mice receiving a standard diet with additional HF complement containing SBA‑15 twice a week significantly decreased in weight and body fat composition. • The reduction in body weight and body fat composition exerted by SBA‑15 was attributed to the 110 Å pore size and its ability to encapsulate lipases. • The pore size of mesoporous silicas can be tailored to achieve reduction in body weight and fat composition. mesoporous nanoparticles.
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