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Development of thioflavin-modified mesoporous silica framework for amyloid fishing
Accepted Manuscript Development of Thioflavin-modified Mesoporous Silica Framework for Amyloid Fishing Vivekanandan Viswanathan, Gopal Murali, Sakthivel Gandhi, Priyadharshini Kumaraswamy, Swaminathan Sethuraman, Uma Maheswari Krishnan PII: DOI: Reference:
S1387-1811(14)00317-5 http://dx.doi.org/10.1016/j.micromeso.2014.05.045 MICMAT 6591
To appear in:
Microporous and Mesoporous Materials
Received Date: Revised Date: Accepted Date:
19 October 2013 13 January 2014 28 May 2014
Please cite this article as: V. Viswanathan, G. Murali, S. Gandhi, P. Kumaraswamy, S. Sethuraman, U.M. Krishnan, Development of Thioflavin-modified Mesoporous Silica Framework for Amyloid Fishing, Microporous and Mesoporous Materials (2014), doi: http://dx.doi.org/10.1016/j.micromeso.2014.05.045
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Development of Thioflavin-modified Mesoporous Silica Framework for Amyloid Fishing Vivekanandan Viswanathan, Gopal Murali, Sakthivel Gandhi, Priyadharshini Kumaraswamy, Swaminathan Sethuraman, Uma Maheswari Krishnan*
Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) School of Chemical & Biotechnology SASTRA University, Thanjavur – 613 401, India
________________________________________________________________________ *Corresponding Author Prof. Uma Maheswari Krishnan Ph. D. Deakin Indo–Australia Chair Professor Associate Dean for the Departments of Chemistry, Bioengineering & Pharmacy Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) School of Chemical & Biotechnology SASTRA University, Thanjavur – 613 401 TamilNadu, India Ph.: (+91) 4362 264101 Ext: 3677 Fax: (+91) 4362 264120
E–mail: [email protected]
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Abstract Amyloid beta peptide accumulation in the brain poses a serious threat during Alzheimer’s disease. Various strategies to remove or disrupt these plaques with nanoparticles is an emerging area for the treatment for neurodegenerative diseases. The present work attempts to develop a novel strategy to remove the plaques using magnetic field by employing magnetic nanoparticles conjugated with a plaque–targeting ligand. Thioflavin–S (TF–S) was covalently linked to magnetic iron oxide incorporated mesoporous silica, SBA–15 and the material was characterized using microscopic and spectroscopic techniques. This magnetic conjugate (IO– SBA–TF–S) was found to display the ability to remove in vitro, KLVFF peptide, a recognition motif in β–Amyloid that has been implicated in plaque formation. This system represents a potential smart system that could herald in the next generation therapeutic strategy for Alzheimer’s disease and related disorders. Keywords Magnetic nanoparticles, SBA–15, amyloid fishing, thioflavin
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1. Introduction Aggregation and misfolding of specific proteins is a seminal occurrence in numerous neurodegenerative diseases like Alzheimer’s (AD) and prion diseases [1]. However studies and research over decades has provided considerable evidence on the pathogenesis of the disease and hence new therapeutic targets have emerged. Information on the fundamental basis of pathogenesis of the disease surfaced with the identification of proteins that comprises the lesions characteristic of Alzheimer’s are β–amyloid (Aβ) in senile plaques [2] and tau in neurofibrillary tangles [3,4]. Evidences suggest that Aβ aggregation is a critical trigger that initiates a chain of events resulting in tauopathy, neural dysfunction and dementia [5-7]. These plaques form at the synaptic terminal making nervous conduction severed [5].
Disparate methodologies to control and prevent aggregation of these plaques have been demonstrated and practiced but with scant efficacy. Tramiprosate, a glycosaminoglycan analogue that directly binds to the monomeric Aβ, reduces aggregation and helps in the clearance [8,9]. An altered trafficking approach where an antibody against Aβ is employed to opsonize, and aid receptor mediated transcytosis at the blood brain barrier has also been attempted [10]. Heparin, gelsolin and other molecules that trap Aβ in the blood and mitigate accumulation in the brain have been investigated using animal models [11]. Other Aβ reducing therapies that have been reported include inhibition of β–/γ– secretases or activation of α–secretase, activation of neprilysin–like proteases and regulation of Aβ precursor protein translation by phenserine [12]. More promising treatment strategies that have been attempted include use of anti–inflammatory drugs [13-15], metal chelators [16], antioxidants [17] and even the insulin resistance drug rosiglitazone [18]. Attempts to design drugs that disrupt plaque formation have met with limited success [19, 20]. Beta sheet blockers such as Soto
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peptide and Tjernberg peptides have been reported to prevent progression of plaque formation but remain to be clinically evaluated [21].
Crossing the impregnable blood–brain barrier (BBB) has been a challenge that further limits the use of these therapeutic molecules. In this context, the use of nanoparticles offers an incredible therapeutic edge by mimicking lipoproteins and aides selective uptake through the blood–brain barrier by surfactant modification [22-24]. Nanoparticles prepared from synthetic and natural polymers ranging in size between 10 and 1000 nm show potential to cross the blood brain barrier [25,26]. Polysorbate–80 like surfactant-coated nanoparticles of size less than 300 nm has the ability to transport drug through BBB [22-24] and shows enhanced biodistribution, therapeutic efficacy, and lessened drug toxicity [26].
The application of magnetic nanoparticles in medicine and biology is varied and has become quintessential in the recent times due to their diverse nature and tunable properties such as specificity, magnetophoretic mobility, cytotoxicity, surface–to–volume ratio, shape and size [27]. These nanoparticles have been employed in the process of cell sorting, radio nuclei delivery, drug delivery, targeting studies, and contrast enhancing in MRI [28-35]. A recent study has reported the destruction of tumor cells via artificially induced hyperthermia mediated by magnetic nanoparticles where the main process involved being the heat produced by the magnetic nanoparticle when subjected to an external AC magnetic field. The particles were observed to generate heat up to temperature of 42°C [36]. In the context of Alzheimer’s disease, magnetic nanoparticles have been primarily used as magnetic contrast enhancers to visualize the plaques with high resolution through MRI [37-39]. The present work attempts to develop a novel strategy to remove the plaques using magnetic field by employing magnetic nanoparticles conjugated with a plaque–targeting ligand, thioflavin–S.
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The ligand selection was based on its specificity to bind to the beta sheets in the amyloid plaques as well as its fluorescent properties that aids sensitive detection. Mesoporous silica containing iron oxide was employed as the magnetic system due to its amenability for chemical modification and biocompatibility.
2. Materials and methods 2.1. Materials H2N–KLVFF–CONH2, a pentapeptide with >95% purity was purchased from Bioconcept Labs Pvt. Ltd (Gurgaon, India). Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagles Medium (DMEM) phosphate buffered saline (PBS) solution and antibiotics (Penicillin– Streptomycin (P/S)) were purchased from Gibco, USA. CellTiter 96® AQueous one solution was purchased from Promega, USA.
2.2. Synthesis of iron oxide doped mesoporous silica (IO–SBA–15) The IO–SBA–15 was synthesized using the cooperative formation mechanism as described by Gandhi et al [40]. The method uses tetraethylorthosilicate (TEOS) as the silica source and triblock
copolymer
polyethyleneoxide–polypropyleneoxide–polyethyleneoxide
(P–123,
Sigma–Aldrich Chemicals, USA) that acts as the structure-directing agent. The medium was rendered acidic using aqueous solution of concentrated HCl and FeCl3 was used as the iron source. Five grams of P–123 was added to 60 mL of water and stirred till a clear solution is obtained. pH was maintained below 1 by 2 N HCl and the solution was stirred for 2 h and 9 g of TEOS was added along with equimolar ratio of FeCl3 and stirred at room temperature. It was then refluxed at 80 °C for a period of 24 h. The precipitate thus obtained was cooled, filtered and washed with deionized water for several cycles; then dried and calcined at 550 °C for 6 h.
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2.3. Chemical modification of IO–SBA–15 Two hundred and fifty milligrams of the IO–SBA–15 synthesized as mentioned above was dispersed in 40 mL of toluene and 1 mM of aminopropyltriethoxysilane (APTES, Sigma Aldrich, USA) was added. The above setup was kept for stirring under reflux at 100 °C for 24 h. The precipitate formed was dried in vacuum. The chemical reaction involved is depicted in Scheme 1. To the precipitate obtained in step 1, 1 mM of methylmethacrylate (MMA, Merck, Germany) was added after dispersion in 40 mL of methanol.
After stirring for 24 h at room
temperature, the precipitate obtained was dried in vacuum. The reaction involved in this stage is depicted in Scheme 2. In the final stage, N–hydroxysuccinimide (NHS, Merck, India) (1 mg/mL), 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC, Merck, India) (1 mg/mL) and 10 mL methanol was added to the above product and stirred. Then 1 mM of thioflavin–S (Sigma Aldrich, USA) dissolved in 30 mL of methanol was added drop–wise and stirred at room temperature for 24 h. The final product obtained was centrifuged and washed for several cycles with methanol to remove excess thioflavin–S. Scheme 3 shows the reaction involved in the formation of thioflavin–S linked IO–SBA–15.
2.4. Characterization of the organically modified IO–SBA–15 The mesoporous silica samples were analyzed using Fourier transform infrared spectroscopy (FT–IR). FT–IR analysis was carried out between 4000 and 450 cm–1 at a resolution of 4 cm– 1
and averaging 25 scans. The ultrafine structural features of the mesoporous samples were
evaluated using field emission transmission electron microscopy (FE–TEM), JEM 2100F, JEOL, Japan. The removal of organic moieties and the presence of silica network were
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determined by evaluating the weight loss with increase in temperature using thermogravimetry (SDT Q600, TA Instruments, USA). The samples were analyzed over a temperature range of 0 °C to 1000 °C at a heating rate of 10 °C / minute under nitrogen atmosphere. The fluorescence of the IO-SBA-TF-S samples was analyzed using spectrofluorimetry (LF45, Perkin Elmer, USA). The fluorescent IO-SBA-TF-S was also imaged using laser scanning confocal microscopy (FV1000, Olympus, Japan).
2.5. Separation efficiency of the IO-SBA-TF-S The separation of the beta structures formed by Ab
–modified IO–SBA–15
was investigated using a pentapeptide segment KLVFF from Ab
determination of the separation efficiency, 1 mg/mL of KLVFF peptide was incubated in phosphate buffer solution for 24 h to facilitate the formation β–sheets structure similar to Aβ plaques, which occurs in the pathogenesis of Alzheimer’s disease [41, 42]. After incubation, IO–SBA–TF–S was added to it at different peptide (IO–SBA–TF–S) ratios of 1:10, 1:15 and 1:25. A NdFeB magnet was placed adjacent to the wall of the container and left undisturbed for 8 hours to ensure complete separation of the magnetic particles. The supernatant was then removed and the fluorescence was measured.
2.6. In vitro studies IMR 32 human neuroblastoma cells procured from National Centre for Cell Sciences (NCCS), Pune, India were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10 % Fetal Bovine Serum (FBS, Gibco, USA) and 1 % penicillin/streptomycin (Gibco, USA). The culture was maintained at 37 C in 5 % carbon dioxide incubator. Cell cytotoxicity was quantified using 3–(4,5–dimethylthiazol–2–yl)–5–
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(3–carboxymethoxyphenyl)–2–(4–sulfophenyl)–2H–tetrazolium (MTS) assay (Cell Titer 96® AQueous One solution, Promega, USA). Ten thousand cells were seeded in a 96 well plate. After 24 h, the cells were incubated with the IO-SBA-TF-S for 24 h. After incubation, the medium was removed and the cells were washed with phosphate buffered saline (PBS) to remove any non–adherent cells. 200 L of serum free media and 20 L of MTS reagent were added and incubated at 37C for 2 hours. The reaction was stopped by the addition of 25 L of 10% sodium dodecyl sulfate (SDS) solution. The absorbance was then measured at 490 nm using a multimode reader (Infinite 200M, Tecan®, USA). Untreated cells served as control.
2.7. Statistical analysis Analysis of Variance (one–way ANOVA) was performed to determine the statistical significance (p < 0.05) for determination of separation efficiency and MTS assay (n = 3).
3. Results and Discussion 3.1. Chemical modification of IO–SBA–15 with thioflavin–S The functionalization of IO–SBA–15 with APTES, methyl methacrylate and thioflavin–S was investigated using FT-IR spectroscopy. Table 1 summarizes the major vibration bands obtained in the FT-IR spectra of the IO–SBA–15 sample after each stage of chemical modification.
All three samples exhibit bands between 1080 and 1090 cm–1 indicating a Si–O–Si network that is characteristic of mesoporous silica samples [40]. The –OH bend is observed between 1400 and 1410 cm–1 in all samples. The presence of a vibration band between 555 and 562 cm–1 in all three samples may be attributed to the Fe–O bond indicating the presence of iron oxide in the mesoporous samples. The presence of a band around 798 cm–1 may be attributed
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to the Si–C stretching vibrations suggesting the conjugation of organic moieties with the silica framework. Methylene stretching vibrations are observed at 2945 cm–1 for the APTES modified sample, 2930 cm–1 for the MMA modified sample and at 2927 cm–1 for the thioflavin–S linked sample. This further confirms the presence of organic moieties in the mesoporous sample. The APTES modified sample exhibits a vibration band at 1623 cm–1 arising due to the bending vibration of a primary amine group. The stretching vibration of the primary amine group appears at 3433 cm–1 confirming the presence of the –NH2 terminal group in the APTES modified sample. The MMA modified IO–SBA–15 sample (IO–SBA– 15–APTES–MMA) shows an additional band at 1718 cm–1 due to the stretching of ester carbonyl group. The occurrence of a band at 3447 cm–1 may be attributed to the stretching vibration of secondary amine indicating the conjugation of the methyl methacrylate moiety with the amine terminus of the APTES modified IO–SBA–15. The IO-SBA-TF-S sample shows a vibration band at 1634 cm–1 characteristic of the amide carbonyl stretch. The band at 1557 cm–1 may be assigned to the bending vibrations of the amide –NH– bond. Bands at 1347 cm–1 due to S=O asymmetric stretch and 697 cm–1 due to the out–of–plane bending of aromatic ring (=C–H bond) confirm the conjugation of thioflavin–S with the iron oxide containing mesoporous silica network.
3.2. Thermogravimetric analysis (TGA) Figure 1 shows the thermogravimetric profiles of IO–SBA–15, thioflavin–S and IO-SBA-TFS. The initial weight loss observed in the case of IO–SBA–15 between 100–150 °C can be attributed to the removal of moisture from the mesoporous network. Beyond this temperature range, no significant loss in the weight is discernible. On the contrary, the thermogravimetric profile of thioflavin–S shows distinct weight loss regions around 110 °C due to removal of moisture and around 400 °C, 580 °C and 700 °C due to step–wise degradation of the aromatic
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rings in thioflavin. The IO-SBA-TF-S shows an initial weight loss of 5 % around 100 °C owing to the loss of moisture followed by a progressive weight loss between 400–600 °C indicating the presence of organic moieties due to the conjugation with thioflavin.
The conjugation of thioflavin with the mesoporous silica was further confirmed by confocal microscopy and fluorescence spectroscopy. Figure 2A shows the laser scanning confocal images of the IO-SBA-TF-S, Figure 2B shows the emission intensity of the sample in the absence of pentapeptide KLVFF and Figure 2C shows the emission intensity of the sample in the presence of pentapeptide KLVFF. Intense green fluorescence observed from the IOSBA-TF-S is observed confirming the presence of thioflavin in the sample. Thioflavin has the ability to bind with beta sheet forming peptides and proteins. The KLVFF sequence is present in the amyloid b peptide and hence can serve as a recognition element for amyloid b[4]
Hence
incubation of thioflavin with KLVFF will result in a shift in the fluorescence emission of the thioflavin moiety from 430 nm to 510 nm. The emission profile of the IO-SBA-TF-S sample on incubation with the pentapeptide KLVFF exhibits a maximum at 510 nm when excited at 440 nm. This emission is characteristic of the thioflavin binding to the pentapeptide. These results confirm the conjugation of thioflavin with the IO–SBA–15.
3.3. Morphology characterization The transmission electron micrographs of the IO–SBA–15 and the IO-SBA-TF-S are shown in Figure 3. The transmission electron micrograph of IO–SBA–15 shows the characteristic highly oriented mesoporous framework of SBA–15.
The iron oxide loaded in the
mesoporous matrix is observed as dark spots distributed throughout the mesoporous structure. In the thioflavin-conjugated samples, the ultrafine mesoporous network is not clearly visible
10
due to masking of the mesoporous structure by the organic moieties conjugated to the mesoporous silica – APTES, MMA and finally thioflavin. However, the presence of iron oxide is still visible as dark spots distributed throughout the sample.
3.4. X–ray photoelectron spectral analysis of the IO-SBA-TF-S The presence of iron oxide in the mesopores of IO-SBA-TF-S was further confirmed using x– ray photoelectron spectroscopy (XPS). Figure 4 shows the total scan and element scan spectra recorded for the IO-SBA-TF-S. The survey spectrum indicated the presence of carbon, oxygen, silicon, nitrogen and iron (Figure 4A). This confirms the presence of organic moieties and iron in the silica matrix.
The element scan spectrum of carbon shows a peak at 284.9 eV, which corresponds to C–C bonds present in the organic substituents (Figure 4B). The element scan spectrum of silicon shows a peak at 103.5 eV, which is distinctly different from the binding energy of the elemental silicon (99.4 eV) and silicon nitride (101.7 eV) (Figure 4C). The observed binding energy of 103.5 eV corresponds to SiO2. Figure 4D shows the element scan spectrum of nitrogen, which exhibits a binding energy peak at 399.9 eV that may correspond to C–NH2 bond or N–Si2O moiety. The element scan spectrum of oxygen shows an asymmetric peak at 532.8 eV, which may be attributed to the oxygen in SiO2 (Figure 4E). However, peak resolution shows two peaks – one centered around 532.8 eV and a smaller peak centered around 530.2 eV that corresponds to a metal oxide, which in this case is iron oxide. The binding energy for iron 2p is observed at 710.7 eV and another peak appears at 723.2 eV (Figure 4F). These peaks are characteristic of the high spin Fe2O3, which splits as a complex multiplet. Hence, it can be inferred that the iron oxide is in Fe2O3 form in the mesoporous matrix.
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3.5. Evaluation of the magnetic properties of the chemically modified samples The transmission electron micrographs confirm the presence of iron oxide in the thioflavinmodified samples. However, it is possible that the presence of the organic substituents in the silica matrix may mask the magnetic properties of the iron oxide. To confirm that the iron oxide present in the IO-SBA-TF-S exhibited magnetic properties, the saturation magnetization was recorded at room temperature using vibration scanning magnetometer (VSM). Figure 5 shows the plot of magnetic moment against the magnetic field for the IOSBA-TF-S.
The saturation magnetization of the IO-SBA-TF-S is about 6 emu/g. This value is only slightly lower than those reported for IO–SBA–15, which ranges between 8–10 emu/g [43]. Thus, IO-SBA-TF-S can be used for magnetic applications.
3.6. Evaluation of the ‘fishing’ efficiency of the IO–SBA–TF-S Figure 6 shows the image of the separation of the IO-SBA-TF-S from a solution containing KLVFF peptide on application of magnetic field. It was clearly observed that application of a magnetic field causes complete separation of the IO-SBA-TF-S (Figures 6 A & B). To establish whether the KLVFF present in the solution has indeed been removed by the IOSBA-TF-S, the clear solution was quantified for residual KLVFF. Figure 6C shows the efficiency of the IO-SBA-TF-S to separate KLVFF from the solution. The ratio of the peptide and IO-SBA-TF-S was varied from 1:10, 1:15 and 1:25. The removal of KLVFF from the solution may have occurred due to its interactions with the thioflavin-S moiety linked with the mesoporous matrix or through adsorption into the mesopores. To identify whether the fishing efficiency was due to adsorption, the mesoporous sample after separation with the
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magnetic field was dispersed in an aqueous medium and left for 48 h with continuous shaking to desorb any peptide that might have adsorbed in the mesoporous matrix. The separation efficiency was corrected by subtracting the concentration of the desorbed KLVFF. A slight reduction in the separation efficiency was obtained, which was however, not statistically significant. This confirms that the separation of the peptide was predominantly due to its interaction with the thioflavin moiety. The separation efficiency ranged between 65–80% for different ratios of peptide to mesoporous sample. The pores in the IO-SBA-TF-S play a significant role for the incorporation of iron oxide. It may be possible that free amino groups resulting from incomplete reaction between aminopropyl groups and methyl methacrylate could interact with the peptide. Similar possibilities have been reported in literature on mesoporous silica modified with aminopropyl groups that tend to associate through extensive hydrogen bonding leading to occlusion of the pores [44]. However, in the present study, there exists a remote possibility of free amino groups in the mesoporous IO-SBA-TF-S because of the optimized ratio of the reactants adopted in the reaction. Also, the FTIR spectrum of the thioflavin-S modified mesoporous silica (IO-SBA-TF-S) does not reveal characteristic bending vibration of primary amine around 1620 cm-1 suggesting absence of any free amino group. Hence the interactions of KLVFF are expected to be with the thioflavin moiety in the mesoporous sample facilitating its separation on application of an external magnetic field.
3.7. Cell viability studies The influence of IO-SBA-TF-S on cell viability is an important parameter that can determine its further applications in vivo. Figure 7 shows the cell viability studies carried out using IMR-32 human neuroblastoma cells. It was observed that the IO-SBA-TF-S did not bring about any significant decrease in the cell viability thereby suggesting its suitability for further investigations towards in vivo applications.
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The concept of magnetic separation of amyloid plaques i.e., ‘fishing’ of the plaques can be a novel strategy that can be explored further as a possible stratagem against Alzheimer’s disease (Figure 8). The property of thioflavin–S moiety to selectively bind to amyloid plaques has been well–documented [38]. Further, thioflavin–S does not bind to amyloid monomers unlike thioflavin–T and hence is more specific in binding the plaques [45]. Thus, it can serve as the ‘bait’ to bind the plaques. The use of iron oxide loaded mesoporous silica confers the ability to ‘reel’ in the ‘fish’ i.e., plaques. Moreover, the ability of iron oxide loaded mesoporous silica to serve as a magnetic contrast enhancer had been demonstrated earlier by our group [40]. This can enable visualization of the plaques through magnetic resonance imaging.
After separation of the plaques, the question of their fate post–separation emerges. Though separation of the plaques alone may not constitute complete cure for Alzheimer’s disease, the mesoporous matrix may be used to load drugs that can bring about disruption of the aggregates that have been ‘fished’ using the IO-SBA-TF-S. This smart strategy may serve as a complete package for alleviating plaque–associated complications in Alzheimer’s disease.
4. Conclusion The present work demonstrates for the first time the development of a thioflavin modified iron oxide loaded mesoporous silica and its promise for separation of amyloid fibrils as a possible stratagem to reduce the plaque load in Alzheimer’s disease. The work employed a pentapeptide KLVFF present in the amyloid beta that has been implicated as a recognition element in the formation of plaques through aggregation. Future studies employing amyloid beta in in vivo models may serve to validate this concept.
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Acknowledgement The authors wish to acknowledge FIST programme, Department of Science & Technology (SR/FST/LSI–058/2010
&
SR/FST/LSI–327/2007)
and
SASTRA
University
for
infrastructural support.
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Figure Captions: Figure 1. Thermogravimetric profiles of IO–SBA–15, thioflavin–S and thioflavin–modified IO–SBA–15 Figure 2. [A] Laser Scanning Confocal Image of thioflavin conjugated IO–SBA–15; [B] Emission intensity of the thioflavin conjugated IO–SBA–15 in the absence of KLVFF; [C] Emission intensity of the thioflavin conjugated IO–SBA–15 with KLVFF when excited at 440 nm Figure 3. Transmission electron micrographs of [A] IO–SBA–15 and [B] Thioflavin conjugated IO–SBA–15 Figure 4. [A] Survey spectrum of thioflavin conjugated IO–SBA; [B] element scan of Si; [C] element scan of C; [D] element scan of O; [E] element scan of N; [F] element scan of Fe Figure 5. Magnetization profile of thioflavin–modified IO–SBA–15 recorded at room temperature Figure 6. Image of thioflavin conjugated IO–SBA–15 in PBS containing KLVFF peptide [A] Before application of magnetic field; [B] After application of magnetic field and [C] efficiency of thioflavin conjugated IO–SBA–15 to separate KLVFF Figure 7. Viability of IMR 32 cells after 24 h of incubation with thioflavin conjugated IO– SBA–15 Figure 8. Cartoon depicting the Concept of amyloid ‘fishing’ Scheme 1: Introduction of amine functional group to mesoporous silica (IO–SBA–15) through reaction with aminopropyltriethoxy silane (APTES) Scheme 2: Conjugation of methyl methacrylate to APTES modified mesoporous silica Scheme 3: Covalent linking of thioflavin–S to MMA modified IO–SBA–APTES List of Table Table 1. Vibration frequencies of organically modified IO–SBA–15
19
Vibration frequency (cm–1) APTES MMA Thioflavin–S modified conjugated linked sample 560 555 562 – – 697 798 798 797 1090 1093 1082 – – 1347 1400 1409 1410 – – 1557 1623 – – – – 1634 – 1718 – 2945 2930 2927 3433 – – – 3447 –
Bond involved
Fe–O stretch Out–of–plane bend of aromatic ring =C–H Si–C stretch Si–O–Si stretch –S=O asymmetric stretch –OH bend –NH– (amide) bend –NH2 bend –C=O (amide) stretch –C=O (ester) stretch –CH2– stretch –NH2 stretch –NH– (secondary amine) stretch
20
Scheme 1
Scheme 2
Scheme 3
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Highlights
A novel strategy to remove amyloid plaques using magnetic field
Thioflavin-S was covalently linked to magnetic mesoporous silica for the first time
Magnetic conjugate remove KLVFF peptide that has been implicated in Aβ formation
Graphical abstract
S1387-1811(14)00317-5 http://dx.doi.org/10.1016/j.micromeso.2014.05.045 MICMAT 6591
To appear in:
Microporous and Mesoporous Materials
Received Date: Revised Date: Accepted Date:
19 October 2013 13 January 2014 28 May 2014
Please cite this article as: V. Viswanathan, G. Murali, S. Gandhi, P. Kumaraswamy, S. Sethuraman, U.M. Krishnan, Development of Thioflavin-modified Mesoporous Silica Framework for Amyloid Fishing, Microporous and Mesoporous Materials (2014), doi: http://dx.doi.org/10.1016/j.micromeso.2014.05.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of Thioflavin-modified Mesoporous Silica Framework for Amyloid Fishing Vivekanandan Viswanathan, Gopal Murali, Sakthivel Gandhi, Priyadharshini Kumaraswamy, Swaminathan Sethuraman, Uma Maheswari Krishnan*
Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) School of Chemical & Biotechnology SASTRA University, Thanjavur – 613 401, India
________________________________________________________________________ *Corresponding Author Prof. Uma Maheswari Krishnan Ph. D. Deakin Indo–Australia Chair Professor Associate Dean for the Departments of Chemistry, Bioengineering & Pharmacy Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) School of Chemical & Biotechnology SASTRA University, Thanjavur – 613 401 TamilNadu, India Ph.: (+91) 4362 264101 Ext: 3677 Fax: (+91) 4362 264120
E–mail: [email protected]
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Abstract Amyloid beta peptide accumulation in the brain poses a serious threat during Alzheimer’s disease. Various strategies to remove or disrupt these plaques with nanoparticles is an emerging area for the treatment for neurodegenerative diseases. The present work attempts to develop a novel strategy to remove the plaques using magnetic field by employing magnetic nanoparticles conjugated with a plaque–targeting ligand. Thioflavin–S (TF–S) was covalently linked to magnetic iron oxide incorporated mesoporous silica, SBA–15 and the material was characterized using microscopic and spectroscopic techniques. This magnetic conjugate (IO– SBA–TF–S) was found to display the ability to remove in vitro, KLVFF peptide, a recognition motif in β–Amyloid that has been implicated in plaque formation. This system represents a potential smart system that could herald in the next generation therapeutic strategy for Alzheimer’s disease and related disorders. Keywords Magnetic nanoparticles, SBA–15, amyloid fishing, thioflavin
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1. Introduction Aggregation and misfolding of specific proteins is a seminal occurrence in numerous neurodegenerative diseases like Alzheimer’s (AD) and prion diseases [1]. However studies and research over decades has provided considerable evidence on the pathogenesis of the disease and hence new therapeutic targets have emerged. Information on the fundamental basis of pathogenesis of the disease surfaced with the identification of proteins that comprises the lesions characteristic of Alzheimer’s are β–amyloid (Aβ) in senile plaques [2] and tau in neurofibrillary tangles [3,4]. Evidences suggest that Aβ aggregation is a critical trigger that initiates a chain of events resulting in tauopathy, neural dysfunction and dementia [5-7]. These plaques form at the synaptic terminal making nervous conduction severed [5].
Disparate methodologies to control and prevent aggregation of these plaques have been demonstrated and practiced but with scant efficacy. Tramiprosate, a glycosaminoglycan analogue that directly binds to the monomeric Aβ, reduces aggregation and helps in the clearance [8,9]. An altered trafficking approach where an antibody against Aβ is employed to opsonize, and aid receptor mediated transcytosis at the blood brain barrier has also been attempted [10]. Heparin, gelsolin and other molecules that trap Aβ in the blood and mitigate accumulation in the brain have been investigated using animal models [11]. Other Aβ reducing therapies that have been reported include inhibition of β–/γ– secretases or activation of α–secretase, activation of neprilysin–like proteases and regulation of Aβ precursor protein translation by phenserine [12]. More promising treatment strategies that have been attempted include use of anti–inflammatory drugs [13-15], metal chelators [16], antioxidants [17] and even the insulin resistance drug rosiglitazone [18]. Attempts to design drugs that disrupt plaque formation have met with limited success [19, 20]. Beta sheet blockers such as Soto
3
peptide and Tjernberg peptides have been reported to prevent progression of plaque formation but remain to be clinically evaluated [21].
Crossing the impregnable blood–brain barrier (BBB) has been a challenge that further limits the use of these therapeutic molecules. In this context, the use of nanoparticles offers an incredible therapeutic edge by mimicking lipoproteins and aides selective uptake through the blood–brain barrier by surfactant modification [22-24]. Nanoparticles prepared from synthetic and natural polymers ranging in size between 10 and 1000 nm show potential to cross the blood brain barrier [25,26]. Polysorbate–80 like surfactant-coated nanoparticles of size less than 300 nm has the ability to transport drug through BBB [22-24] and shows enhanced biodistribution, therapeutic efficacy, and lessened drug toxicity [26].
The application of magnetic nanoparticles in medicine and biology is varied and has become quintessential in the recent times due to their diverse nature and tunable properties such as specificity, magnetophoretic mobility, cytotoxicity, surface–to–volume ratio, shape and size [27]. These nanoparticles have been employed in the process of cell sorting, radio nuclei delivery, drug delivery, targeting studies, and contrast enhancing in MRI [28-35]. A recent study has reported the destruction of tumor cells via artificially induced hyperthermia mediated by magnetic nanoparticles where the main process involved being the heat produced by the magnetic nanoparticle when subjected to an external AC magnetic field. The particles were observed to generate heat up to temperature of 42°C [36]. In the context of Alzheimer’s disease, magnetic nanoparticles have been primarily used as magnetic contrast enhancers to visualize the plaques with high resolution through MRI [37-39]. The present work attempts to develop a novel strategy to remove the plaques using magnetic field by employing magnetic nanoparticles conjugated with a plaque–targeting ligand, thioflavin–S.
4
The ligand selection was based on its specificity to bind to the beta sheets in the amyloid plaques as well as its fluorescent properties that aids sensitive detection. Mesoporous silica containing iron oxide was employed as the magnetic system due to its amenability for chemical modification and biocompatibility.
2. Materials and methods 2.1. Materials H2N–KLVFF–CONH2, a pentapeptide with >95% purity was purchased from Bioconcept Labs Pvt. Ltd (Gurgaon, India). Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagles Medium (DMEM) phosphate buffered saline (PBS) solution and antibiotics (Penicillin– Streptomycin (P/S)) were purchased from Gibco, USA. CellTiter 96® AQueous one solution was purchased from Promega, USA.
2.2. Synthesis of iron oxide doped mesoporous silica (IO–SBA–15) The IO–SBA–15 was synthesized using the cooperative formation mechanism as described by Gandhi et al [40]. The method uses tetraethylorthosilicate (TEOS) as the silica source and triblock
copolymer
polyethyleneoxide–polypropyleneoxide–polyethyleneoxide
(P–123,
Sigma–Aldrich Chemicals, USA) that acts as the structure-directing agent. The medium was rendered acidic using aqueous solution of concentrated HCl and FeCl3 was used as the iron source. Five grams of P–123 was added to 60 mL of water and stirred till a clear solution is obtained. pH was maintained below 1 by 2 N HCl and the solution was stirred for 2 h and 9 g of TEOS was added along with equimolar ratio of FeCl3 and stirred at room temperature. It was then refluxed at 80 °C for a period of 24 h. The precipitate thus obtained was cooled, filtered and washed with deionized water for several cycles; then dried and calcined at 550 °C for 6 h.
5
2.3. Chemical modification of IO–SBA–15 Two hundred and fifty milligrams of the IO–SBA–15 synthesized as mentioned above was dispersed in 40 mL of toluene and 1 mM of aminopropyltriethoxysilane (APTES, Sigma Aldrich, USA) was added. The above setup was kept for stirring under reflux at 100 °C for 24 h. The precipitate formed was dried in vacuum. The chemical reaction involved is depicted in Scheme 1. To the precipitate obtained in step 1, 1 mM of methylmethacrylate (MMA, Merck, Germany) was added after dispersion in 40 mL of methanol.
After stirring for 24 h at room
temperature, the precipitate obtained was dried in vacuum. The reaction involved in this stage is depicted in Scheme 2. In the final stage, N–hydroxysuccinimide (NHS, Merck, India) (1 mg/mL), 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC, Merck, India) (1 mg/mL) and 10 mL methanol was added to the above product and stirred. Then 1 mM of thioflavin–S (Sigma Aldrich, USA) dissolved in 30 mL of methanol was added drop–wise and stirred at room temperature for 24 h. The final product obtained was centrifuged and washed for several cycles with methanol to remove excess thioflavin–S. Scheme 3 shows the reaction involved in the formation of thioflavin–S linked IO–SBA–15.
2.4. Characterization of the organically modified IO–SBA–15 The mesoporous silica samples were analyzed using Fourier transform infrared spectroscopy (FT–IR). FT–IR analysis was carried out between 4000 and 450 cm–1 at a resolution of 4 cm– 1
and averaging 25 scans. The ultrafine structural features of the mesoporous samples were
evaluated using field emission transmission electron microscopy (FE–TEM), JEM 2100F, JEOL, Japan. The removal of organic moieties and the presence of silica network were
6
determined by evaluating the weight loss with increase in temperature using thermogravimetry (SDT Q600, TA Instruments, USA). The samples were analyzed over a temperature range of 0 °C to 1000 °C at a heating rate of 10 °C / minute under nitrogen atmosphere. The fluorescence of the IO-SBA-TF-S samples was analyzed using spectrofluorimetry (LF45, Perkin Elmer, USA). The fluorescent IO-SBA-TF-S was also imaged using laser scanning confocal microscopy (FV1000, Olympus, Japan).
2.5. Separation efficiency of the IO-SBA-TF-S The separation of the beta structures formed by Ab
–modified IO–SBA–15
was investigated using a pentapeptide segment KLVFF from Ab
determination of the separation efficiency, 1 mg/mL of KLVFF peptide was incubated in phosphate buffer solution for 24 h to facilitate the formation β–sheets structure similar to Aβ plaques, which occurs in the pathogenesis of Alzheimer’s disease [41, 42]. After incubation, IO–SBA–TF–S was added to it at different peptide (IO–SBA–TF–S) ratios of 1:10, 1:15 and 1:25. A NdFeB magnet was placed adjacent to the wall of the container and left undisturbed for 8 hours to ensure complete separation of the magnetic particles. The supernatant was then removed and the fluorescence was measured.
2.6. In vitro studies IMR 32 human neuroblastoma cells procured from National Centre for Cell Sciences (NCCS), Pune, India were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10 % Fetal Bovine Serum (FBS, Gibco, USA) and 1 % penicillin/streptomycin (Gibco, USA). The culture was maintained at 37 C in 5 % carbon dioxide incubator. Cell cytotoxicity was quantified using 3–(4,5–dimethylthiazol–2–yl)–5–
7
(3–carboxymethoxyphenyl)–2–(4–sulfophenyl)–2H–tetrazolium (MTS) assay (Cell Titer 96® AQueous One solution, Promega, USA). Ten thousand cells were seeded in a 96 well plate. After 24 h, the cells were incubated with the IO-SBA-TF-S for 24 h. After incubation, the medium was removed and the cells were washed with phosphate buffered saline (PBS) to remove any non–adherent cells. 200 L of serum free media and 20 L of MTS reagent were added and incubated at 37C for 2 hours. The reaction was stopped by the addition of 25 L of 10% sodium dodecyl sulfate (SDS) solution. The absorbance was then measured at 490 nm using a multimode reader (Infinite 200M, Tecan®, USA). Untreated cells served as control.
2.7. Statistical analysis Analysis of Variance (one–way ANOVA) was performed to determine the statistical significance (p < 0.05) for determination of separation efficiency and MTS assay (n = 3).
3. Results and Discussion 3.1. Chemical modification of IO–SBA–15 with thioflavin–S The functionalization of IO–SBA–15 with APTES, methyl methacrylate and thioflavin–S was investigated using FT-IR spectroscopy. Table 1 summarizes the major vibration bands obtained in the FT-IR spectra of the IO–SBA–15 sample after each stage of chemical modification.
All three samples exhibit bands between 1080 and 1090 cm–1 indicating a Si–O–Si network that is characteristic of mesoporous silica samples [40]. The –OH bend is observed between 1400 and 1410 cm–1 in all samples. The presence of a vibration band between 555 and 562 cm–1 in all three samples may be attributed to the Fe–O bond indicating the presence of iron oxide in the mesoporous samples. The presence of a band around 798 cm–1 may be attributed
8
to the Si–C stretching vibrations suggesting the conjugation of organic moieties with the silica framework. Methylene stretching vibrations are observed at 2945 cm–1 for the APTES modified sample, 2930 cm–1 for the MMA modified sample and at 2927 cm–1 for the thioflavin–S linked sample. This further confirms the presence of organic moieties in the mesoporous sample. The APTES modified sample exhibits a vibration band at 1623 cm–1 arising due to the bending vibration of a primary amine group. The stretching vibration of the primary amine group appears at 3433 cm–1 confirming the presence of the –NH2 terminal group in the APTES modified sample. The MMA modified IO–SBA–15 sample (IO–SBA– 15–APTES–MMA) shows an additional band at 1718 cm–1 due to the stretching of ester carbonyl group. The occurrence of a band at 3447 cm–1 may be attributed to the stretching vibration of secondary amine indicating the conjugation of the methyl methacrylate moiety with the amine terminus of the APTES modified IO–SBA–15. The IO-SBA-TF-S sample shows a vibration band at 1634 cm–1 characteristic of the amide carbonyl stretch. The band at 1557 cm–1 may be assigned to the bending vibrations of the amide –NH– bond. Bands at 1347 cm–1 due to S=O asymmetric stretch and 697 cm–1 due to the out–of–plane bending of aromatic ring (=C–H bond) confirm the conjugation of thioflavin–S with the iron oxide containing mesoporous silica network.
3.2. Thermogravimetric analysis (TGA) Figure 1 shows the thermogravimetric profiles of IO–SBA–15, thioflavin–S and IO-SBA-TFS. The initial weight loss observed in the case of IO–SBA–15 between 100–150 °C can be attributed to the removal of moisture from the mesoporous network. Beyond this temperature range, no significant loss in the weight is discernible. On the contrary, the thermogravimetric profile of thioflavin–S shows distinct weight loss regions around 110 °C due to removal of moisture and around 400 °C, 580 °C and 700 °C due to step–wise degradation of the aromatic
9
rings in thioflavin. The IO-SBA-TF-S shows an initial weight loss of 5 % around 100 °C owing to the loss of moisture followed by a progressive weight loss between 400–600 °C indicating the presence of organic moieties due to the conjugation with thioflavin.
The conjugation of thioflavin with the mesoporous silica was further confirmed by confocal microscopy and fluorescence spectroscopy. Figure 2A shows the laser scanning confocal images of the IO-SBA-TF-S, Figure 2B shows the emission intensity of the sample in the absence of pentapeptide KLVFF and Figure 2C shows the emission intensity of the sample in the presence of pentapeptide KLVFF. Intense green fluorescence observed from the IOSBA-TF-S is observed confirming the presence of thioflavin in the sample. Thioflavin has the ability to bind with beta sheet forming peptides and proteins. The KLVFF sequence is present in the amyloid b peptide and hence can serve as a recognition element for amyloid b[4]
Hence
incubation of thioflavin with KLVFF will result in a shift in the fluorescence emission of the thioflavin moiety from 430 nm to 510 nm. The emission profile of the IO-SBA-TF-S sample on incubation with the pentapeptide KLVFF exhibits a maximum at 510 nm when excited at 440 nm. This emission is characteristic of the thioflavin binding to the pentapeptide. These results confirm the conjugation of thioflavin with the IO–SBA–15.
3.3. Morphology characterization The transmission electron micrographs of the IO–SBA–15 and the IO-SBA-TF-S are shown in Figure 3. The transmission electron micrograph of IO–SBA–15 shows the characteristic highly oriented mesoporous framework of SBA–15.
The iron oxide loaded in the
mesoporous matrix is observed as dark spots distributed throughout the mesoporous structure. In the thioflavin-conjugated samples, the ultrafine mesoporous network is not clearly visible
10
due to masking of the mesoporous structure by the organic moieties conjugated to the mesoporous silica – APTES, MMA and finally thioflavin. However, the presence of iron oxide is still visible as dark spots distributed throughout the sample.
3.4. X–ray photoelectron spectral analysis of the IO-SBA-TF-S The presence of iron oxide in the mesopores of IO-SBA-TF-S was further confirmed using x– ray photoelectron spectroscopy (XPS). Figure 4 shows the total scan and element scan spectra recorded for the IO-SBA-TF-S. The survey spectrum indicated the presence of carbon, oxygen, silicon, nitrogen and iron (Figure 4A). This confirms the presence of organic moieties and iron in the silica matrix.
The element scan spectrum of carbon shows a peak at 284.9 eV, which corresponds to C–C bonds present in the organic substituents (Figure 4B). The element scan spectrum of silicon shows a peak at 103.5 eV, which is distinctly different from the binding energy of the elemental silicon (99.4 eV) and silicon nitride (101.7 eV) (Figure 4C). The observed binding energy of 103.5 eV corresponds to SiO2. Figure 4D shows the element scan spectrum of nitrogen, which exhibits a binding energy peak at 399.9 eV that may correspond to C–NH2 bond or N–Si2O moiety. The element scan spectrum of oxygen shows an asymmetric peak at 532.8 eV, which may be attributed to the oxygen in SiO2 (Figure 4E). However, peak resolution shows two peaks – one centered around 532.8 eV and a smaller peak centered around 530.2 eV that corresponds to a metal oxide, which in this case is iron oxide. The binding energy for iron 2p is observed at 710.7 eV and another peak appears at 723.2 eV (Figure 4F). These peaks are characteristic of the high spin Fe2O3, which splits as a complex multiplet. Hence, it can be inferred that the iron oxide is in Fe2O3 form in the mesoporous matrix.
11
3.5. Evaluation of the magnetic properties of the chemically modified samples The transmission electron micrographs confirm the presence of iron oxide in the thioflavinmodified samples. However, it is possible that the presence of the organic substituents in the silica matrix may mask the magnetic properties of the iron oxide. To confirm that the iron oxide present in the IO-SBA-TF-S exhibited magnetic properties, the saturation magnetization was recorded at room temperature using vibration scanning magnetometer (VSM). Figure 5 shows the plot of magnetic moment against the magnetic field for the IOSBA-TF-S.
The saturation magnetization of the IO-SBA-TF-S is about 6 emu/g. This value is only slightly lower than those reported for IO–SBA–15, which ranges between 8–10 emu/g [43]. Thus, IO-SBA-TF-S can be used for magnetic applications.
3.6. Evaluation of the ‘fishing’ efficiency of the IO–SBA–TF-S Figure 6 shows the image of the separation of the IO-SBA-TF-S from a solution containing KLVFF peptide on application of magnetic field. It was clearly observed that application of a magnetic field causes complete separation of the IO-SBA-TF-S (Figures 6 A & B). To establish whether the KLVFF present in the solution has indeed been removed by the IOSBA-TF-S, the clear solution was quantified for residual KLVFF. Figure 6C shows the efficiency of the IO-SBA-TF-S to separate KLVFF from the solution. The ratio of the peptide and IO-SBA-TF-S was varied from 1:10, 1:15 and 1:25. The removal of KLVFF from the solution may have occurred due to its interactions with the thioflavin-S moiety linked with the mesoporous matrix or through adsorption into the mesopores. To identify whether the fishing efficiency was due to adsorption, the mesoporous sample after separation with the
12
magnetic field was dispersed in an aqueous medium and left for 48 h with continuous shaking to desorb any peptide that might have adsorbed in the mesoporous matrix. The separation efficiency was corrected by subtracting the concentration of the desorbed KLVFF. A slight reduction in the separation efficiency was obtained, which was however, not statistically significant. This confirms that the separation of the peptide was predominantly due to its interaction with the thioflavin moiety. The separation efficiency ranged between 65–80% for different ratios of peptide to mesoporous sample. The pores in the IO-SBA-TF-S play a significant role for the incorporation of iron oxide. It may be possible that free amino groups resulting from incomplete reaction between aminopropyl groups and methyl methacrylate could interact with the peptide. Similar possibilities have been reported in literature on mesoporous silica modified with aminopropyl groups that tend to associate through extensive hydrogen bonding leading to occlusion of the pores [44]. However, in the present study, there exists a remote possibility of free amino groups in the mesoporous IO-SBA-TF-S because of the optimized ratio of the reactants adopted in the reaction. Also, the FTIR spectrum of the thioflavin-S modified mesoporous silica (IO-SBA-TF-S) does not reveal characteristic bending vibration of primary amine around 1620 cm-1 suggesting absence of any free amino group. Hence the interactions of KLVFF are expected to be with the thioflavin moiety in the mesoporous sample facilitating its separation on application of an external magnetic field.
3.7. Cell viability studies The influence of IO-SBA-TF-S on cell viability is an important parameter that can determine its further applications in vivo. Figure 7 shows the cell viability studies carried out using IMR-32 human neuroblastoma cells. It was observed that the IO-SBA-TF-S did not bring about any significant decrease in the cell viability thereby suggesting its suitability for further investigations towards in vivo applications.
13
The concept of magnetic separation of amyloid plaques i.e., ‘fishing’ of the plaques can be a novel strategy that can be explored further as a possible stratagem against Alzheimer’s disease (Figure 8). The property of thioflavin–S moiety to selectively bind to amyloid plaques has been well–documented [38]. Further, thioflavin–S does not bind to amyloid monomers unlike thioflavin–T and hence is more specific in binding the plaques [45]. Thus, it can serve as the ‘bait’ to bind the plaques. The use of iron oxide loaded mesoporous silica confers the ability to ‘reel’ in the ‘fish’ i.e., plaques. Moreover, the ability of iron oxide loaded mesoporous silica to serve as a magnetic contrast enhancer had been demonstrated earlier by our group [40]. This can enable visualization of the plaques through magnetic resonance imaging.
After separation of the plaques, the question of their fate post–separation emerges. Though separation of the plaques alone may not constitute complete cure for Alzheimer’s disease, the mesoporous matrix may be used to load drugs that can bring about disruption of the aggregates that have been ‘fished’ using the IO-SBA-TF-S. This smart strategy may serve as a complete package for alleviating plaque–associated complications in Alzheimer’s disease.
4. Conclusion The present work demonstrates for the first time the development of a thioflavin modified iron oxide loaded mesoporous silica and its promise for separation of amyloid fibrils as a possible stratagem to reduce the plaque load in Alzheimer’s disease. The work employed a pentapeptide KLVFF present in the amyloid beta that has been implicated as a recognition element in the formation of plaques through aggregation. Future studies employing amyloid beta in in vivo models may serve to validate this concept.
14
Acknowledgement The authors wish to acknowledge FIST programme, Department of Science & Technology (SR/FST/LSI–058/2010
&
SR/FST/LSI–327/2007)
and
SASTRA
University
for
infrastructural support.
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Figure Captions: Figure 1. Thermogravimetric profiles of IO–SBA–15, thioflavin–S and thioflavin–modified IO–SBA–15 Figure 2. [A] Laser Scanning Confocal Image of thioflavin conjugated IO–SBA–15; [B] Emission intensity of the thioflavin conjugated IO–SBA–15 in the absence of KLVFF; [C] Emission intensity of the thioflavin conjugated IO–SBA–15 with KLVFF when excited at 440 nm Figure 3. Transmission electron micrographs of [A] IO–SBA–15 and [B] Thioflavin conjugated IO–SBA–15 Figure 4. [A] Survey spectrum of thioflavin conjugated IO–SBA; [B] element scan of Si; [C] element scan of C; [D] element scan of O; [E] element scan of N; [F] element scan of Fe Figure 5. Magnetization profile of thioflavin–modified IO–SBA–15 recorded at room temperature Figure 6. Image of thioflavin conjugated IO–SBA–15 in PBS containing KLVFF peptide [A] Before application of magnetic field; [B] After application of magnetic field and [C] efficiency of thioflavin conjugated IO–SBA–15 to separate KLVFF Figure 7. Viability of IMR 32 cells after 24 h of incubation with thioflavin conjugated IO– SBA–15 Figure 8. Cartoon depicting the Concept of amyloid ‘fishing’ Scheme 1: Introduction of amine functional group to mesoporous silica (IO–SBA–15) through reaction with aminopropyltriethoxy silane (APTES) Scheme 2: Conjugation of methyl methacrylate to APTES modified mesoporous silica Scheme 3: Covalent linking of thioflavin–S to MMA modified IO–SBA–APTES List of Table Table 1. Vibration frequencies of organically modified IO–SBA–15
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Vibration frequency (cm–1) APTES MMA Thioflavin–S modified conjugated linked sample 560 555 562 – – 697 798 798 797 1090 1093 1082 – – 1347 1400 1409 1410 – – 1557 1623 – – – – 1634 – 1718 – 2945 2930 2927 3433 – – – 3447 –
Bond involved
Fe–O stretch Out–of–plane bend of aromatic ring =C–H Si–C stretch Si–O–Si stretch –S=O asymmetric stretch –OH bend –NH– (amide) bend –NH2 bend –C=O (amide) stretch –C=O (ester) stretch –CH2– stretch –NH2 stretch –NH– (secondary amine) stretch
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Scheme 1
Scheme 2
Scheme 3
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Highlights
A novel strategy to remove amyloid plaques using magnetic field
Thioflavin-S was covalently linked to magnetic mesoporous silica for the first time
Magnetic conjugate remove KLVFF peptide that has been implicated in Aβ formation
Graphical abstract