In-vivo and In-vitro Monitoring of Amyloid Aggregation via BSA

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In-vivo and In-vitro Monitoring of Amyloid Aggregation via BSA@FGQDs Multimodal Probe Maryam Yousaf, Muhammad Ahmad, Ijaz Ahmad Bhatti, Abdul Nasir, Dr Murtaza Hasan, Xian Jian, Kourosh Kalantar-Zadeh, and Nasir Mahmood ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01216 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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In-vivo and In-vitro Monitoring

of

Amyloid

Aggregation via BSA@FGQDs Multimodal Probe Maryam Yousafa,b, ǂ, Muhammad Ahmadc, ǂ, Ijaz Ahmad Bhattia, Abdul Nasirc, Murtaza Hasand, Xian Jiane, Kourosh Kalantar-zadehf,*, Nasir Mahmoodg,* aDepartment

bCAS

of Chemistry, University of Agriculture Faisalabad, 38040, Pakistan

Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center

for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, P. R. China cDepartment

of Structure and Environmental Engineering, University of Agriculture

Faisalabad, 38040, Pakistan dDepartment

of Biochemistry & Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia

University of Bahawalpur, Bahawalpur 63100, Pakistan eSchool

of Materials and Energy, National Engineering Research Centre of Electromagnetic

Radiation Control Materials, University of Electronic Science and Technology, Chengdu 611731, P.R. China fSchool

of Chemical Engineering, University of New South Wales (UNSW), 2052 Kensington,

New South Wales, Australia gSchool

ǂ These

of Engineering, RMIT University, 3001 Melbourne, Victoria, Australia

authors contributed equally to this work

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KEYWORDS:

19F

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magnetic resonance imaging; multimodal probe; contrast agent;

fluorographene quantum dots; amyloid fibrillation monitoring

ABSTRACT: Early detection of peptide aggregates intermediates is quite challenging because of their variable and complex nature as well as due to lack of reliable sensors for diagnosis. Herein, we report monomers and oligomers specified fluorescence and magnetic resonance imaging (MRI) multimodal probe based on bovine serum albumin capped fluorine functionalized graphene quantum dots (BSA@FGQDs). This probe enables in-vitro fluorescence based monitoring of human islet amyloid polypeptide (hIAPP), insulin and amyloid β(1–42) (Aβ42) monomers and oligomer during fibrillogenesis dynamic. Up to 90% fluorescence quenching of BSA@FGQDs probe upon addition of amyloid monomers/oligomers was observed due to static quenching and non-radiative energy transfer. Moreover, BSA@FGQDs probe shows 10 times higher signals in detecting amyloid intermediates and fibrils than that of conventional thioflavin dye. Negative ΔG° value (-36.21 kJ/mol) indicates spontaneous interaction of probe with peptide. Theses interaction are hydrogen bonding and hydrophobic as proved by thermodynamic parameters. Visual binding clues of BSA@FGQDs with different morphological states of amyloid protein was achieved through electron microscopy. Furthermore, intravenous and intracranial injection of BSA@FGQDs probe in Alzheimer model mice brain enabled in-vivo detection of amyloid plaques in live mice brain by

19F

MRI through contrast enhancement. Our proposed probe not only

effectively monitor in-vitro fibrillation kinetics of number of amyloid proteins with higher sensitivity and specificity than thioflavin dye, but also presence of 19F center makes BSA@FGQDs effective probe as a non-invasive and non-radiative in-vivo amyloid plaques detection probe.


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Spontaneous misfolding of amyloid β(1–42) (Aβ42) and human islet amyloid polypeptide (hIAPP) protein into amyloid fibrils is correlated to the pathology of neurodegerative and progressive disorders such as Alzheimer disease (AD) and type 2 diabetes (T2D), respectively.1-2 The hIAPP aggregation not only leads to the progression of T2D but also contributes in the development of AD as hIAPP fibrils and oligomers, formed during T2D, cross the blood brain barriers (BBB), leading to neurological defects. T2D is controlled by insulin injection, which itself can be aggregated and poses serious issues for T2D treatment.3-5 It is therefore necessary to inhibit the insulin aggregation in order to control T2D that may otherwise further lead to the development of AD. A variety of methods, based on spectroscopy and microscopy techniques, have been reported to monitor amyloid aggregation. 6-7 The most simple, and conventional way of real time monitoring of amyloid fibrillation is fluorescence imaging and spectroscopy, where amyloid aggregates are stained with fluorophores such as thioflavin T and Congo red.7-9 These fluorophores show enhanced fluorescence upon binding with hydrophobic β sheets of amyloid fibrils.10-11 However, these fluorescence methods may result in poor and false results due to the accumulation of multiple fluorophores in hydrophobic regions other than amyloid fibrils.12 Amyloidogensis process involves a number of intermediate products i.e., monomers, oligomers, protofibrils that are matured into fibrils and possess significant cytotoxicity. These monomers, oligomers and protofibrils play an important role in amyloid aggregation. However, due to lack of reliable methods, they are directly undetectable. Currently employed detection methods can only detect amyloid fibrils during log phase of amyloidogensis process that make it difficult to identify true toxic aggregates. Therefore, it is imperative to design an effective and non-invasive probe that can detect the monomers, oligomers and protofibrils involved in amyloidogensis process.9, 13


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Few efforts have been made to monitor amyloid intermediates by storing them in dhexafluoroisopropanol followed by analysis with small angle neutron scattering and dynamic light scattering techniques, which are very complex systems and have limitations for in-vivo applications. Alternatively, fluorescence based resveratrol@GO composites have been reported for ex-vivo labelelling of Aβ42.14-15 In reality, yet simple but sensitive detection methods should be devised that require less complicated facilities and processes. Such methods are highly desired for real time monitoring of amyloid intermediates concentrations at the time of generation and aggregation. Carbon materials (carbon nanotubes, fullerenes and graphene), mainly graphene based nanomaterials could be potential candidates for amyloid fibrillation monitoring because of their unique physico-chemical, fluorescence and biological properties.16 Among one dimensional, two dimensional and zero dimensional (graphene quantum dots (GQDs)) graphene nanomaterials, GQDs possess high water dispersibility, larger surface area with rich surface chemistry, biologically friendly nature, exceptional photostability and strong fluorescence.16-18 Based on these superior properties, GQDs has been used to detect various analytes including metal ions, anions and organic molecules and also in magnetic imaging.19-21 In these systems, GQDs were used for its superb detection ability due to the interaction induced energy transfer and strong concomitant photoluminescence quenching phenomenon. Electronic properties and surface functional groups of GQDs have been efficiently modified by inclusion of electron withdrawing or donating heteroatoms likes nitrogen, sulphur, boron and halogens (F, Cl).22-24 Among these heteroatoms, fluorine (F) atom could easily modify the chemical, electrical and structural features of GQDs because of its high electronegativity.25 Furthermore, F atom due to its strong electron withdrawing effect reduces electron density, thus


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reducing the chances of GQDs interactions with singlet oxygen products during irradiation process, resulting in enhanced fluorescence and photostability of GQDs. Further, F atom also induces magnetic properties by creating a paramagnetic center in graphene. 26-27 Herein, we report F functionalized GQDs (FGQDs) to monitor intermediate species involved in an amyloid aggregation process, its reaction dynamics and in-vivo monitoring of amyloid plaques via fluorescence spectroscopy and

19F

MRI, respectively. Surface of FGQDs was capped with

bovine serum albumin (BSA@FGQDs probe) to prevent FGQDs from aggregation and increase the amino, carboxylic and sulphur functionalities for binding over its surface. BSA@FGQDs probe specifically binds with the amyloid intermediates by π–π staked conjugated network and carboxyl/amino groups. Binding between BSA@FGQDs probe and peptide causes photoluminescence (PL) variation of BSA@FGQDs probe either by resonance energy transfer or by static quenching mechanism. Moreover, blood brain permeability and presence of 19F center makes BSA@FGQDs probe suitable for metal free in-vivo amyloid detection probe, which proves that BSA@FGQDs probe is both effective for monitoring in-vitro amyloid fibrillation kinetics of variety of amyloid proteins with higher sensitivity and specificity than thioflavin dye and also offers a non-invasive and non-radiative probe for in-vivo amyloid detection.

EXPERIMENTAL METHODS Detail description of FGQDs synthesis and characterization is provided in Supporting Information (SI). Synthesis of BSA capped FGQDs probe BSA capped FGQDs was prepared by mixing FGQDs solution (0.4 mg/mL) with BSA in 33:1 mass ratio followed by incubation at room temperature for 5-20 min. Unreacted BSA and FGQDs


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were separated by dialyzing with dialysis bags and centrifuge filters. Resulting purified mixture of BSA and FGQDs was named as BSA@FGQDs that was later on used as an ex-situ probe. Reaction time between BSA and FGQDs was confirmed by measuring FGQDs fluorescence intensity at an emission wavelength of 498 nm. Furthermore, binding between BSA and FGQDs was confirmed by measuring fluorescence spectra of FGQDs at different fluorescence excitation wavelengths (360-520 nm) using spectrophotometer (SpectraMax i3, Molecular Devices, USA) and by HT7700 transmission electron microscope (TEM) for biological (Hitachi, Ltd., 80 kV, Japan). Peptides pre-treatment for amyloid fibrils preparation hIAPP and Aβ42 lyophilized powder were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (1 mg/mL) under constant agitation overnight to break the already formed bonds and completely convert them into monomers. After this, the respective solutions were divided into small aliquots and stored at -20 °C as stock solutions for further use. The working solution of peptide (10 µM) was prepared by evaporating HFIP with the freeze dryer and reconstituting peptide in Milli-Q water. Whereas, to prepare insulin solution, insulin powder (3 mg/mL) was dissolved in in 0.1 M NaCl/HCl acidic solution (pH 1.6), followed by filtration using 0.2 μm filter. Final concentration of insulin solution was spectrophotometrically determined at 278 nm. In-vitro monitoring of amyloid aggregation using BSA@FGQDs probe For the formation of amyloid aggregates of hIAPP and Aβ42, respective peptide solution (10 μM) was incubated at 37 °C under constant stirring at 150 rpm. Whereas to prepare insulin aggregates, 500 μM of insulin solution was prepared in acidic buffer (1.6 pH) and incubated at 65 °C without stirring. Acidic solution was used for accelerating the process of insulin fibrillation. For ex-situ fibrillation kinetics monitoring, 80 μL of hIAPP and Aβ42 aliquot’s were taken out from incubation mixture at particular time intervals (for hIAPP 0, 5, 10, 15, 20, 40, 60, 80, 100, 120


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min, while for Aβ42 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 490 min), followed by mixing with BSA@FGQDs (120 μL) in a 96-well black microtiter plate (Nunc, Roskilde, Denmark) and incubation (30 seconds) with constant stirring in spectrophotometer at room temperature (SpectraMax i3, Molecular Devices, USA) followed by spectral measurements at an emission wavelength of 498 nm. While for insulin fibrillation monitoring, aliquots (80 μL) were taken out from incubation mixture after set time intervals (0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360 min) and diluted with Milli-Q water up to 500× followed by mixing with 120 μL of BSA@FGQDs probe and incubation at room temperature for 3 min followed by spectral measurements. 50 μM ThT solution was used as a control in all ex-situ experiments. Briefly, ThT solution (20 μL) was mixed with incubated peptide solution (80 μL) in a 96-well black microtiter plate (Nunc, Roskilde, Denmark) and diluted with Milli-Q water up to 200 μL working volume followed by ThT fluorescence measurement at an emission wavelength of 482 nm. Other conditions were similar as adopted for the BSA@FGQDs probe. Study of interaction between BSA@FGQDs probe and peptides using fluorescence quenching Interaction between BSA@FGQDs probe and peptides were investigated by measuring fluorescence quenching of BSA@FGQDs probe upon the addition of different concentrations (025 µM) of amyloid peptides monomeric/oligomeric species (quencher). Change in fluorescence intensity of BSA@FGQDs probe was measured using F-4600 fluorescence spectrophotometer (Hitachi, Japan). This fluorescence quenching data was used for plotting Stern–Volmer plot in order to explain weather the fluorescence quenching of BSA@FGQDs probe solution is either due to static quenching effect or dynamic quenching effect. Quenching rate constant (Kq) was also calculated by Stern–Volmer plot in the presence and absence of the quencher (amyloid peptides


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monomeric/oligomeric species) in BSA@FGQDs probe. Moreover, fluorescence quenching data was employed to find out the values of available number of binding sites of BSA@FGQDs probe and binding constant. Further, for the measurement of the values of life time decay constants (τ) of BSA@FGQDs probe, before and after addition of quencher, time-resolved spectrofluorometer (Perkin Elmer Instruments) at an excitation wavelength of 365 nm was used. Afterwards, concentrations in the order of 0 to 220 µg/mL of the quencher was added in BSA@FGQDs probe solution followed by fluorescence quenching measurements using F-4600 fluorescence spectrophotometer (Hitachi, Japan) at an excitation wavelength of 365 nm that was used for the calculation of percentage fluorescence quenching efficiency using the following equation:

% 𝐐𝐮𝐞𝐜𝐧𝐜𝐡𝐢𝐧𝐠 𝐞𝐟𝐟𝐢𝐜𝐢𝐞𝐧𝐜𝐲 =

PL intensity of BSA@FGQDs probe without quencher ― PL intensity of BSA@FGQDs probe with quencher PL intensity of BSA@FGQDs probe without quencher

× 100

(1)

In-vivo detection of amyloid aggregates in AD mice with F19MRI The AD and age controlled normal mice (12–24 weeks) were anesthetized before mounting at stereotactic instrument and injected with 10 µL of BSA@FGQDs probe solution (500 µg/mL), both types of mice were also subjected to

19F

and 1H MRI pre-scan before injecting them with

BSA@FGQDs probe. Moreover, 200 µL of BSA@FGQDs probe (1mg/mL) was intravenously administrated to AD and age controlled normal mice (12–24 weeks) by continuous infusion for 15 min and scanned after 40 min of injection. Mice were analyzed using Biospec 70/20 USR AV 3HD MRI. Note: the details of in-vitro cytotoxicity in human cell lines and in-vivo biodistribution in mice for BSA@FGQDs probe is provided in supporting information.


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RESULTS AND DISCUSSION Binding mechanism between BSA and FGQDs in BSA@FGQDs probe The FGQDs are prepared according to the described method and well-characterized prior to further use as shown in Figure S1. The TEM and high resolution TEM (HRTEM) images clearly show that as-synthesized FGQDs are of the mean value of ~2.9 nm in size and well crystalline having a lattice spacing of 0.214 nm (Figure S1a-b). The existence of core level of F (~688 eV) in x-ray photoelectron spectroscopy (XPS) spectrum (Figure S1c) and strong peak of C-F bond at 1080 cm-1 in Fourier transform infrared (FTIR) spectrum (Figure S1d) confirmed the doping of fluorine in GQDs. Atomic percent of fluorine (F) atoms in FGQDs was 19.24 %. These F atoms were efficiently doped throughout the GQDs. Further FGQDs show strong green fluorescence emission band at 498 nm (Figure S1e), green fluorescence can be seen by naked eyes (the inset of Figure S1e) making it a promising fluorescence probe. Moreover, fitting of tri exponentials curve t1=1.6 ns (fast component) and t2=3.3 ns and t3=5.9 ns (two slow components) to fluorescence life time decay curve of FGQDs (Figure S1f) show excitation-dependent PL emissions of FGQDs. After confirming the proper growth of FGQDs, amino functionalized BSA was decorated on the FGQDs through constructing a bond between amino group of BSA and carboxyl/flouro groups of FGQDs. The binding mechanism to construct FGQDs@BSA probe was confirmed by TEM (Figure 1), two different types of bindings exist between FGQDs and BSA. In one, several FGQDs surround the BSA molecules forming the FGQDs@BSA probe (Figure 1a). While, in another one, conformational changes (unfolding/denaturation) in secondary structure of BSA occurred in the presence of FGQDs favors adsorption of BSA over FGQDs surface, resulting in the increased FGQDs size (Figure 1b) from the mean value of ~2.9 to ~5.9 nm (Figure 1c), suggesting a likely coating of BSA protein over FGQDs.


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These conformational changes in secondary structure of BSA i.e., unfolding of BSA protein in the presence of/upon adsorption over FGQDs structure, can be clearly seen in circular dichroism (CD) spectrum of BSA protein (Figure S2a). In normal CD spectra of BSA at neutral pH (7.4), two characteristic minima are seen at 208 and 222 nm. However, in the presence of FGQDs a decline in α helical character is likely. In fact, the BSA secondary structure may not be completely deformed but instead slightly shifted. Such structural perturbance and unfolding, due to the presence of negatively charged particles, have been quoted in previous studies, where mainly hydrophobic and electrostatic interactions have been involved in destabilizing protein structure.28 In our case, the observed changes in secondary structure of BSA protein is possibly attributed to H-bonded and electrostatic interactions between –COO/–NH2 groups present on tryptophan, aspartate and glutamate residues of BSA and –COO/–NH2 groups of FGQDs. These interactions are confirmed by quenching of BSA PL after the formation of the BSA@FGQDs probe (Figure S2b). BSA shows fluorescence due to the tryptophan residue, and in the presence of FGQDs this fluorescence is quenched as an evidence regarding the involvement of tryptophan residue in binding with FGQDs. BSA and FGQDs binding and BSA structural alteration were further confirmed by FTIR spectra (Figure S2c). In the absence of FGQDs, normal structure of BSA corresponds to two bands namely amide I (1600 cm-1) and amide II (1500 cm-1) associated with native and unfolded conformation of BSA. Slight shifts from 1600 to 1650 cm-1 and from 1500 to 1560 cm-1, along with reduction in peak intensity in amide I and amide II bands, respectively in the presence of FGQDs, confirmed the surface binding and subsequent conformational changes in BSA. Conclusively, the minimal structural alteration in BSA structure can be due to unfolding of domains I and II, whereas overall tertiary structure of BSA is retained even after binding with FGQDs. Moreover, the hydrophobic


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interaction, followed by H-bonding and electrostatic interaction with FGQDs, may cause minimal structural deformation of BSA but instead forms highly effective multi-modal probe BSA@FGQDs.

Figure 1. (a) TEM image of FGQDs@BSA probe in which yellow arrows point at attached FGQDs, (b) a representative TEM image of BSA@FGQDs probes and (c) particle size distribution of BSA@FGQDs based on images similar to Figure 1b.

In-vitro monitoring of amyloid fibrillation by BSA@FGQDs probe In our previous study, we found that FGQDs upon interaction with hIAPP monomeric/oligomeric species inhibit hIAPP aggregation.29 Thus, it is hypothesized that PL properties of FGQDs must be influenced by these interactions, which facilitate the possibility of using BSA@FGQDs as a fluorescence probe for monitoring amyloidosis processes. To confirm this hypothesis, effect of amyloid monomer and fibrils over PL response of BSA@FGQDs probe were evaluated using three model amyloid peptides namely Aβ42, hIAPP and insulin because of common biophysical and structural properties of their fibrils.30 It is obvious from Figure 2a,b that PL of BSA@FGQDs probe was quenched (up to 90%) upon the addition of Aβ42, hIAPP and insulin monomers. Whereas no remarkable effect over the PL response of BSA@FGQDs probe was observed upon


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adding Aβ42, hIAPP and insulin fibrils, ascertaining different interaction of BSA@FGQDs probe with monomers and fibrils. Furthermore, a continuous decrease in PL of BSA@FGQDs probe upon increment in monomeric concentration (0-300 μg/mL) is obvious in Figure 2c. An obvious decrease in the PL intensity of BSA@FGQDs probe (Figure 2c) upon increasing the concentration of monomers presented a relatively wide detection range of the BSA@FGQDs probe. This distinct PL behavior for monomers and fibrils ascertain the use BSA@FGQDs as a new fluorescence probe for monitoring amyloid monomer concentrations involved in the progression of amyloid aggregation. A simulation experiment was performed to confirm the feasibility of monitoring of amyloid fibrillation dynamics using BSA@FGQDs probe (Figure 2d). The PL response of BSA@FGQDs probe was measured at an emission wavelength of 498 nm after mixing it with the mixture containing increasing fibrillar percentage and decreasing monomers concentrations of amyloids (Aβ42, hIAPP and insulin). A continuous increase in BSA@FGQDs probe PL intensity (70 times) with the increase in fibrillar percentage confirmed the feasibility and sensitivity of monitoring fibrillation dynamics using BSA@FGQDs probe.


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Figure 2. (a) Variation in the PL response of BSA@FGQDs probe solution (400 μg/mL) in response to monomeric and fibrillar forms of Aβ42, hIAPP and insulin. (b) Snapshots of BSA@FGQDs mixture of monomeric and fibrillar form of peptides taken under UV light illumination (365 nm). (c) Plot of changes in the PL intensity of BSA@FGQDs upon addition of different concentrations of monomers and (d) simulation experiment with varying percentage of fibrillar forms of Aβ42, hIAPP and insulin at an excitation wavelength of 498 nm.

Additionally, the ability of BSA@FGQDs probe to monitor fibrillation kinetics was compared with ThT by performing the ex-situ time-dependent fluorescent monitoring of amyloid fibrillation. Fibrillation samples were withdrawn at different time intervals (as shown in Figure 3a,c,e), mixed with probe and ThT followed by the PL measurement of BSA@FGQDs probe and ThT. A continuous increase in the PL intensity of FGQDs@BSA probe (up to 80 %) with


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quite obvious three distinct amyloid growth phases (nucleation, elongation and saturation phase), which is involved in the progression of fibrils growth (Figure 3a,c,e), is proof-of-concept that FGQDs@BSA probe fluorescence can efficiently measure kinetics of amyloid aggregation even with more accuracy than the standard ThT (Figure 3b,d,f). It is seen from the fibrillation kinetics curves (Figure 3) that BSA@FGQDs probe is superior in comparison to conventional dye ThT. The hIAPP fibrillated very fast within 120 min mostly with no observable nucleated phase when monitored though ThT (Figure 3b) but a distinct lag phase of ⁓5-10 min can be clearly seen in Figure 3a when monitored by FGQDs@BSA probe, showing strong sensitivity of FGQDs@BSA probe for amyloid intermediates than ThT. This difference in fibrillation kinetics monitoring behavior of BSA@FGQDs probe (Figure 3c,e) and ThT (Figure 3d,f) is more obvious in case of insulin and Aβ42 detection, especially during the nucleation phase. The t1/2 (the time required for the formation of 50 % fibrils) calculated for two curves of Aβ42 and insulin as ~150 and ~100 min, for BSA@FGQDs probe (Figure 3c,e), while ~80 and ~50 min for ThT (Figure 3d,f), respectively. This significant difference in calculated t1/2 for the two processes is ascribed to two different detection mechanisms. The ThT monitor amyloid aggregation process by binding with β-sheets of amyloid peptides,14 therefore, continuous increase in ThT PL intensity with passage of time is attributed to increased β-sheets contents that might be fibrillar contents. Whereas BSA@FGQDs probe detection mechanism is based upon binding with monomeric/oligomeric contents, where, at nucleation phase mainly monomeric aggregates are present. These aggregates form oligomers, which are then matured into amyloid fibrils. Thus, besides the early detection and growth trend of the fibrils, the curve of BSA@FGQDs probe also provides the information of the oligomers in the fibrillization process. These qualities make BSA@FGQDs probe a more sensitive


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approach as it can detect different species involved in the fibrillation process. These results makes BSA@FGQDs probe a potential candidate for amyloid fibrillation detection.

Figure 3. Real time fluorescence based study of fibrillation kinetics via BSA@FGQDs (a,c,e) and ThT (b,d,f), (hIAPP (a,b), Aβ4210 µM (c,d) and insulin 500 µM (e,f)).

Binding of BSA@FGQDs probe with different morphological states of amyloid aggregates


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A variable PL response of BSA@FGQDs probe during fibrillation kinetics suggested binding interactions of BSA@FGQDs with different states of amyloid proteins. To test this prediction, morphological studies were carried as shown in Figure 4. TEM image taken at the nucleation stage shows interaction between BSA@FGQDs probe and hIAPP (27.58 and 38 nm) (Figure 4a) and Aβ42 (28 nm) oligomers (Figure 4e).31,32 Yellow circles in Figure 4a and e highlight the BSA@FGQDs probe and oligomers complex, whereas yellow and red arrows point towards BSA@FGQDs probe and oligomers, respectively. Whereas increment in BSA@FGQDs particle size up to 4-8 nm was observed (Figure 4c) upon binding with insulin monomers, complying that insulin monomeric species might have surrounded the BSA@FGQDs probe, resulting in increased particle sizes. As previously mentioned, BSA@FGQDs probe is a mixture of FGQDs and BSA, therefore to discriminate between BSA particle and peptide monomers/oligomers, TEM micrograph of BSA was taken. Particle size was measured (Figure S3) and compared with particle size of oligomeric species of peptides. The BSA particles are having an average size of 23 nm, which is smaller than the size of that observed for peptide oligomers attached with BSA@FGQDs probe. Furthermore, BSA@FGQDs probe can also binds with hIAPP, insulin and Aβ42 fibrils, as can be deduced from TEM images taken at elongation phase (Figure 4b,d,f). Amyloid proteins contains both positively and negatively charged groups that led to the electrostatic interactions with the BSA@FGQDs probe due to the presence of oppositely charged groups that facilitated complex formation followed by electron transfer, that ultimately quenched the PL intensity of BSA@FGQDs probe.33 The amyloid fibrils also possess charged groups and aromatic compounds, therefore, interaction of amyloid fibrils with the BSA@FGQDs probe cannot be completely ruled out. The faster self-assembly of fibrils may results in reduced number of binding sites for BSA@FGQDs probe, however, BSA@FGQDs probe completely covered all the available vacant


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sites, leading to appropriate interaction of amyloid fibrils with the BSA@FGQDs probe, presented by the arrows (arrows pointed towards attached BSA@FGQDs probe) in Figure 4b,d,f. Thus, morphological studies have confirmed that BSA@FGQDs probe has a strong interaction with various intermediates of amyloid, which assist in their detection at each phase with clear distinction.


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Figure 4. TEM images showing binding interaction of BSA@FGQDs probe with hIAPP, insulin and Aβ42 during nucleation (a,c,e) and elongation phase (b, d, f) of fibrillation process. Scale bars in the insets of image d and e are 20 nm.


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To further understand the binding interaction between BSA@FGQDs probe and amyloid peptide, AFM images were taken for BSA@FGQDs probe, with and without the addition of amyloid peptide monomers/oligomeric species and fibrils. Spherical shaped FGQDs could be clearly seen in Figure 5(a) that upon the addition of BSA, binding with it form a changed morphology BSA@FGQDs probe (Figure 5(b)). Upon the addition of peptide solution (mixture of oligomers and fibrils), BSA@FGQDs probe co-localized both oligomers and fibrils (Figure 5(c-e)). The possible formation of BSA@FGQDs@peptide oligomers complex is shown by black circles in Figure 5(c-e). Oligomeric species were identified by size measurements that were ~38, ~30 and ~25 nm for hIAPP, Aß42 and insulin oligomers, respectively. Rest of the image possibly shows the binding between BSA@FGQDs and fibrils. Because of the fast aggregation of hIAPP clusters, dense fibrillar network with attached BSA@FGQDs probe can be seen in Figure 5(c), whereas due to the slow aggregation of Aß42 (Figure 5(d)) with few fibrils, some oligomeric species attached onto BSA@FGQDs probe are obvious. Insulin aggregation/fibrillation was also fast due to the acidic experimental condition adopted for insulin fibrillation. Therefore, only few oligomers (black circle, ~25 nm in size), bound with BSA@FGQDs probe, are seen while the rest are mostly attached to the fibrils (Figure 5(e)). These AFM results are consistent with the observation from the TEM micrographs, confirming binding interactions of BSA@FGQDs probe with different morphological states of amyloids.


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Figure 5. AFM images of (a) FGQDs, (b) BSA@FGQDs probe, (c) BSA@FGQDs probe@hIAPP oligomers (black circles)/fibrils, (d) BSA@FGQDs probe@Aß42 oligomers (black circles)/fibrils and (e) BSA@FGQDs probe@insulin.

Study of interaction between BSA@FGQDs probe and peptides using fluorescence quenching The interactions between BSA@FGQDs probe and amyloid peptides monomeric/oligomeric species were studied by measuring fluorescence quenching in the presence of different concentrations of amyloid peptides monomeric/oligomeric species. Fluorescence intensity of BSA@FGQDs probe decreases upon continuous increase in the concentration of peptides monomeric/oligomeric species (Figure S4) without any significant shift in emission wavelength or change in peak shape, demonstrating intrinsic fluorescence quenching of probe. Quenched


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fluorescence shows the formation of BSA@FGQDs@peptide complex as surrounding peptides change/block the microenvironment around the probe. Herein by assessing fluorescence of the interaction, we have described fluorescence quenching mechanism, binding constant of BSA@FGQDs@peptide complex and number of binding sites available over BSA@FGQDs probe for peptides.

Fluorescence quenching mechanism Fluorescence quenching is mainly due to static quenching effect (SQE), dynamic quenching effect (DQE) and resonant energy transfer (RET).34 It is speculated that monomers/oligomers form a complex

with

BSA@FGQDs

probe’s

surface,

resulting

in

the

formation

of

BSA@FGQDs@peptide complex (Scheme 1). This complex absorbed the energy that was released in the form of non-radiative decay, therefore, could not be observed in emission spectra of BSA@FGQDs@peptide complex.


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Scheme 1. Schematic representation of quenching mechanism of BSA@FGQDs in presence of various peptides.

Further, fluorescence quenching may be either due to SQE or DQE or by both. DQE arises due to formation of an excited state complex between quencher and fluorophore resulting in non-radiative decay, while SQE is due to formation of non-fluorescence complexes at ground state due to orbital overlapping or charge transfer. Stern–Volmer equation can well explain mechanism and extent of SQE or DQE as:35

Fo F

(2)

= 1 + KSV[Q] = 1 + Kq 𝜏o[Q]


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where Fo and F are fluorescence intensities of fluorophore in the absence and presence of quencher (peptide), respectively. KSV and Kq are Stern–Volmer and bimolecular quenching rate constants, respectively, and 𝜏o is fluorescence life time decay constant that is 5.9 ns in the absence of quencher. A linear plot between Q and Fo/F is obtained that shows either SQE and/or DQE is responsible for PL quenching of BSA@FGQDs probe (Figure 6a). The SQE and DQE can be differentiated on the basis of dependency over lifespan measurement, viscosity and temperature. Values of Ksv and Kq calculated at different temperatures are given in Table 1, Ksv is calculated from the slop of graph, whereas values of Kq is calculated by using the following equation:36

(3)

Ksv = Kq 𝜏0

Table 1. Values of Kq and Ksv for BSA@FGQDs@peptide complex for various peptide complexes at different temperatures. Ksv (μM-1)

Ksv (μM-1)

Ksv (μM-1)

Kq (M-1 S-1)

Kq (M-1 S-1)

Kq (M-1 S-1)

(298 K)

(303 K)

(308 K)

(298 K)

(300 K)

(308 K)

hIAPP

0.00056

0.00020

0.00016

9.4×1011

8.1×1011

7.1×1011

Aβ42

0.0028

0.0015

0.0010

4.7×1010

3.3×1010

2.7×1010

Insulin

0.0016

0.0014

0.0012

2.7×1010

1.7×1010

0.6×1010

Peptide

The decreasing Ksv and Kq values with increase in temperature suggest that quenching is purely static, ruling out dynamic quenching. Because in case of DQ binding constant values increases with increase in diffusion, maximum value reported for binding constant is 1×1010 M-1s-1 , whereas


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Kq quite higher than maximum possible value are obtained ruling out DQE.36 We further calculated the static quenching by noting time-resolved fluorescence values of BSA@FGQDs probe solution before and after addition of peptide. In case of SQE excited state lifetime remains constant, while for DQE τ0/τ ratio varied proportional to the F0/F ratio. The τo value in the absence of peptide was 5.9 ns, which is slightly changed to 5.8, 5.7 and 5.6 ns upon addition of peptide implying that SQE is responsible for BSA@FGQDs probe PL quenching.

Calculation of BSA@FGQDs probe’s number of binding sites The available number of binding sites (n) over BSA@FGQDs probe were calculated by applying Scatchard equation, assuming independent interaction of peptide monomer/oligomers with BSA@FGQDs probe as:37 log

F0 ― F F

(4)

= log Kb +nlog [Q]

where Kb is the binding constant. The intercept and the slope of Figure 6b represent the values of Kb and n respectively. The estimated number of binding sites of BSA@FGQDs for peptide is equal to 1 ruling out the presence of fractional and higher than 1 binding site. These results confer that a single independent binding sites is available over BSA@FGQDs probe for binding of single monomer/oligomer of peptide.


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2.8

(a)

0.4

log[Fo-F/F]

-0.4

2.0

-0.8

1.6

-1.2

1.2 0.8

(b)

0.0

2.4

log[F/Fo]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

-1.6 0

5

10

Q

15 [M]

20

25

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

log[Q]

BSA@FGQDs+hIAPP

BSA@FGQDs+A

BSA@FGQDs+Insulin

Figure 6. (a) Stern-Volmer plot of FGQD@BSA@peptide conjugate and (b) number of binding sites 𝐹0 ― 𝐹

calculation by 𝑙𝑜𝑔

𝐹

versus log[peptides concentration, μM] plot.

Extent of fluorescence quenching by amyloid peptide (quencher) was further determined by calculating the percentage of quenching efficiency. From Figure S6 it is seen that fluorescence quenching efficiency by the quencher increases with the increase in the quencher concentrations from 0 to 220 µg/mL. At 220 µg/mL concentration of quencher (hIAPP, Aß42 and insulin monomers/oligomeric species) fluorescence of BSA@FGQDs probe was quenched up to 80% (hIAPP), 91% (Aß42) and 90 % (insulin). Efficient quenching of BSA@FGQDs probe could be attributed to binding with quencher either due to hydrogen bonding or hydrophobic interaction that causes non-radiative decay of electrons. Evaluation of binding forces Mainly hydrogen bonding, electrostatic, hydrophobic, or van der Waals forces drive the interaction between the macromolecules (proteins) and small molecules.38 Thermodynamic parameters like change in Gibbs free energy (ΔG°), entropy (ΔS°) and enthalpy (ΔH°) provide valuable information


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Page 26 of 36

about these interactions. The ΔH° and ΔS°˂ 0 indicate hydrophobic interaction, while ˃ 0 reflect the formation of van der Waals force or hydrogen bonding, whereas ΔH°⁓ 0 and ΔS°> 0 suggest electrostatic force. Van’t Hoff equation is used for calculating thermodynamic parameters as:

lnKb =

― ΔH° RT

+

ΔS°

(5)

R

where Kb is binding constant, R is gas constant and T is absolute temperature. ΔG° was calculated using ΔG° = ΔH° ― TΔS°

.

Negative values of ΔG° (-36.21 kJ/mol) indicate that binding between probe and peptide monomers is spontaneous and thermodynamically favorable. The negative value of ΔH° (-55.21 kJ/mol) suggests hydrogen bonding and/or van der Waals forces, while positive value of ΔS° (64.25 kJ/mol) is an evidence for hydrophobic interaction. 4, 39-41 In-vivo detection of amyloid aggregates in AD mice with F19MRI In-vivo studies were carried out to capture amyloid ß plaques in living animals by detecting 19F nucleus–derived magnetic resonance signals using

19F-containing

compound that specifically

labels amyloid ß plaques in the brain. Presence of F atom gives a paramagnetic center to FGQDs, making them suitable for magnetic imaging through 19F MRI.42 The BSA@FGQDs probe within 40 min pass blood brain barrier upon intravenously injecting to old mice (6-8 weeks) providing the possibility to detect amyloid fibrils in brain after certain modifications (Figure S5). Later, BSA@FGQDs probe was injected in AD mice, amyloid plaques signals were obtained in T2-weighted 1H and 19F MRI, whereas no signals were achieved in T1-weighted 1H and 19F MRI scan. In AD mice brain, strong signals of

19F

were achieved as compared to the normal mice

(Figure 7a), especially in the hippocampal region, where there is maximum localization of the amyloid plaques (Figure 7b) confirming successful attachment of our probe with amyloid plaques.


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Furthermore, administration of BSA@FGQDs probe enhanced the contrast of amyloid plaques (Figure 7c and d), thus increased the reliability of amyloid ß plaques detection. Contrast enhancement might be due to decrease in T2 relaxation time, it can be speculated that paramagnetic center (F) interacts with the applied magnetic field and produce its own magnetic field. This insitu induced magnetic field by FGQDs causes in homogeneity that interacts with the protons, resulting a reduced relaxation time followed by enhanced contrast.43 Herein, 19F MRI images were generated by averaging two 1-h frames to detect amyloid ß plaques acquired through a series of 1-h scans. Therefore, before trials on human begin, the process needs slight modification in the hardware and software of MRI for the sake of sensitivity and specificity. Moreover, a better signalto-noise ratio can be achieved by the use of surface coil radio-frequency receiver arrays. High spatial resolution used for AD mice brain here is not required for the plaques detection in human’s brain. Currently used metal-based MRI contrast agents are quite expensive, while carbon-based FGQDs will be a better choice both from economical aspects and toxicological issues, making MRI based detection inexpensive and more reliable. Further, low toxicity measured by in-vitro cytotoxicity in human cell lines and in-vivo biodistribution in mice for BSA@FGQDs probe as shown in Figure S7 and S8, respectively, confirming these probes have no side effects. Thus, we believe that as-developed multi-modal BSA@FGQDs probe can be implemented for real applications and helpful in early detection of the target diseases.


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Figure 7. 19FMRI signals of BSA@FGQDs probe in normal mice brain (a) and detection of in-vivo amyloid plaques (indicated by arrows) in AD mice. Comparison of 1H MRI signals before (c) and after (d) BSA@FGQDs probe injection.

CONCLUSIONS In summary, we developed a BSA@FGQDs multi-modal fluorescence and MRI based probe, which can detect amyloid peptide monomers/oligomers, perform the real time monitoring of dynamics of amyloid monomers into amyloid fibrillation and in-vivo label amyloid β aggregates in AD mice brain. The BSA@FGQDs as a new fluorescent probe can monitor amyloid protein


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fibrillation with more sensitivity compared to conventional dye ThT and its ability to bind with the amyloid monomers made it superior to ThT. Monitoring of amyloid aggregation dynamic and monomers/oligomers by BSA@FGQDs probe is based upon hydrophobic, electrostatic, H-bonded and π−π-stacking interactions as proved by morphological and thermodynamics studies. Upon the interaction between probe and amyloid monomers/oligomers, PL of FGQDs@BSA probe is changed instantly, either quenched or released, and this PL quenching was well explained by static quenching mechanism that is proved by the Stern-Volmer plot. The BSA@FGQDs probe can cross BBB that leads to the detection of amyloid β plaques in the brain of AD mice by MRI imaging with higher contrast than a conventional benchmark. Imaging agents that can detect Aβ monomers in-vitro and in-vivo like as-developed BSA@FGQDs probe are believed to be essential for disease prevention, diagnosis, and medical treatment monitoring and are therefore greatly needed.

ASSOCIATED CONTENT Supporting Information Supporting information contains part of experimental methods, TEM, HRTEM, particle size distribution curves, XPS spectrum, FTIR spectrum, PL spectra and in-vivo 19F MRI scan images. AUTHOR INFORMATION Corresponding Author Dr. Nasir Mahmood ([email protected]) Prof. Kourosh Kalantar-Zadeh ([email protected])

Acknowledgement


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The research was performed with the collaboration of the National Natural Science Foundation, China, Peking University, National Center for Nanoscience and Technology, University of Agriculture Faisalabad, Pakistan. The authors are very thankful to all institutions for providing the support and infrastructure to complete the project. Conflict of Interest Authors declare no conflict of interest. Ethical Approval All animal experiments were performed according to the protocol approved by Institutional Animal Care and all mice were place in hygienic conditions (pathogen-free) to avoid animal’s diseases. References 1.

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In-vivo and In-vitro Monitoring of Amyloid Aggregation via BSA

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