Article Cite This: Mol. Pharmaceutics 2018, 15, 3356−3368
Calmidazolium Chloride and Its Complex with Serum Albumin Prevent Huntingtin Exon1 Aggregation Virender Singh,† R. N. V. Krishna Deepak,‡ Bhaswati Sengupta,€ Abhayraj S. Joshi,† Hao Fan,‡,§ Pratik Sen,€ and Ashwani Kumar Thakur*,† †
Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, India Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, India ‡ Bioinformatics Institute, 30 Biopolis Street, Matrix #07-01, Singapore 138671 § Department of Biological Sciences, National University of Singapore, Singapore 117545
Mol. Pharmaceutics 2018.15:3356-3368. Downloaded from pubs.acs.org by UNIV OF BRITISH COLUMBIA on 01/03/19. For personal use only.
€
S Supporting Information *
ABSTRACT: Huntington’s disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing an expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules for HD, which is currently untreatable. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Because it is hydrophobic, CLC was adsorbed to the vial surface and could not sustain an inhibition effect for a longer duration. The use of bovine serum albumin (BSA) prevented CLC adsorption by forming a BSA−CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, the NT17 part of Httex1. Furthermore, biocompatible CLC-loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells. KEYWORDS: Huntington’s disease, polyglutamine aggregation, calmidazolium chloride, adsorption, serum albumin and peptides yielding oligomer, protofibril, and fibril formation,20 we selected a molecule with a known antiaggregation effect on other amyloid-forming proteins. Taking aromatic interaction-mediated inhibition as a reference, we chose to test calmidazolium chloride (CLC), which is known to modulate amyloid-β (1−40) aggregation to prevent mature fiber formation.21 CLC is a hydrophobic molecule and carries a positive charge with an imidazolium ring at the center and a large extended hydrophobic region at the periphery. It is a known calmodulin (CaM) antagonist22,23 and also blocks the transient receptor potential cation channel subfamily V member 1 (TRPV1) channel to alter calcium transport to relieve pain.24 CaM is one of the known targets in HD, which binds to mutant huntingtin (mHtt) and regulates transglutaminase 2 dependent crosslinking of Htt.25 Testing a molecule which could show a bispecific property (i.e., target Httex1 aggregation as well as block CaM association with mHtt and transglutaminase) might have high therapeutic value.25,26 In this article, we show the ability of a hydrophobic molecule calmidazolium chloride (CLC) to prevent Httex1 aggregation. To overcome the problem of loss of CLC to vial surface
1. INTRODUCTION With an increase in life expectancy over the years, there is an unexpected surge in the incidence of age-related ailments, particularly neurodegenerative diseases.1,2 Huntington’s disease (HD) is one late-age-onset disease caused by the CAG triplet expansion mutation in the exon 1 of Huntingtin gene.3,4 This results in expanded polyglutamine (polyQ) in Huntingtin (Httex1) protein, which forms aggregates in neurons and causes cellular toxicity.3 Also, recent studies have elucidated the role of the flanking N-terminus-17 amino acid (NT17) of Httex1 to accelerate the polyQ aggregation kinetics. NT17 tends to form an amphipathic helix which self-associates to alter polyQ aggregation pathway toward non-nucleated pathway via oligomer formation. Thus, polyQ-rich Httex1 peptide aggregation and its aggregation steps are promising therapeutic targets in HD.5−8 Reducing its aggregation with peptide inhibitors like NT17, QBP1, and P42 is known to mitigate the cellular toxicity.9−11 These inhibitors have not been used in clinical developments because of difficulty in delivery across the blood brain barrier (BBB).12 Structurebased design research on small-molecule inhibitors is limited in HD because of the intrinsic disorder of Httex1.13,14 Based on the research on other intrinsically disordered peptides like amyloid-β15 and synuclein,16 aromatic interactions (π···π stacking) are proved to play an important role in the aggregation inhibition.16−19 Considering the similarities in some of the steps of aggregation among aggregating proteins © 2018 American Chemical Society
Received: Revised: Accepted: Published: 3356
April 11, 2018 June 16, 2018 July 6, 2018 July 6, 2018 DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
Article
Molecular Pharmaceutics
concentration of DMSO was kept at 10% (v/v) in the aggregation reaction. For BSA−CLC complex formation, constituents were added in the following order: first acidified water (pH 3 adjusted with TFA) was added followed by PBS, 0.05% (v/v) sodium azide, 5 μM BSA, and lastly, 100 μM CLC. The mixture was mixed gently, and then peptide was added. Httex1 peptide aggregation reaction was monitored by taking small aliquots of sample intermittently at different time points. Samples were centrifuged at 119 500g for 30 min at 4 °C. The top 70% of supernatant was taken, and formic acid (final concentration 20% v/v) was added to stop the ongoing aggregation reaction. Samples were analyzed using RP-HPLC at 215 nm wavelength on an Agilent HPLC 1260 infinity system. ZORBAX eclipse plus C18 rapid resolution column (4.6 × 100 mm, 3.5 μm) was used with the column thermostat set at 25 °C. Water and acetonitrile with 0.05% (v/v) TFA were used as mobile phase at 1 mL min−1 flow rate. To determine the effect on the initial phase of aggregation, Httex1 aggregation was set at three different concentrations (15, 20, and 25 μM) in the presence of CLC (100 μM). For comparison of the delay in aggregation rate, the reaction was monitored for an initial 30% loss of monomer and concentration was plotted against square of time (s2). The slopes obtained from the plot define the aggregation rate for the initial phase of Httex1 aggregation. 2.4. CLC Recovery from Httex1 Aggregates. For determining the presence of CLC in the Httex1 aggregates, Httex1 was allowed to aggregate in the presence of CLC (100 μM). Aggregates were washed eight times with water (resuspending aggregates and centrifuging intermittently) until all loosely bound CLC was removed. After the eighth wash, the pellet was dissolved in formic acid (20% v/v) to dissolve all aggregates. CLC was characterized by using RPHPLC. 2.5. Vial Surface Adsorption Assay. To monitor CLC loss to the vial surface, 100 μM CLC was added to PBS (pH 7.4) and incubated at 37 °C. Samples were analyzed using RPHPLC. The two-group comparison was performed using unpaired student’s t test, and the difference was considered statistically significant when p < 0.05. 2.6. Dynamic Light Scattering (DLS). Particle size and diffusion coefficient analysis was performed on Malvern Zetasizer Nano ZS90 equipped with He−Ne laser operating at a wavelength of 633 nm and the scattering angle of 90°. Both buffer and peptide solution were filtered through 0.22 μm syringe filter before mixing. All the DLS measurements were performed at 25 °C. The size of a particle is calculated from the translational diffusion coefficient by using the Stokes− Einstein equation:
adsorption, we developed a serum-albumin-based novel assay where serum albumin (bovine or human origin) forms a complex with CLC to prevent its adsorption to the vial surface. With the help of biophysical techniques and molecular docking, we characterized the serum albumin−CLC complex formation. This complex shows better inhibition of Httex1 aggregation by forming a ternary complex with NT17 of Httex1 peptide. For cell culture studies, we have synthesized and characterized the biocompatible CLC-loaded serum albumin nanoparticles. We have demonstrated that these nanoparticles are able to reduce the polyQ aggregate burden in HD-150Q cells, suggesting their potential for HD therapeutic and could be evaluated in preclinical studies.
2. MATERIALS AND METHODS 2.1. Materials. Calmidazolium chloride (CLC, 1-[bis(4chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4dichlorobenzyloxy)phenethyl]imidazolium chloride) was purchased from Sigma. Httex1 (MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPKK) and Q35 (KKQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQKK) peptides were purchased from Keck biotechnology resource laboratory, Yale University, New Haven, CT, U.S.A. NT17 (MATLEKLMKAFESLKSF) peptide, bovine serum albumin (BSA), human serum albumin (HSA), coumarin-343, 4dimethylamino pyridine (DMAP), N,N-dicyclohexylcarbodiimide, trifluoroacetic acid (TFA), 1,1,1,3,3,3-Hexafluoro-2Propanol (HFIP), sodium azide, formic acid, dimethyl sulfoxide (DMSO)-d6, Ponasterone A (PNST), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), p-nitrophenyl coumarin ester (NPCE), and dialysis membrane tubing (12 kDa cutoff), were purchased from Sigma-Aldrich (Bangalore, India). Acetonitrile (HPLC grade), centrifugal filter units (Amicon Ultra, 10 kDa cutoff), ethanol (99.9% purity), tritonX 100, glutaraldehyde (25% aqueous solution), DMSO, disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Merck, India. N6,2′-ODibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbcAMP) was purchased from Sisco Research Laboratories (SRL). Trypsin was purchased from New England Biolabs. All chemicals were used as received. In all of the experiments, 0.22 μm filtered Milli-Q water (18 MΩ cm) was used unless stated otherwise. 2.2. Peptide Disaggregation. All three peptides (i.e., Httex1, Q35, and NT17) were purified, lyophilized, and disaggregated as per published protocols.7 Briefly, 0.1 mg of peptide was dissolved in 1 mL of TFA: HFIP (1:1) and kept at room temperature for 12 h in the dark. The solvent was evaporated under the purge of nitrogen and vacuum-dried in the desiccator for 2 h for vacuum drying. 2.3. Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) Analysis. A disaggregated peptide was dissolved in acidified water (pH 3 adjusted with TFA) followed by centrifugation at 303 824g (Thermo S80-AT2 fixed angle rotor) for 4 h at 4 °C. A peptide aggregation reaction was carried out in phosphate buffer saline (PBS, pH 7.4) with 0.05% (v/v) sodium azide as an antimicrobial agent. Sample was incubated at 37 °C in a microcentrifuge tube (polypropylene, Tarsons). The peptide aggregation reaction was set up in the absence and presence of 100 μM CLC. The CLC stock (1 M) was prepared in DMSO, and the final
d(H) =
kT 3πηD
(1)
where d(H): hydrodynamic diameter; D: translational diffusion coefficient; k: Boltzmann’s constant; T: absolute temperature; η: viscosity. 2.7. Tryptophan Fluorescence Quenching Measurements. The fluorescence intensities were recorded with a PerkinElmer LS 55 fluorescence spectrometer using a lowvolume cuvette (120 μL) with 1 cm path length. The tryptophan was excited at λex 295 nm with a slit width of 10 nm for excitation and 5 nm for emission, and the scan speed was kept at 500 nm min−1. BSA concentration was kept at 1 3357
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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Molecular Pharmaceutics μM for tryptophan quenching. CLC dissolved in DMSO was titrated to 130 μL of protein solution (PBS pH 7.4), to give a final CLC concentration in the range of 0−20 μM. Fluorescence measurements were recorded at 25 °C. The dilution at the end of titration was 2.3% (v/v) and accounted for the calculation of ligand concentration. To confirm no interference from CLC in tryptophan fluorescence, UV absorbance spectra was recorded for 1 M CLC and found to be negligible at 295 nm. The change in fluorescence emission intensity was measured immediately after mixing CLC or DMSO to the protein. A normalized fluorescence spectrum for BSA was plotted for CLC titrations. Data was analyzed by plotting the relative fluorescence intensities (F0 − F)/F0 at 354 nm versus increasing concentrations of CLC (μM). Here, F0 and F are the fluorescence intensities of BSA in the absence and presence of CLC, respectively. Three independent titration experiments were performed, and data was plotted as mean ± SD. The equilibrium dissociation constant (Kd) was determined using a one-site saturation module of simple ligand binding tool of Sigma plot 12.0. For BSA and BSA−CLC complex interaction with NT17 peptide, the slit width was kept at 10 nm for excitation and 7 nm for emission, and the scan speed was 600 nm min−1. BSA− CLC complex was prepared by adding 5 μM CLC to 1 μM BSA (PBS pH 7.4, 5% DMSO). BSA−CLC complex was titrated with NT17 peptide and change in tryptophan fluorescence was monitored. For determining NT17 interaction with only BSA, 1 μM BSA was titrated with NT17 solution, and the change in tryptophan fluorescence was monitored. 2.8. Fluorescence Correlation Spectroscopy (FCS). An in-house assembled FCS instrument was used for all measurements. It consists of an inverted confocal microscope (Olympus IX-71, Japan) equipped with a 60× water immersion objective with numerical aperture (NA) 1.2 (UplanSApo, Olympus, Japan) to focus the 405 nm excitation light, from a 5 mW CW laser source (PSU-III-FDA, Optoelectronics Tech. Co. Ltd., China). The protein sample was put on the top of a coverslip (Blue Star, Polar Industrial Corporation) placed on the sample platform. The laser light was focused into the sample at a distance of 40 μm from the upper surface of the coverslip. The emitted photons were collected by the same objective and focused on a multimode fiber patch cord of 25 μm diameter (M67L01 25 μm 0.10 NA, ThorLabs, U.S.A.) after passing through a dichroic mirror (ZT405rdc, Chroma Tech. Corp., U.S.A.) and an emission filter (FSQ-GG455, U.S.A.). A fluorescence signal was then directed toward a photon-counting module (SPCM-AQRH13-FC, Excelitas Tech. Inc., Canada) through the fiber patch chord and then to a correlator card (Flex99OEM-12/E, Correlator.com, U.S.A.) to generate the autocorrelation function, G(τ). Finally, autocorrelation curves were displayed using LabView (National Instruments, U.S.A.) software. Details of autocorrelation function and equations are provided in the Supporting Information. The samples were prepared in 50 mM PBS (pH 7.4), and the concentration of NPCE tagged HSA was kept at 50 nM. All the measurements were done at 298 ± 1 K. Data analysis was performed as described earlier.27−30 2.9. Computational Docking of CLC to BSA. The structure of calmidazolium (CLC; ZINC 4262457) was obtained from the ZINC12 database.31,32 To identify the possible binding sites in BSA, CLC was computationally docked to BSA (PDB: 4F5S)33 using SwissDock.34−36 Using
the web server of SwissDock (http://www.swissdock.ch/ docking), the docking was done for BSA without specifying particular binding site to avoid any sampling bias. SwissDock performs calculations in the CHARMM force field. The most favorable BSA−CLC binding mode was interpreted on the basis of the cluster with the best FullFitness score. A greater negative score represents a better fit for a favorable binding mode. The binding conformation of CLC within the binding pocket in BSA identified using SwissDock was refined by further docking, using GOLD.37 GOLD uses a genetic algorithm to explore the full range of ligand conformational flexibility.37 2.10. Synthesis and Purification of BSA Nanoparticles. BSA nanoparticles were synthesized as per published protocol with minor modifications.38 BSA was dissolved in water (pH 8.5) at a concentration of 50 mg mL−1. To 500 μL of BSA, 2 mL of ethanol was added at the rate of 1 mL min−1 under constant stirring at 800 rpm at 25 °C. The suspension was allowed to stir for 20 min and 8% glutaraldehyde (0.6 μL per mg of BSA) was added. The suspension was stirred continuously for another 18−20 h. The resulting albumin nanoparticles were purified by three cycles of ultracentrifugation (16 000g, 20 min, 25 °C) followed by redispersion of the pellet in 2.5 mL of water. Each redispersion step was performed in an ultrasonication bath (MSE 150 ultrasonicator) for 5 min. Finally, empty BSA nanoparticles (ENPs) were resuspended in 2.5 mL of water (0.02% sodium azide) and stored at 4 °C. For CLC-loaded BSA nanoparticles (CNPs), CLC (100 μM) was dissolved in ethanol and added to BSA solution as described for empty nanoparticles. To optimize maximum encapsulation and loading, different concentrations of CLC were used (i.e., 25, 200, and 200 μM. Similarly, two different concentrations of BSA were tested (i.e., 50 and 75 mg mL−1). 2.11. Scanning Electron Microscopy. BSA nanoparticles (8 mg mL−1) were diluted 500 times with 0.2 μm filtered water. An aliquot was placed on a glass coverslip pasted on carbon tape attached to the copper stub and air-dried at room temperature overnight. The samples were sputtered with gold for 60 s under argon gas atmosphere (automatic sputter coater) and analyzed with a field emission electron microscope with an upper detector (FE-SEM, JSM-7100F; JEOL) at 10 kV. The size of the BSA-NPs was examined using the ImageJ software. 2.12. In Vitro CLC Release. An aliquot of 5 mg of CLC nanoparticles (CNP) were suspended in 300 μL of PBS pH 7.4 (0.05% v/v sodium azide) in a 1.5 mL microcentrifuge tube. The tube was incubated at 37 °C under constant stirring at 175 rpm. The sample was withdrawn at 24 h and centrifuged at 16 000g for 20 min. Supernatant was analyzed using RPHPLC. For trypsin digestion of CNPs, trypsin was used at concentration of 1.25 μg mg−1 of CNP. Released CLC was analyzed after 24 h using RP-HPLC. 2.13. Cell Viability Assay. Cell viability in the presence of various concentrations of CLC and BSA nanoparticles was analyzed using MTT assay. The HD-150Q (kindly gifted by Dr. Nihar Ranjan Jana, NBRC, India) cells were seeded (2500 cells/well) in a 96-well plate. After 24 h, cells were treated with 1 mM dbcAMP for differentiation. To induce expression of mHtt150Q-EGFP, cells were incubated with 10 μM PNST for 24 h. Neuro-2a cells were incubated with ENPs and CNPs (800 μg mL−1 concentration) after differentiation and tNmhHtt-150Q-EGFP expression. After 24 h of treatment, 3358
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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Figure 1. Calmidazolium chloride (CLC) modulates Httex1 aggregation. (a) Chemical structure of CLC (ZINC 4262457). (b) Httex1 peptide aggregation inhibition by CLC. Httex1 peptide (20 μM) aggregation kinetics in the absence (black) and presence (red) of 100 μM CLC (n = 4). Aliquots of sample were centrifuged at respective time point and monomer content of Httex1 peptide in the absence and presence of CLC was determined using RP-HPLC. (c) Aggregation rate of Httex1 at different concentrations in the presence of 100 μM CLC. Plot represents the first 30% of Httex1 aggregation reaction, and slope represents the corresponding aggregation rate. (d) Detection of the presence of CLC in Httex1 aggregates using RP-HPLC. Aggregates of Httex1 incubated with CLC were washed eight times with water, and final aggregates were solubilized in formic acid (20% v/v). CLC released from aggregates was analyzed by RP-HPLC, and a representative chromatogram is shown with a CLC peak at 12.6 min.
20 μL of MTT solution was added into each well, and the incubation was continued for another 4 h at 37 °C. After 4 h, the medium was removed from wells carefully, and 200 μL of DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm using PerkinElmer plate reader and cell viability was represented as a percentage relative to the untreated control cells. 2.14. Effect of CLC on Htt Aggregation in HD-150Q Cells. In vitro effect of CLC against aggregation was analyzed in Neuro-2a cells expressing tNhtt-EGFP-150Q (HD-150Q).39 2500 HD-150Q cells were seeded in each well of a 24-well plate containing a glass coverslip. After 24 h, cells were treated with 1 mM dbcAMP for differentiation. To induce expression of tNhtt-EGFP-150Q, cells were incubated with 10 μM PNST for 24 h. After PNST treatment, cells were incubated with only medium (control), ENPs (control), and CNPs (250 μg mL−1 CLC concentration) for 24 h. On the following day, cells were fixed using 4% paraformaldehyde, stained with DAPI, and put on a glass slide for microscopic analysis. Images were captured using 10× and 40× objectives using ultraviolet (UV) and green filters, merged using adobe photoshop CS6. For quantification of tNhtt-EGFP-150Q aggregates, images were analyzed using ImageJ software (V1.50i). For each image, the total number of green fluorescence protein (GFP) positive puncta was counted representing total number of aggregates. Similarly, the total number of DAPI-stained nucleus were counted, representing the total number of cells. Results were represented as percentage of cells containing aggregates obtained by dividing GFP puncta count by DAPI count.
2.15. Hemolytic Assay. Blood was collected from three healthy mice in anticoagulant-coated AcCuvet-Plus K3-EDTA tube as per approved protocol (IITK/IAEC/2017/1071). Blood was immediately centrifuged at 1000g for 10 min at 4 °C, and the plasma along with the buffy coat was aspirated carefully. Red blood cells (RBC) were washed thrice with 0.9% saline. RBCs were then diluted to 1/10th of their original volume (10%v/v) in PBS pH 7.4. Next, 60 μL of RBCs suspension (10% v/v) was then added to 140 μL of PBS (pH 7.4) followed by 100 μL of nanoparticles to obtain 2% (v/v) RBCs suspension. PBS pH 7.4 was used as a negative control, and 1% Triton-X 100 was used as a positive control (100% hemolysis). RBCs suspension containing ENPs and CNPs (800 μg mL−1) was incubated at 37 °C for 1 h and centrifuged at 1000g for 10 min to separate RBCs. Then 200 μL of supernatant from each tube was transferred to separate wells in a 96-well plate, and absorbance was measured at 540 nm. Hemolysis was characterized by disappearance of the RBCs pellet and appearance of free hemoglobin in the supernatant. yz ij A540 sample − A540 negative control zz = jjj j A540 positive control − A540 negative control zz { k
% hemolysis
× 100
(2)
2.16. Statistics. Statistics were performed in Sigma plot 12.0. For two-sample comparison, an unpaired student t test was performed. For all pairwise multiple comparisons and comparisons against control, the differences between the groups were evaluated with one-way analysis of variance 3359
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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Figure 2. CLC adsorption to the vial surface. (a) CLC loss to vial surface during Httex1 peptide aggregation reaction. Soluble CLC content was determined by RP-HPLC at each time point (n = 2). (b) CLC loss to the vial surface adsorption in PBS (pH 7.4). CLC solubilized in 100% DMSO served as a positive control with no loss to the vial surface. Adsorbed CLC was recovered with DMSO wash from the vial, previously incubated with CLC in PBS (n = 4, * p < 0.05, ns − not significant, pairwise comparison was done using one-way ANOVA with Holm−Sidak posthoc test). (c) Schematic representation of CLC adsorption to the vial surface. Red circles represent CLC. (d) The particle size of CLC in the aqueous solution shown as number percent was determined by DLS. CLC was used at three different concentrations.
action was earlier reported for peptide inhibitors.7 Furthermore, to confirm possible interaction of CLC with Httex1 before its spontaneous aggregation, we checked CLC colocalization with aggregates. The presence of CLC in formic-acid-solubilized aggregates confirmed CLC Httex1 interaction (Figure 1d). Similar inhibition trends and inhibitor colocalization with Httex1 aggregates were reported for peptide aggregation inhibitors.6 Thus, CLC interacts with the Httex1 peptide to target the initial event (NT17 peptide selfassociation) during its spontaneous aggregation. However, aggregation inhibition observed was short-lived, and the effect was lost after the initial delay. 3.2. CLC Adsorption to Vial Surface Diminish Its Aggregation Inhibitory Activity. We observed only 7−10 μM of CLC in the supernatant of Httex1 reaction after 3 h (Figure 2a). In addition, CLC recovered from the aggregates accounted for the partial CLC amount, which prompted us to look for the cause responsible for its loss. Literature suggests that hydrophobic molecules tend to adsorb nonspecifically to the vial (plastic/glass) surface.41,42 Because CLC is hydrophobic, we suspected that it might not be stable in solution for long and might adsorb to the hydrophobic surface of the reaction vial. To investigate this, CLC was separately incubated in the vial containing 100% DMSO, an ideal solvent for solubilizing hydrophobic molecules. No loss of CLC was observed from the solution after 24 h of incubation in DMSO, while 80% of CLC was lost when incubated in PBS (Figure 2b). CLC adsorption to the vial surface was further confirmed when the vial surface was rinsed with DMSO, and the lost CLC was recovered. Because of the hydrophobic nature of CLC, it preferred the vial surface over solution (Figure 2c). Adsorption kinetics showed a rapid
(ANOVA) followed by a Holm−Sidak posthoc test. p < 0.05 was considered to be statistically significant.
3. RESULTS AND DISCUSSION 3.1. CLC Modulates Httex1 Aggregation. Calmidazolium chloride (CLC), a hydrophobic molecule was used here to check its aggregation inhibitory potential against Httex1 (Figure 1a). Literature suggests that targeting the amphipathic face of NT17 domain of Httex1 may prevent its aggregation.6,40 Thus, the use of hydrophobic CLC might help it to bind to the hydrophobic face, probably via aromatic interactions and might abrogate Httex1 aggregation. The Httex1 peptide follows nonnucleated spontaneous aggregation kinetics via oligomer formation,7 as a first step (Figure 1b) toward complete aggregation. CLC significantly delayed this aggregation of the Httex1 peptide for initial 15 h but inhibition effect receded thereafter. Httex1 alone follows downhill aggregation kinetics,7 but the presence of CLC probably added a kinetic barrier to this spontaneous aggregation. The effect induced by CLC was further confirmed by determining the aggregation rate for 30% of initial aggregation at different concentrations of Httex1. At 15 μM concentration of Httex1, the presence of CLC resulted in a slower aggregation rate compared with that of 20 and 25 μM concentrations (Figure 1c). These results hint toward the possible interaction of CLC with Httex1 during an early stage of the aggregation reaction. Interestingly, CLC failed to inhibit aggregation of Q35 peptide which was without NT17 domain (Figure S1). This corroborates well with the published literature where no aggregation inhibition was observed on polyQ in the presence of CLC.21 This suggests toward NT17interaction-mediated antiaggregation activity of CLC. A similar 3360
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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Figure 3. Serum albumin prevents CLC adsorption to the vial surface. (a) Effect of vial surface modification (chemical and physical) and the addition of protein in solution on CLC adsorption after 24 h of incubation. CLC content in solution was determined using RP-HPLC (n = 3). *p < 0.05, ns − not significant, all pairwise comparisons were done using one-way ANOVA with Holm−Sidak posthoc test. (b) Particle size (diameter in nanometer) of CLC, BSA and BSA−CLC in PBS was determined by DLS. Change in hydrodynamic diameter of BSA−CLC complex formation shown as number percent. (c) Intrinsic tryptophan fluorescence quenching by CLC binding to BSA. Decrease in fluorescence emission of BSA (1 μM) on titration with CLC. (d) Effect of increasing concentration of CLC on BSA tryptophan fluorescence. Relative fluorescence intensity represented as ((F0 − F)/F0). Dissociation constant (Kd) was determined using Sigma Plot 12.0 (n = 3, mean ± SD).
adsorption until 5 h followed by a decline after 10 h (Figure S2). This is in congruence with Httex1 aggregation inhibition data wherein CLC lost its activity at the later stages of aggregation reaction (Figure 1b). CLC formed submicron sized particles in PBS as observed by DLS (Figure 2d), similar to other hydrophobic molecules under similar conditions.43 To our surprise, adsorption of CLC did not take place in water pH 5.5 and phosphate buffer pH 7 (without salt) (Figure S3a). However, adsorption took place in both PBS (pH 7) with 150 mM NaCl and water (pH 5.5) containing 150 mM NaCl. This was also confirmed by the increase in particle size and scattered light intensity (kcps) (Figure S3b). Thus, ionic strength contributes majorly to CLC surface adsorption. The presence of NaCl resulted in the formation of large particle size in aqueous solution indicating self-assembly of CLC. This was further confirmed by 1H NMR, where aryl protons of CLC showed an upfield shift, indicating π−π stacking induced self-association of CLC when present in aqueous solution (Figure S4). Although the self-association phenomenon of the small molecule is not completely understood, it was reported for a few dyes and many lipophilic molecules.44−46,43 Thus, data suggests that the observed loss in inhibition potency of CLC could be attributed to the nonavailability of CLC in solution. Because it was unavoidable to omit salt from the buffer for the present study to mimic in vivo conditions, we tested other ways to avoid vial surface adsorption of CLC. First, we tested the vial with hydrophilic surface modification or coating with protein to avoid adsorption. A similar loss of CLC was
observed in the case of eppendorf Lobind vial and BSA-coated vial in comparison to tarson microcentrifuge vial used in this study (Figure 3a). Coating the vial surface with BSA is a wellknown technique to prevent protein surface adsorption, but it failed in the case of CLC. This could be due to possible loss of CLC to the adsorbed BSA on the vial surface. To check this hypothesis, we used BSA in solution to have free BSA molecules to bind to the adsorption-prone CLC. A significant decrease was observed in CLC adsorption to the vial at 5 and 100 μM BSA concentrations (Figure 3a). Even 5 μM BSA was able to diminish CLC vial surface adsorption. The observed phenomenon was specific to BSA, as lysozyme could not prevent adsorption. Serum albumin was effective irrespective of the origin of protein (i.e., bovine (BSA) or human (HSA; Figure S5). The presence or absence of fatty acid did not affect the antiadsorption property of BSA (Figure S5). Also, for 100 μM CLC, 1 μM BSA prevented 60% adsorption after 96 h at 37 °C, and 5 μM prevented 80% surface adsorption and preferred for further experiments (Figure S6). BSA/CLC complex in the ratio of 1:10 is capable of preventing adsorption completely while BSA/CLC ratio of 1:50 prevented adsorption partially. 3.3. CLC Complex with BSA Rescues Its Vial Surface Adsorption. DLS data revealed the emergence of a new peak with an intermediate size when compared with BSA and CLC alone (Figure 3b). This could be due to the complex formation which was further characterized by a combination of biophysical and computational methods. 1H NMR spectroscopy showed the changes in the chemical shifts of amide 3361
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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Figure 4. Serum albumin−CLC molecular docking. (a) Clusters of docked conformations of CLC (shown in sphere representation) on BSA (shown in ribbon representation) obtained following docking using SwissDock. The regions of the BSA colored red, green, and blue correspond to the domain I, II, and III, respectively. The clusters shown in yellow and magenta colors correspond to CLC binding modes within the S1 and S2 pockets, respectively. (b) The refined binding conformation of CLC (shown in sphere representation and yellow color) in the S1 pocket of BSA with a GoldScore of 77.33 (Top 10 average GoldScore = 70.55). (c) Close-up of CLC (ball-and-stick) in the S1 pocket of BSA showing residues forming noncovalent interactions with CLC. Cation···π and π···π interactions between CLC and residues of S1 pocket of BSA are shown using dotted lines.
Figure 5. Serum albumin−CLC complex delays Httex1 peptide aggregation. (a) Aggregation kinetics of Httex1 peptide (20 μM) in the absence (black) and presence of 100 μM CLC (green), 5 μM BSA (red), and 100 μM CLC + 5 μM BSA (complex- blue). At each time point, an aliquot was centrifuged, and Httex1 monomer content was determined in supernatant using RP-HPLC (n = 4). (b) Comparison of diffusion coefficient of Httex1 in the absence and presence of BSA−CLC complex. Diffusion coefficient of Httex1 alone and in the presence of BSA−CLC complex at 0 and 48 h were determined by DLS (n = 4). *p < 0.05, statistical analysis was done using t test. (c) Fluorescence correlation spectroscopy (FCS) of fluorophore (NPCE) tagged serum albumin (HSA) in absence and presence of CLC. Normalized autocorrelation functions of NPCE tagged HSA at increasing concentrations of CLC. (d) FCS of NPCE-tagged HSA−CLC complex in the absence and presence of NT17 peptide. Normalized autocorrelation functions of NPCE tagged HSA in complex with CLC at increasing concentrations of NT17.
fluorescence quenching due to binding of CLC (Figure 3c). Also, a slight blue shift in the emission maximum of both BSA and HSA indicates binding of CLC that resulted in a more hydrophobic environment around tryptophan.47,48 It yielded a binding constant (Kd) of 0.78 ± 0.06 μM for BSA−CLC complex (Figure 3d). Similar tryptophan fluorescence quenching was observed for HSA (Figure S9a) with Kd of 2.48 ± 0.42 μM for HSA−CLC complex (Figure S9b) representing weak CLC binding. The small difference in Kd for
hydrogen and aryl protons of BSA with increasing CLC concentration (Figure S7). It could be possible that the aromatic ring of CLC interacts with either aromatic amino acids through π−π interaction or with positively charged residues of BSA through cation···π interactions. Extent of this interaction was further revealed by the fluorescence quenching of tryptophan of serum albumin (Figure S8). Titration of BSA with CLC resulted in a gradual decrease in emission intensity with increasing CLC concentration, indicating intrinsic 3362
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instead of BSA as the latter cannot be tagged with a single fluorophore. It is also to be noted that the three-dimensional structure of HSA and BSA is very similar. The autocorrelation curves of HSA tagged with NPCE were recorded at different CLC concentrations (Figure 5c). The single diffusion equation fails to fit the data. Instead, we observed two diffusion components, one for unbound HSA and the other for HSA− CLC complex. There was a regular increment of the diffusion time for HSA−CLC complex with increasing concentration of CLC (Figure 5c). Also, percent fraction of HSA−CLC complex was found to increase with increasing CLC concentration (Figure S10a). Further, we checked the direct interaction of NT17 with serum albumin−CLC via formation of a ternary complex using FCS. We used NT17 peptide for FCS experiment as it does not aggregate and serves as a good model peptide for mechanistic studies. At constant HSA (50 nM) and CLC (5 μM) concentration, an increase in diffusion time of HSA with increasing concentration of NT17 was observed (Figure 5d), which indicates the formation of the ternary HSA−CLC-NT17 complex. It is notable that for 20 μM of NT17, the diffusion time of the ternary complex (HSA:50 nM−CLC:5 μM−NT17:20 μM) is 7835 ± 635 μs, whereas for the HSA−CLC complex of the same composition, the value of the diffusion time is found to be 4335 ± 360 μs. Percent fraction of such HSA−CLC-NT17 complex was also found to increase with increasing NT17 concentration (Figure S10b). Formation of this ternary HSA−CLC-NT17 complex might be responsible for the inhibitory effect seen in the case of Httex1. Since NT17 participate in the oligomer formation to accelerate the aggregation, nonavailability of NT17 for oligomerization due to ternary complex formation possibly delayed the aggregation kinetics. In order to capture one of the binding modes of NT17 peptide to BSA and BSA−CLC complex, we used α-helical NT17 peptide for docking.54−56 Four out of the top five resultant transformations occupied the S1 pocket, similar to CLC, while the other was found docked in the S2 pocket. The best-scoring transformation (NT17 in S1) was further refined using FiberDock 57 and is illustrated in Figure S11a. Interestingly, in the presence of CLC, NT17 peptide tends to occupy S2 pocket formed by domain I, II, and III together (Figure S11b). NT17 peptide binding to BSA involves different interactions (i.e., salt-bridge: K15NT17···D450BSA, cation···π: F11NT17···R217BSA, F11NT17···K294BSA and multiple intermolecule hydrophobic contacts). This binding mode could be one of the ways by which BSA−CLC complex interacts with the NT17 peptide of Httex1 to make a ternary complex. On the basis of our experimental data and docking observations, it could be safely concluded that BSA−CLC complex is capable of forming a ternary complex to prevent Httex1 aggregation. CLC in free and serum albumin bound state (BSA−CLC complex) prevents NT17 self-association probably by targeting the amphipathic helix of NT17.40,58 Interaction of CLC and BSA−CLC with NT17 renders it to form oligomers which in turn inhibit Httex1 aggregation. However, further experimental validation is required to prove this interaction and complete inhibition mechanism. 3.6. BSA Nanoparticles as Formulation of CLC. Because of the hydrophobic nature of CLC, use in cell culture assays and in vivo studies is limited due to nonspecific adsorption and possible membrane disruption. Further, we used 10% DMSO (v/v) for in vitro studies and at this concentration DMSO is known to be toxic to mammalian
HSA and BSA could be due to the availability of different binding sites for CLC.49 3.4. Computational Docking Analysis of CLC Binding to Serum Albumin. Putative binding pockets for CLC on BSA were identified by docking using the SwissDock web server.36 Docking predictions primarily place CLC at the interdomain interfaces of BSA with a majority of docked poses clustered within the cleft formed by domain I and III (22/32 clusters). A small number of clusters (5/32) also occupy a pocket formed by the regions of domains I, II, and III (Figure 4a). We refer to the former as the S1 pocket and the latter as S2. It is interesting to note that both the S1 and S2 pockets are highly polar with side chains of many polar and charged residues projecting into them. As ∼70% of CLC docked clusters are predicted within the S1 pocket, we identified S1 to be the primary binding site for CLC and refined its binding conformation using GOLD.37 The resultant best-scoring conformation of CLC in the S1 pocket of BSA is shown (Figure 4b) with some of the key interactions highlighted in Figure 4c. From the docking results, it could be suggested that the interaction of CLC with the S1 pocket of BSA can be stabilized by noncovalent interactions such as cation···π, between the basic residues projecting into the S1 pocket (R458, R435, R185, R144, and K114) and the aromatic moieties of CLC. These basic residues also form multiple polar contacts with the chlorine atoms of CLC. The cationic imidazolium moiety of CLC could also favorably interact with Glu424, potentially forming a salt-bridge under physiological conditions. Also, His145 of BSA interacts with CLC through π···π interaction. Taken together, experimental and docking results clearly indicate that serum albumin forms a complex with CLC in aqueous solution. Complex is stabilized by multiple noncovalent interactions with binding affinity in the range of micromolar concentration. This complex abrogates the adsorption of CLC to the vial surface, thus making it available in solution and may act on Httex1 to suppress aggregation. 3.5. Inhibition of Httex1 Peptide Aggregation by BSA−CLC Complex. To ascertain that CLC in complex with BSA still retains the aggregation inhibitory property, Httex1 aggregation was set in the absence or presence of CLC, BSA and BSA−CLC complex. A delay in aggregation kinetics of Httex1 was observed in the presence of CLC, BSA, and BSA− CLC complex (Figure 5a). Interestingly, a 25-fold decrease in Httex1 aggregation was found in the presence of BSA−CLC complex as compared to only 10-fold decrease for BSA alone and a 4-fold decrease in case of CLC alone. Aggregation inhibition by serum albumin is not surprising as it is earlier shown to directly interact with the amyloid-β and inhibit its aggregation.50−52 The presence of BSA−CLC complex reduces the aggregate formation ability of Httex1 and results in the formation of smaller assemblies as shown by a large diffusion coefficient (Figure 5b). Both CLC alone and BSA−CLC complex showed an impact on the initial aggregation phase (Figure 5a). It is known that NT 17 domain flanking polyQ forms an amphipathic helix which triggers an oligomerization event to accelerate the aggregation kinetics of Httex1.7 Inhibiting this event at the time of NT17 self-association diminishes the ability of Httex1 to form aggregates.6,53 So, in order to get more details on the behavior of serum albumin−CLC complex in the presence of NT17 peptide in solution, fluorescence correlation spectroscopy (FCS) was used. For FCS study, we used HSA 3363
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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Figure 6. Characterization of BSA nanoparticles. Scanning electron microscopy image of (a) empty BSA nanoparticles (ENP) and (b) CLC-loaded BSA nanoparticles (CNP). Inset shows single near-spherical BSA nanoparticle. Scale bar is 1 μm (10 000× magnification), and for the inset, the scale is 100 nm (50 000× magnification). (c) Average particle size (d.nm) of nanoparticles (ENPs and CNPs) based on SEM image analysis using ImageJ. n = 60, *p < 0.05, comparison was done using t test. (d) Autofluorescent CLC-loaded BSA nanoparticles showing both green and red fluorescence. (e) CLC loading on BSA nanoparticles confirmed using RP-HPLC analysis. Representative chromatograms of CLC, ENPs, and CNPs are shown. The peak at ∼15 min represents CLC which is present in CNPs and absent in ENPs. (f) Effect of trypsin on CLC release from BSA nanoparticles. CLC released from CNPs after 24 h of incubation in the absence and presence of trypsin was determined using RP-HPLC (n = 2). *p < 0.05, comparison was done using t test.
cells.59 To avoid these problems, we encapsulated CLC in BSA nanoparticles. Since CLC showed the binding affinity for BSA, we opted for the albumin nanoparticle-based approach for the preparation of CLC-loaded BSA nanoparticle formulation. Further, the formulation in the form of nanoparticles might show better solution stability as compared to BSA−CLC complex alone.60 CLC-loaded BSA nanoparticles were prepared by an established desolvation technique followed by glutaraldehyde cross-linking (Figure S12a,b).38 We obtained nanoparticles with a Z-average size of 124 ± 2 nm for empty nanoparticles (ENP) and 134 ± 4 nm for CLC-loaded nanoparticles (CNP) (Table S1). Both ENPs and CNPs showed PDI < 0.1 and zeta potential of −22 and −15, respectively. BSA nanoparticles showed stability with no comparable change in size and PDI over 15 days when stored at 4 °C (Table S1). Zeta potential showed a slight change for both ENP and CNP after 15 days of storage. Both empty (Figure 6a) and loaded nanoparticles (Figure 6b) obtained were spherical in shape as shown by SEM. An average size for ENPs and CNPs was 99 ± 17 and 120 ± 14, respectively, which matches with the hydrodynamic size determined by DLS (Figure 6c). CLC-loaded nanoparticles showed significantly large size in both DLS and SEM as compared to ENPs. Also, the resultant BSA nanoparticles were autofluorescent with both green and red fluorescence (Figure 6d) due to electronic transitions (π···π* of CC bond and n···π* of CN bond.61 Different concentrations of CLC and BSA were optimized in order to improve encapsulation efficiency. We could achieve 23% loading efficiency at 100 μM CLC concentration and 50 mg mL−1 BSA concentration, with 2 μg CLC per mg of BSA in nanoparticles (Table S2). The BSA nanoparticles showed a
change in secondary structure when compared with free BSA as observed by CD and FTIR spectroscopy (Figure S13a,b). On the contrary, ENP and CNP showed little change in secondary structure, which indicates CLC loading did not alter BSA structure to a large extent (Figure S13a,b). CLC loading into CNPs was confirmed by dissolving CNPs in DMSO and analyzing the supernatant by RP-HPLC (Figure 6e). To simulate in vivo release of CLC, CNPs were incubated at 37 °C with shaking (175 rpm) in the absence and presence of trypsin. We observed 4% CLC release from CNPs in 24 h, while CLC release increased by 2-fold when CNPs were digested with trypsin (Figure 6f). 3.7. Effect of CNPs on Httex1 Aggregation in HD150Q Cells. To check the applicability of CNPs to the in vivo studies, we tested their biocompatibility and efficacy in Huntington’s disease mammalian cell culture model. After successful synthesis and characterization of CNPs, the toxicity of CNPs to HD-150Q cells was determined by MTT assay. CNPs showed no significant cellular toxicity on HD-150Q (Figure 7a). Also, both ENPs and CNPs showed good hemocompatibility (Figure 7b). In the case of positive control Triton-X 100, complete hemolysis was observed as seen by the red-colored supernatant due to complete hemoglobin release due to membrane disruption by the surfactant (Figure 7b inset). No hemolysis was observed for isotonic PBS (negative control) and RBCs formed pellet after centrifugation (Figure 7b inset). Similar to the negative control, no hemoglobin was released in case of ENPs and CNPs treated RBCs. Having biocompatible CLC-loaded nanoparticles, we further checked the efficacy of CNPs to modulate Httex1 aggregation in HD150Q cells expressing tNhtt-EGFP-150Q. 3364
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Figure 7. CLC-loaded nanoparticles prevent aggregate formation in HD-150Q cells. (a) Effect of CLC-loaded nanoparticles on cell viability. Cell toxicity was determined using MTT assay by incubating CNPs with HD-150Q. n = 4, ns − not significant, comparison was done using t test. (b) Hemocompatibility of BSA nanoparticles was determined using hemolysis assay. 1% Triton-X 100 was used as a positive control, and PBS was used as a negative control. The ENPs and CNPs were suspended at 800 μg mL−1 (n = 3), and RBC lysis was determined by absorbance at 540 nm. An inset shows the image of samples after centrifugation with complete hemolysis in case of Triton-X 100 while RBC pellet in PBS, ENPs, and CNPs. *p < 0.05, ns − not significant, comparison with control was done using one-way ANOVA with Holm−Sidak posthoc test. (c) Representative fluorescent images of Neuro-2a cells expressing tNhtt-EGFP-150Q. Aggregates are shown by green fluorescence (GFP) and nucleus by blue fluorescence (DAPI). Untreated cells were considered as control (c). (d) ENP treated cells. (e) CNP (250 μg mL−1 CLC concentration) treated cells. Scale bar for c, d, and e is 50 μm. (f) Effect of BSA nanoparticles treatment on Neuro-2a cells expressing tNhtt-EGFP-150Q. Percentage of cells containing aggregates in the absence and presence of nanoparticles. n = 15, *p < 0.05, pairwise comparison was done using one-way ANOVA with Holm−Sidak posthoc test. (g) Schematic representation of CLC action on Httex1 aggregation. (1) CLC-loaded BSA nanoparticle formulation on digestion with trypsin (2) releases CLC to prevent Httex1 aggregation. (3) BSA−CLC complex forms a ternary complex with NT17 of Httex1 to inhibit its aggregation.
nanoparticle formulation of paclitaxel (Abraxane) was approved for oncotherapy.60
Htt-150Q expression results in the formation of green fluorescent aggregates when induced with PNST (Figure 7c). Both ENPs and CNPs were incubated with HD-150Q cells for 24 h after inducing expression. Cells treated with ENPs (Figure 7d) and CNPs (Figure 7e) for 24 h postinduction showed fewer green fluorescent puncta in comparison to control. Quantification of GFP puncta per cell revealed an inhibitory effect of both ENPs and CNPs, with CNPs showing better efficiency in aggregation inhibition (Figure 7f). Effect shown by ENPs is in accordance with the published literature and our in vitro result where BSA alone also showed an inhibitory effect (Figure 5a).50−52 CNPs treatment of HD-150Q cells replicated the antiaggregation effect of CLC observed in vitro. CNPs could now be tested in a rodent HD model for in vivo efficacy of CLC as a part of preclinical study for this therapeutic formulation against HD. On the basis of a similar concept of hydrophobic drug binding to serum albumin,
4. CONCLUSIONS To summarize, a hydrophobic molecule calmidazolium chloride was tested for aggregation modulation of Httex1 as a potential therapeutic molecule for Huntington’s disease. CLC targets the initial step of Httex1 aggregation, which is a potential therapeutic target in HD. We speculate the involvement of aromatic interactions of CLC with a hydrophobic face of NT17 domain of Httex1 to prevent its aggregation. Since CLC adsorbs to the vial surface during aggregation reaction, we have developed a serum albuminbased assay to prevent adsorption of CLC while simultaneously monitoring aggregation kinetics (Figure 7g). The BSA−CLC complex formation and characterization were done with the help of tryptophan fluorescence, FCS, DLS, NMR, 3365
DOI: 10.1021/acs.molpharmaceut.8b00380 Mol. Pharmaceutics 2018, 15, 3356−3368
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analyzed FCS experiments. ARJ performed mammalian cell culture experiments. RNVKD carried out the docking studies overseen by HF. VS and AKT analyzed and interpret all the data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The work is supervised and directed by AKT.
and molecular docking studies. The increased inhibitory potential of serum albumin−CLC complex was achieved due to a ternary complex formation with NT17, which is involved in oligomer formation during aggregation (Figure 7g). The serum-albumin-based nanoparticle formulation of CLC was developed to overcome the limitations of poor aqueous solubility of CLC, DMSO cytotoxicity, and CLC adsorption to the culture dish. CLC-loaded BSA nanoparticles with size
