Dendritic Effect of Ligand-Coated Nanoparticles: Enhanced

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Langmuir 2009, 25, 2339-2347

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Dendritic Effect of Ligand-Coated Nanoparticles: Enhanced Apoptotic Activity of Silica-Berberine Nanoconjugates Mahantappa Halimani,† S. Prathap Chandran,‡ Sudhir Kashyap,§ V. M. Jadhav,‡ B. L. V. Prasad,*,‡ Srinivas Hotha,*,§ and Souvik Maiti*,† Proteomics and Structural Biology Unit, Institute of Genomics and IntegratiVe Biology, Mall Road, New Delhi 110 007, and Materials Chemistry DiVision and DiVision of Organic Chemistry, National Chemical Laboratory, Pune 411 008, India ReceiVed August 26, 2008. ReVised Manuscript ReceiVed December 5, 2008 We describe the synthesis and biological characterization of a novel prototype, namely, silica nanoconjugates bearing a covalently linked berberine, a plant alkaloid known to have antiproliferative activity. The effect of synthesized nanoconjugates on cell proliferation, the cell cycle profile, and apoptosis in the human cervical carcinoma cell line (HeLa), human hepatocellular liver carcinoma cell line (HepG2), and human embryonic kidney (HEK) 293T cell line has been studied and compared with the results obtained for free berberine. Our results show that all the nanoconjugates display higher antiproliferative activity than free berberine. The ability of these nanoconjugates to inhibit cellular proliferation is mediated by the cell cycle arrest at the G1 phase. Moreover, silica nanoconugates caused selective apoptotic arrest with a higher efficiency than free berberine followed by apoptotic cell death as shown by quantitative fluorescence-activated cell sorting analyses. Efficiency of the nanoconjugates increases upon an increase in the linker chain length, demonstrating the distinct role of the spacer chain that conjugates nanoparticles and ligands. The actual reason to show enhanced efficiency by the nanoconjugates has not been elucidated in the present study; however, we hypothesize that an increase in local concentration due to the confinement of a ligand on the nanosurface (“dendritic” effect) might have led to the observed effect.

Introduction The field of nanotechnology in recent years has motivated researchers to develop nanomaterials (NMs) for biomedical applications especially in drug delivery and imaging. Efficacy in these applications is largely determined by their ability to avoid clearance by the reticulo endothelial system (RES) and localize in regions of maximum therapeutic value, both of which are determined by the surface chemistry of the NMs.1,2 Moreover, they can be readily fabricated with dimensions comparable to those of biological macromolecules.3 Additionally, possible synthetic control of the surface facilitates fine-tuning of the structure and dynamics of the surface of the NMs. For example, peptide-functionalized nanoparticles (NPs) have been constructed to act as artificial proteins and enzymes,4,5 glyconanoparticles have been used as useful models of cell adhesion,6,7 and a variety of functionalized particles have been used for recognition in * To whom correspondence should be addressed. (S.M.) Phone: +91 11 2766 6175. Fax: +91 11 2766 7174. E-mail: [email protected]. (B.L.V.P.) Phone: +91 20 2590 2013. Fax: +91 20 2590 2601. E-mail: l.bhagavatula@ ncl.res.in. (S.H.) Phone: +91 20 2590 2401. Fax: +91 20 2590 2601. E-mail: [email protected]. † Institute of Genomics and Integrative Biology. ‡ Materials Chemistry Division, National Chemical Laboratory. § Division of Organic Chemistry, National Chemical Laboratory. (1) Otsuka, H.; Nagasaki, Y.; Kataoka, K. AdV. Drug. DeliVery ReV. 2003, 55, 403. (2) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Langmuir 2004, 20, 112857. (3) Hostetler, M. J.; Wingate, J. E.; Zhong, C. J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 17. (4) Pengo, P.; Baltzer, L.; Pasquato, L.; Scrimin, P. Angew. Chem., Int. Ed. 2007, 46, 400. (5) Pengo, P.; Polizzi, S.; Pasquato, L.; Scrimin, P. J. Am. Chem. Soc. 2005, 127, 1616. (6) de la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Canada, J.; Fernandez, A.; Penades, S. Angew. Chem., Int. Ed. 2001, 40, 2257. (7) de la Fuente, J. M.; Eaton, P.; Barrientos, A. G.; Menendez, M.; Penades, S. J. Am. Chem. Soc. 2005, 127, 6192.

aqueous media.8-10 Similarly, nanoparticle-protein interactions have found promising applications in modulation of enzymatic activity,11,12 biosensing,13 separation,14 and production of hybrid materials.15 Silica-based nanoparticles are one of the most used NMs as they provide a unique platform to accomplish many functions at the nanoscale.16 The surfaces of silica nanoparticles are chemically reactive, thus offering a practical method for introducing multiple functionalities into the same nanoparticle. Unlike polymer-based nanoparticles, these robust inorganic materials can tolerate many organic solvents.17 Silica-based materials have been successfully used as drug-delivery vectors,18,19 gene transfection reagents,20 cell markers,21 and carriers of molecules.22 There are several reports describing the advantages of ligandfunctionalized nanoparticles.11,12 For example, amino acidfunctionalized gold clusters have been shown to modulate the (8) Shenhar, R.; Rotello, V. M. Acc. Chem. Res. 2003, 36, 549. (9) Sastry, M.; Rao, M.; Ganesh, K. N. Acc. Chem. Res. 2002, 35, 847. (10) Nam, J.-M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932. (11) Verma, A.; Nakade, H.; Simard, J. M.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 10806. (12) You, C.-C.; Agasti, S. S.; De, M.; Knapp, M. J.; Rotello, V. M. J. Am. Chem. Soc. 2006, 128, 14612. (13) Tsai, C.-S.; Yu, T.-B.; Chen, C.-T. Chem. Commun. 2005, 4273. (14) Hong, R.; Emrick, T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 13572. (15) Xu, C. J.; Xu, K. M.; Gu, H. W.; Zhong, X. F.; Guo, Z. H.; Zheng, R. K.; Zhang, X. X.; Xu, B. J. Am. Chem. Soc. 2004, 126, 3392. (16) Halas, N. J. ACS Nano 2008, 2, 179. (17) Stein, A.; Melde, B. J.; Schroden, R. C. AdV. Mater. 2000, 12, 1403. (18) Arruebo, M.; Galan, M.; Navascues, N.; Tellez, C.; Marquina, C.; Ibarra, M. R.; Santamaria, J. Chem. Mater. 2006, 18, 1911. (19) Arruebo, M.; Fernandez-Pacheco, R.; Irusta, S.; Arbiol, J.; Ibarra, M. R.; Santamaria, J. Nanotechnology 2006, 17, 4057. (20) Radu, D. R.; Lai, C. Y.; Jeftinija, K.; Rowe, E. W.; Jeftinija, S.; Lin, V. S.-Y. A. J. Am. Chem. Soc. 2004, 126, 13216. (21) Lin, Y. S.; Tsai, C. P.; Huang, H. Y.; Kuo, C. T.; Hung, Y.; Huang, D. M.; Chen, Y. C.; Mou, C. Y. Chem. Mater. 2005, 17, 4570. (22) Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.; Jeftinija, S.; Lin, V. S.-Y. J. Am. Chem. Soc. 2003, 125, 4451.

10.1021/la802761b CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

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catalytic behavior of R-chymotrypsin (ChT) towards cationic, neutral, and anionic substrates.12 The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the active site and release of product through electrostatic interactions, leading to the substrate specificities. Another study describes the complete enzyme inhibition by positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) of different chain lengths (C8 and C11) when they bind to β-galactosidase through complementary electrostatic interactions. This inhibition can be reversed in vitro by intracellular concentrations of glutathione (GSH), the main thiol component of the cell.11 The restoration of activity depends on the chain length of the monolayer. The activity of enzyme bound to particles with a C8 monolayer was completely restored by intracellular concentrations (1-10 mM) of GSH. In contrast, no restoration was observed for enzyme bound to the C11 particles at any of the concentrations studied. From these studies it is apparent that the function of ligand-functionalized nanoparticles can be tuned through the structure of the monolayer. In this paper, we report functionalization of silica nanoparticles with berberine attached through a variable linker of different chain lengths C2 to C6 (silica-berberine nanoconjugates, SNC_Cn) using “click” chemistry. Berberine is a natural isoquinoline alkaloid that has been found in medicinally important plants including Coptis chinensis (coptis or goldenthread), Berberis aquifolium (Oregon grape), Berberis Vulgaris (barberry), and Coscinium fenestratum.23,24 Berberine possesses a wide range of biochemical and pharmacological activities including antidiarrheal, antiarrhythmic, and antitumor activities.25,26 The effect of synthesized nanoconjugates on cell proliferation, the cell cycle profile, and apoptosis/necrosis in the human cervical carcinoma cell line (HeLa), human hepatocellular liver carcinoma cell line (HepG2), and human embryonic kidney (HEK) 293T cell line has been studied. Studies identified that the nanoconjugates are more effective in antiproliferative activity and show higher induction of apoptosis in vitro compared to free berberine. Moreover, the effect becomes prominent for the nanoconjugates with a linker of C6 chain length.

Experimental Section Unless otherwise noted, materials for syntheis were obtained from commercial suppliers and were used without further purification. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], acridine orange, and trypsin/EDTA were procured from Sigma Chemical (St. Louis, MO), and all other chemicals were of analytical grade and purchased from Sigma Chemical. The annexin Vconjugated 7-aminoactinomycin D (7-ADD) Apoptosis Detection Kit (Nexin Apoptosis Detection Kit, PCA-96) was from Guava Technologies, Hayward, CA. Dubelcco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Gibco, Grand Island, NY. Unless otherwise reported all reactions were performed under an argon atmosphere. Removal of solvent in vacuo refers to distillation using a rotary evaporator attached to an efficient vacuum pump. Products obtained as solid or syrup were dried under high vacuum. Analytical thin-layer chromatography was performed on precoated silica plates (F254, 0.25 mm thickness); compounds were visualized by UV light or by staining with anisaldehyde spray. 1H NMR spectra were recorded on 200 or 300 MHz NMR spectrometers. Chemical shifts (δH) are quoted in parts per million and are referenced to the (23) Stermitz, F. R.; Lorenz, P.; Tawara, J. N.; Zenewicz, L. A.; Lewis, K. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1733. (24) Rojsanga, P.; Gritsanapan, W.; Suntornsuk, L. Med. Princ. Pract. 2006, 15, 373. (25) Newman, D. J.; Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003, 66, 1022. (26) Mantena, S. K.; Sharma, S. D.; Katiyar, S. K. Carcinogenesis 2006, 27, 2018.

Halimani et al. peak for tetramethylsilane (internal). Mass spectra were recorded on a Q Star Pulsar LC-MS-MS-TOF machine. UV-vis spectroscopic measurements were carried out on a Jasco model V-570 dual-beam spectrophotometer operated at a resolution of 2 nm. FTIR spectra were recorded on a Perkin-Elmer FTIR spectrophotometer in the diffuse reflectance mode operating at a resolution of 4 cm-1. General Experimental Procedure for the Synthesis of 2a-2e Commercially available berberine (1; 1 g) was heated at 190 °C under vacuum (10-15 mm) for 30 min. The crude product was recrystallized from EtOH to provide 535 mg (56%) of C-9hydroxyberberine whose spectral characteristics matched with those of the reported data.27 A solution of C-9-hydroxyberberine (1 mmol) prepared vide supra and 1,n-dibromoalkanes (20 mmol) in anhydrous DMF (5 mL) was heated at 60 °C for 6 h, and then Et2O (25 mL) was added. The resulting solid was filtered and washed with Et2O (3 × 15 mL), and the crude (bromoalkyl)berberine was redissolved in anhydrous DMF (10 mL) and treated with NaN3 (25 mmol) at 60 °C overnight. The reaction mixture was filtered through a pad of Celite and thoroughly washed with cold DMF (3 × 10 mL). The filtrate was poured into a cold solution of Et2O (50 mL), the resulting precipitate was filtered and washed with Et2O (3 × 15 mL), and the product was recrystallized from EtOH to give 2a-2d. Spectroscopic Characterization Data of Compounds 2a-2d Characterization Data for Compound 2a 1H NMR (200.13 MHz, DMSO-d6-CD3OD (4:1)): δ 3.28 (t, 2 H, J ) 6.4 Hz), 3.92 (t, 2 H, J ) 4.4 Hz), 4.09 (s, 3 H), 4.65 (t, 2 H, J ) 4.4 Hz), 5.12 (t, 2 H, J ) 6.4 Hz), 6.10 (s, 1 H), 6.84 (m, 1 H), 7.33 (s, 1 H), 7.43 (s, 1 H), 7.92 (d, 1 H, J ) 3.92 Hz), 7.98 (s, 1 H), 8.39 (s, 1 H), 10.12 (s, 1 H). Anal. Calcd for C21H19N4O4: C, 69.44; H, 4.89; N, 14.31. Found: C, 69. 94; H, 5.71; N, 14.87. FT-IR (Nujol): 2948.96, 2921.95, 2852.52, 2154.41, 1633.59, 1602.74, 1594.37, 1469.69, 1461.94, 1454.23, 1377.08, 1274.86, 1037.63, 929.63, 721.33, 683.39 cm-1. Characterization Data for Compound 2b 1H NMR (200.13 MHz, DMSO-d6-CD3OD (4:1)): δ 2.28 (m, 2 H), 3.28 (t, 2 H, J ) 5.80 Hz), 4.09 (s, 3 H), 4.21 (m, 1 H), 4.52 (t, 2 H, J ) 6.2 Hz), 5.10 (t, 2 H, J ) 6.2 Hz), 6.10 (s, 2 H), 6.86 (s, 1 H), 7.36 (s, 1 H), 7.45 (s, 1 H), 7.93 (s, 1 H), 7.95 (s, 1 H), 8.42 (s, 1 H), 9.91 (s, 1 H). Anal. Calcd for C22H21N4O4: C, 65.17; H, 5.22; N, 13.82. Found: C, 64. 38; H, 5.93; N, 14.53. FT-IR (Nujol): 2950.89, 2920.03, 2850.59, 2135.05, 1602.74, 1469.69, 1461.94, 1454.23, 1377.08, 1301.86, 1274.86, 1035.70, 927.70, 721.33, 683.39 cm-1. Characterization Data for Compound 2c 1H NMR (200.13 MHz, DMSO-d6-CD3OD (4:1)): δ 1.91 (m, 2 H), 2.05 (m, 2 H), 3.29 (t, 2 H, J ) 6.5 Hz), 3.47 (t, 1 H, J ) 6.6 Hz), 4.09 (s, 3 H), 4.48 (t, 2 H, J ) 6.3 Hz), 5.08 (t, 2 H, J ) 6.3 Hz), 6.11 (s, 2 H), 6.88 (s, 1 H), 7.38 (s, 1 H), 7.48 (s, 1 H), 7.96 (m, 2 H), 8.46 (s, 1 H), 9.86 (s, 1 H). Anal. Calcd for C23H23N4O4: C, 65.86; H, 5.53; N, 13.36. Found: C, 64.93; H, 6.04; N, 13.98. FT-IR (Nujol): 2952.81, 2921.95, 2852.52, 2146.62, 1672.17, 1633.59, 1602.74, 1568.02, 1461.94, 1454.23, 1377.08, 1344.29, 1276.79, 1234.36, 1112.85, 1101.28, 1060.78, 1039.56, 941.20, 871.76 727.11, 683.39 cm-1. Characterization Data for Compound 2d 1H NMR (200.13 MHz, DMSO-d6-CD3OD (4:1)): δ 1.58 (m, 6 H), 2.17 (m, 2 H), 3.33 (m, 3 H), 4.09 (s, 3 H), 4.42 (t, 2 H, J ) 6.7 Hz), 5.04 (t, 2 H, J ) 6.2 Hz), 6.11 (s, 2 H), 6.88 (s, 1 H), 7.43 (s, 1 H), 7.51 (s, 1 H), 7.98 (m, 2 H), 8.51 (s, 1 H), 9.76 (s, 1 H). Anal. Calcd for C25H27N4O4: C, 67.10; H, 6.08; N, 12.52. Found: C, 66. 52; H, 6.47; N, 12.98. FT-IR (Nujol): 2954.74, 2921.95, 2852.52, 2140.84, 1666.38, 1660.60, 1650.95, 1503.30, 1461.94, 1456.16, 1377.08, 1342.36, 1301.86, 1276.79, 1234.36, 1101.28, 1039.56, 721.33, 683.39 cm-1. Synthesis of Silica Nanoparticles (3). Silica nanoparticles were synthesized following the procedure reported by Stober and coworkers.28 Purification of silica nanoparticles was carried out using three rounds of centrifugation (13 000 rpm for 15 min) interspersed with redispersion and washing with ethanol. (27) Stober, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62. (28) Ramesh, R.; Bhat, R. G.; Chandrasekaran, S. J. Org. Chem. 2005, 70, 837.

Dendritic Effect of Ligand-Coated Nanoparticles

Langmuir, Vol. 25, No. 4, 2009 2341 Scheme 1. Synthetic Route for SNCs

Synthesis of Amine-Functionalized Silica Nanoparticles (4). Bare silica nanoparticles were dispersed in absolute ethanol (150 mg in 15 mL) followed by the addition of 2% (w/v) (aminopropyl)triethoxysilane (APTES). The above mixture was stirred for a period of 6 h under reflux conditions. Purification of functionalized silica nanoparticles was carried out using three rounds of centrifugation (13 000 rpm for 15 min) interspersed with redispersion and washing with ethanol. Amine-functionalized silica nanoparticles were subjected to FTIR and TEM characterization after purification. Synthesis of Propargyl-Functionalized Silica Nanoparticles (5). In a typical reaction 250 mg of amine-functionalized silica nanoparticles was dispersed in 50 mL of dry acetonitrile followed by addition of 0.3 mL of propargyl chloroformate.29 The above reaction mixture was stirred for a period of 48 h at room temperature under a nitrogen atmosphere. These silica nanoparticles were purified using multiple rounds of centrifugation and washing with acetonitrile. The size of the silica nanoparticles at this stage was determined to be about ∼55 nm using TEM studies. Purified propargyl-capped silica nanoparticles were characterized using FTIR spectroscopy. Synthesis of Berberine-Capped Silica Nanoparticles 6a-6d In a typical reaction, 100 mg of purified propargyl-capped silica nanoparticles was dispersed in 10 mL of a 1:1 ethanol-water mixture along with CuSO4 (4.7 mg) and sodium ascorbate (3.7 mg).30 This was followed by the addition of 7.8 mg of berberine-9-O-ethylazide (2a), dissolved in 1 mL of water, to the above reaction mixture with vigorous stirring under ambient conditions for 24 h. Purification of 6a was carried out using three rounds of centrifugation interspersed with redispersion and washing with deionized water. Similar procedures were employed for modification of propargyl-capped silica nanoparticles with propylazide (2b), pentylazide (2c), and hexylazide (2d) substituted berberine to give 6b-6d, respectively. The molar concentration of berberine analogues was 10-5 M in all cases. The berberine-functionalized silica nanoparticles thus prepared were subjected to FTIR and UV-vis absorbance characterization after purification. Thermogravimetric Analysis (TGA). The TGA experiments were carried out on a Mettler Toledo TGA instrument (TGA/SDTA 851e) controlled by STARe software (Mettler Toledo GmbH, Switzerland). Dry sample powders were placed in a ceramic crucible and analyzed over the temperature range of 32-800 °C at a rate of 10 °C min-1 under a dry flow of N2 at a rate of 30 mL min-1. Determination of the Conjugated Bereberine Concentration. The concentration of berberine in the nanoconjugates was determined by UV-vis spectroscopy as well as by TGA. UV-vis spectra for nanoconjugates and the same amount of silica nanoparticles were scanned, and the spectrum of pure silica was subtracted from the spectra of the nanoconjugates. The resultant spectra were used to calculate the conjugated berberine concentrations. The concentrations (29) Hotha, S.; Kashyap, S. J. Org. Chem. 2006, 71, 364. (30) Prado, A. G. S.; Airoldi, C. J. Colloid Interface Sci. 2001, 236, 161.

were also calculated from the density values of the berberine derivative per milligram of silica-berberine composite (Table SI-2, Supporting Information) obtained from TGA analysis. Both the concentrations obtained from these two different methods were similar within experimental error. Cell Culture Conditions and Treatments. The HeLa, HepG2, and HEK 293T cell lines were obtained from the NCCS (National Center for Cell Sciences), Pune, India. The cell lines were cultured as monolayers in DMEM supplemented with 10% heat inactivated FBS (Gibco/Invitrogen, Carlsbad, CA), 100 µg/mL streptomycin, and 250 µg/mL amphoterecin B (Sigma Chemical) and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 °C and in an incubator under the conditions as described above. In all treatments, berberine was initially dissolved in a small amount of ethanol and made up to the maximum final concentration of 0.2% (v/v) in the complete cell culture medium. The subconfluent cells (50-60%) were treated with varying concentrations of berberine either free or bound to berberine nanoconjugates and vehicle alone (0.2% ethanol in media) that served as the control. MTT Assay for Cell Viability/Proliferation. The effect of berberine and berberine nanoconjugates on the cell viability/ proliferation was determined using MTT assay. Briefly, 1 × 104 cells/well were plated in 96-well culture plates. After overnight incubation, the cells were treated with varying concentrations (0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 15, 20 µM) of berberine and different SNCs for 24 h. The cells were treated with 10 µL of 5 mg/mL MTT, and the resulting formazan crystals were dissolved in dimethyl sulfoxide (200 µL). The absorbance was recorded at 570 nm with a reference at 650 nm serving as the blank. The effect of berberine on cell viability was assessed as cell viability (%) compared to vehicle-treated control cells, which were arbitrarily assigned 100% viability. DNA Cell Cycle Analysis. Subconfluent cells (50-60%) were treated at the IC50 concentrations of berberine for the respective cell line in complete medium for 24 h. The cells were then harvested, washed with cold phosphate-buffered saline (PBS), and processed for cell cycle analysis. Briefly, the cells (1 × 105) were resuspended in 50 µL of cold PBS to which 450 µL of cold ethanol was added, and the cells were then incubated for 1 h at 4 °C. The cells were centrifuged at 5000g for 5 min, and the pellet was washed with cold PBS, resuspended in 500 µL of PBS, and incubated with 50 µL of RNase (250 µg/mL) for 30 min. The cells were incubated with 10 µL of propidium iodide (5 mg/mL) on ice for 1 h in the dark. The cell cycle distribution of the cells was then determined using the fluorescence-activated cell sorting (FACS) instrument GuavaEasycyte from Guava Technologies. WinMDI 2.8 software was used to analyze flow cytometry data. Apoptotic Cell Death Assay by FACS. Induction of apoptosis in all cell lines caused by berberine and different SNCs was quantitatively determined by flow cytometry using the annexin

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Halimani et al.

Figure 1. (A) Representative TEM images of propargyl-capped silica nanoparticles. The average size is 54.4 ( 2.8 nm. (B) TEM images of silica particles functionalized with 2a.

V-conjugated 7AAD Apoptosis Detection Kit (Guava Technologies) following the manufacturer’s instructions and as previously described. Briefly, subconfluent cells (50-60%) were treated at the IC50 concentrations of berberine for the respective cell line in complete medium for 24 h. The cells were then harvested, washed with PBS, and incubated with annexin V binding reagent added for cellular staining at room temperature for 10 min in the dark. The stained cells were analyzed by FACS using GuavaEasycyte from Guava Technologies. WinMDI 2.8 software was used to analyze flow cytometry data: nonapoptotic cells, annexin V (-) and 7-AAD (-) lower left (LL) quadrant; early apoptotic cells, annexin V (+) and 7-AAD (-) lower right (LR) quadrant; late-stage apoptotic and dead cells, annexin V (+) and 7-AAD (+) upper right (UR) quadrant; nuclear debris, annexin V (-) and 7-AAD (+) upper left (UL) quadrant. Fluorescence Microscopy Imaging. Cell morphology was evaluated by fluorescence microscopy following DAPI staining. Cells were grown on poly-L-lysine-coated coverslips in six-well plates and were treated with different SNCs and controls for 24 h. After incubation, the coverslips were fixed in cold methanol and washed with PBS, stained with DAPI, and mounted on slides. Images were captured using a BX60 microscope (Olympus, Tokyo, Japan) with an eight-bit camera (Dage-MTI, Michigan City, IN) and IP Laboratory software (Scanalytics, Fairfax, VA). Apoptotic cells were identified by features characteristic of apoptosis (e.g., nuclear condensation, formation of membrane blebs, and apoptotic bodies). Acridine Orange Staining. Changes in cell characteristics of apoptosis were examined by fluorescence microscopy of acridine orange-stained cells. The HeLa, HepG2, and HEK 293T cells were treated with vehicle (0.2% ethanol in medium), berberine, or different SNCs at their respective IC50 concentrations in complete medium for 24 h. The monolayer of cells was fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were permeabilized with 0.2% Triton X-100 and 10 µg/mL RNAase A in PBS three times and incubated with 25 µg/mL acridine orange for 30 min. The apoptotic nuclei were detected under 200× magnification using a fluorescent microscope (Nikon TS 1000) with an excitation filter of 470/490 nm and brier filter of 520/560 nm. A white arrow indicates typical apoptotic nuclear characteristics. Statistical Analysis. The statistical significance of the difference between control and treatment groups was determined by Bonferroni’s multiple comparison tests. A P value of