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Polyamidoamine-decorated Nanodiamonds as a Hybrid Gene Delivery Vector and siRNA Structural Characterization at the Charged Interfaces Dae Gon Lim, Nirmal Rajasekaran, Dukhee Lee, Nam Ah Kim, Hun Soon Jung, Sungyoul Hong, Young Kee Shin, Eunah Kang, and Seong Hoon Jeong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09624 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on September 3, 2017

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ACS Applied Materials & Interfaces

Polyamidoamine-decorated Nanodiamonds as a Hybrid Gene Delivery Vector and siRNA Structural Characterization at the Charged Interfaces Dae Gon Lim1, Nirmal Rajasekaran2,3, Dukhee Lee4, Nam Ah Kim1,3, Hun Soon Jung3, Sungyoul Hong2, Young Kee Shin2, Eunah Kang4*, Seong Hoon Jeong1* 1

College of Pharmacy, Dongguk University-Seoul, Gyeonggi, Republic of Korea

2

College of Pharmacy, Seoul National University, Seoul, Republic of Korea

3

Abion Inc., Seoul, Republic of Korea

4

School of Chemical Engineering and Material Science, Chung-Ang University, Seoul,

Republic of Korea

* To whom correspondence should be addressed. Seong Hoon Jeong, PhD College of Pharmacy Dongguk University – Seoul Goyang, Gyeonggi 10326, Republic of Korea Tel: 82) 10-5679-0621 E-mail: [email protected]

Eunah Kang, PhD School of Chemical Engineering and Material Science Chung-Ang University 221 Heukseok-Dong, Dongjak-Gu, Seoul, Republic of Korea Tel: 82)-2-820-6684 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract Nanodiamonds have been enlightened as a new exogenous material source in biomedical applications. As a new potent form of nanodiamond (ND), polyamidoamine-decorated nanodiamonds (PAMAM-NDs) were prepared for E7 or E6 oncoprotein-suppressing siRNA gene delivery for high risk human papillomavirus (HPV) - induced cervical cancer, such as types 16 and 18. It is critical to understand the physicochemical properties of siRNA complexes immobilized on cationic solid ND surfaces in the aspect of biomolecular structural and conformational changes, as the new inert carbon material can be extended into the application of a gene delivery vector. A spectral study of siRNA/PAMAM-ND complexes using differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD) proved that the hydrogen bonding and electrostatic interaction between siRNA and PAMAMNDs decreased endothermic heat capacity. Moreover, siRNA/PAMAM-ND complexes showed low cell cytotoxicity and significant suppressing effects for forward target E6, E7 oncogenic genes, proving functional and therapeutic efficacy. The cellular uptake of siRNA/PAMAM-ND complexes at 8 hours was visualized by macropinocytes and direct endosomal escape of siRNA/PAMAM-ND complexes. It is presumed that PAMAM-NDs provided a buffering cushion to adjust the pH and hard mechanical stress to escape endosomes. siRNA/PAMAM-ND complexes provide a potential organic/inorganic hybrid material source for gene delivery carriers.

Keywords: Nanodiamonds, siRNA, gene delivery, HPV


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1. Introduction Gene therapy is a therapeutic method to treat genetic defect–related diseases by introducing exogenous genetic materials and modulating the gene expression of target proteins. RNA interference is a gene therapy strategy in which antisense oligonucleotides (typically 15–30 nucleotides) block target RNA, thus reducing the expression level of target disease–causing proteins.1-2

Physicochemical properties of siRNA with relatively low

molecular weight, anionic charge, and hydrophilicity make it less effective at passively diffusing across the anionic cellular plasma membrane due to electrostatic repulsion. Moreover, once therapeutic siRNA vectors are delivered into the cytoplasm via endocytosis, siRNA release is required from the endosomes prior to lysosomal degradation.2 Electrostatic physical complexation between cationic polymeric vectors and anionic siRNA has been one solution to elevate the transfection efficacy and stability without losing therapeutic and silencing abilities.3-7 Cationic polymers with high buffer capacity assist endosome rupture by allowing chloride ions into endosome and increasing osmotic pressure, called “proton sponge effect”.1 Versatile polymers or peptides with high buffering capacities such as polyethylenimine (PEI) and poly (L-lysine) have been widely designed as gene delivery carriers to promote endosome release and respond at weakly acidic conditions of pH 7.2–5.0.

However, significant cytotoxicity was occurred by destabilizing the plasma-

membrane and adhering to the cell surfaces due to their high molecular weight and high positive charge.8

As an alternative carrier, polyamidoamine (PAMAM) has shown the

reduced cytotoxicity, and simultaneously owns the buffer capacity for proton sponge effect, compared to the linear cationic polymer (PEI) and unmodified dendrimers.9-10 Moreover, PAMAM dendrimer can be easily grafted with nanomaterials due to uniform size, controlled molecular weight, and generation-dependent flexible chemistry.11



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Nanodiamonds (NDs) are inert carbon nanomaterials with low cytotoxicity and potential biocompatibility12-13, and thus have been recently investigated as a new exogenous source of a drug delivery carrier (synthetic drugs, proteins, and genes).14-20 The characteristic features of NDs are attractive due to their controlled size, high surface-to-volume ratio, and easy access for surface modification.21-23 NDs coupled with proteins24 or genes25-27 are one interesting focus in the aspect of functionality and the structure of biomolecules.28 The conformational changes of biomolecules on rigid surfaces at nanoscale can be critical for understanding their complex formation. The protein alignment and structural arrangement on the ND surface have been investigated for certain ND surface features.24, 29 Several studies of ND in gene delivery were limited to only the physical adsorption of a linear polymer or the direct siRNA adsorption on a detonated ND surface. In detail, NDs physically adsorbed with low molecular weight polyethylenimine (PEI) or poly(allylamine) showed high transfection abilities with reduced toxicity.25, 27 In addition, detonated cationic bare ND showed a high affinity for siRNA and the capability to deliver siRNA into Ewing sarcoma cells.26 The present study is particularly focused on the physicochemical properties and structural stability of siRNA on a cationic charged PAMAM decorated ND (PAMAM-NDs) surface. The present study is particularly focused on fabrication and characterization of the cationic charged PAMAM decorated ND (PAMAM-NDs) from negatively charged carboxylated nanodiamond (ND-COOH). The surface of ND-COOH with good water dispersity and low cytotoxicity was minimally modified with PAMAM dendrimer in order to exhibit siRNA loading capacity with lower cytotoxicity. The structural stability and conformational changes of siRNA on the PAMAM-

ND surface were investigated at the charged interfaces, relating with the ratio of siRNA to PAMAM-NDs for gene delivery efficacy. PAMAM was chemically conjugated on acyl chloride–functionalized NDs to grant a cationic charge. The physicochemical stability of siRNA complexes immobilized on the rigid core gene vector of PAMAM-decorated NDs was


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characterized with differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). High risk human papillomavirus (HPV)- induced cervical cancer, such as types 16 and 18, are critically affected by two major oncoproteins, E6 and E7.30 In this study, E6 or E7 oncogenic protein–suppressing siRNA/PAMAM-ND complexes were specifically investigated for their cell viability and oncoprotein expression level. Moreover, the transfection efficacy of siRNA was performed using TP53-RE-GFP reporter stable cell lines. 2. Experimental Section Preparation of PAMAM-ND: The commercial products of carboxylated ND (ND-COOH) were kindly supplied by Nanoresource Co., Ltd (Seoul, Korea). Average crystalline size of acid purified nanodiamond is ranged from 4~6 nm. Ethylenediamine core PAMAM dendrimer (3 generation, CAS number 153891-46-4) was purchased from Sigma-Aldrich (MO, USA). The nucleophilic acyl substitution of ND-COOH was carried out with the introduction of SOCl2 to prepare NDs with acyl chlorides (ND-COCl): ND-COOH (100 mg) was dispersed in SOCl2 under nitrogen purge and stirred for 24 hours at 70 °C. After the reaction, ND-COCl was centrifuged and washed with anhydrous tetraydrofuran (THF). The resulting ND-COCl was dried at 60 °C for 24 hours and stored in a vacuum desiccator before further use. PAMAM dendrimer (120 µL) (20 w/v% in methanol) was added to a round flask and dried, forming a film under vacuum conditions for two hours. The dried PAMAM film was dissolved in anhydrous DMF with the addition of pyridine (20 µL). Then, ND-COCl (50 mg) was dispersed into the PAMAM solution and reacted for 24 hours. The PAMAMdecorated NDs (PAMAM-NDs) were filtered and washed with distilled water and lyophilized using a Lyopride 20R lyophilizer (Ilshin Biobase, Gyeonggi, Korea) for further study. Characterization of ND-PAMAM particles: The conjugation of PAMAM to ND-COCl was characterized using a Nicolet iS5 FT-IR spectrophotometer (Thermo Fisher Scientific, MA, USA). The dried powders of ND-PAMAM, PAMAM, and ND-COOH were ground with KBr



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for IR measurements. The FT-IR spectra were obtained for 4000–800 cm-1 with 2 cm-1 resolution and 128 measurements. X-ray photoelectron spectroscopy was performed to examine the surface functional group changes between ND-COCl and PAMAM-ND. XPS measurement was performed with an ESCA 2000 multilab apparatus (VG Microtech, London, UK). Monochromated Mg·Kα X-ray source (1,253.6 eV) and a hemispherical analyzer were used for the experiment. 10 mg of dried ND-COCl and PAMAM-ND was used for the XPS analysis.

The particle size and zeta

potential were measured using a Zetasizer Nano ZS90 (Malvern Instruments, UK) to characterize the dispersive properties of ND-PAMAM and ND-COOH in an aqueous environment. The dispersions of ND-COOH and ND-PAMAM were prepared in distilled water at a 0.1 mg/mL concentration. Zeta potentials were performed with titrations for pH 3.0–9.0 under the condition of 2 min equilibration time at 25 °C, using an MPT-2 autotitrator (0.1 M sodium hydroxide and hydrochloric acid). A disposable sizing cuvette (Sarstedt, Germany) was used for the hydrodynamic radius and a disposable capillary cell (Malvern Instruments, UK) was used for zeta potential with a volume of 1 mL. All measurements were performed at a fixed angle of 90°. The average particle size, polydispersity index (PDI), and zeta potential were obtained from the average of five measurements. The morphology of NDCOOH and ND-PAMAM particles was examined via transmission electron microscopy (TEM). The dispersion of ND-COOH and ND-PAMAM in deionized water was cast dropwise on the lacy carbon grid. Excess water was wiped off and the grid was dried overnight. High resolution-TEM observation was performed using a JEM-3010 (JEOL, Japan) with a 300 kV acceleration voltage. Preparation of PAMAM-ND/siRNA complexes: The PAMAM-ND (2 mg) was dispersed in autoclaved DEPC-treated distilled water (10 mL), using an ultrasonicator (VCX-500, 300W, Sonics & Materials, USA) for 3 min. At pH 4 (1 mM HCl), lyophilized HPV-18 type 426 siRNA (1 µM in DEPC-treated distilled water) was added to PAMAM-ND for dispersion


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with predetermined ratios of 1:0.74, 1:1.48, 1:2.96, 1:4.44, 1:5.93, and 1:7.41. The mixture of PAMAM-ND and siRNA was equilibrated for 30 min to allow for complex formation. The solution was centrifuged at 13,000 rpm for 10 min to separate the unbounded free siRNA from PAMAM-NDs. Then, 30 µL of supernatant was mixed with 30 µL of 1 mg/mL ethidium bromide (EtBr). The binding of the siRNA with PAMAM-NDs was determined from the ratio of unbounded free siRNA to the initial total siRNA, as measured by the EtBr fluorescence intensity. The fluorescence of EtBr was measured using an M3 microplate reader with excitation at 490 nm and emission at 590 nm. Differential Scanning Calorimetry (DSC) Analysis: DSC spectra were measured using a Nano-DSC microcalorimeter (TA Instruments, New castle, DE, USA). Platinum capillary cells (300 µL) were used for reference and sample solutions were filled with determined concentrations of 426 siRNA and PAMAM-NDs (10 µM and 0.135 mg/mL respectively). DEPC-treated distilled water was used as a reference to obtain the baseline. Measurements were carried out with a 1 °C/min scanning rate for 25–90 °C and were performed in triplicate. Thermo compensation curves were obtained by subtracting the baseline from the sample thermograms and evaluating it using a Nanoanalyze 3.6.0 software package provided with the equipment. Circular Dichroism Spectroscopy (CD): Circular dichroism (CD) spectra were obtained to examine the changes in the RNA structure, as related with the ND surface. A Chirascan-plus spectrometer (Applied Photophysics, UK) equipped with a peltier-type temperature controller TC125 (Quantum Northwest Inc., WA, USA) was used to measure the CD spectra in the scanned range 20–90 °C in a quartz cuvette (Helma analytics, Germany, with a 1 mm light path length). The concentration of the ND-PAMAM and siRNA solution was adjusted to 0.1 mg/mL and 10 mM (to 0.135 mg/mL mg/mL and 10 µM, 1:1 ratio in mass), respectively, with a comparison to DEPC-treated distilled water as a reference. The scanning was



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conducted for 190–300 nm wavelength with a 1 nm resolution. All spectra were the average of three accumulated measurements. Cell culture and Transfection: The cervical cancer cell lines (HeLa and CaSki) were purchased from the American Type Culture Collection (Manassas, VA, USA). TP53-RE-GFP reporter stable cell lines (HeLa and CaSki) were established using a lentivirus-based strategy.31 Both cell lines were transduced with a mixture of pGreenFire1-P53-GF-EF1-puro (virus) and TransDux reagent, in accordance with manufacturer’s instructions (System Biosciences, CA, USA). TP53-RE-GFP reporter stable cell lines were kindly provided by Seoul National University (Seoul, South Korea). Cell lines were cultured in DMEM or RPMI1640 (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were seeded on 12-well plates at 0.5 × 105 cells per well at the time of transfection to examine E6/E7 small interfering RNA (siRNA) transfection. E6/E7 siRNA was used to knock down endogenous E6/E7 oncogene expression in cervical cancer cells (Table 1), and cells were transfected with 2’O-methylated E6/E7-specific siRNAs or negative control siRNA (Bioneer, Korea) using DharmaFECT™ siRNA transfection reagent (Dharmacon, CO, USA). The silencing efficiency of the E6/E7 siRNA pool (SP) in combination with PAMAM-NDs 0.01 (1:3.7 ratio) mg/mL and 0.02 (1:7.4 ratio) mg/mL) was checked by western blotting technique, as described earlier.32 Transfected cell lysates were microcentrifuged for 15 min at 14,000 rpm and 4 °C. Supernatant proteins were separated by SDS-PAGE on 12% acrylamide gels and transferred to a polyvinyl difluoride (PVDF) membrane, which was then blocked with 5% skim milk in TBS-T (0.1% Tween-20) for one hour. Membranes were probed with appropriate primary antibody concentrations (TP53 [DO-7], HPV 18-E6 [G-7], HPV 18-E7 [F-7], HPV 18/16-E6 [CIP5], HPV16-E7 [ED-17], β-actin [C-4] was purchased from Santa Cruz Biotechnology, CA, USA), and HRP-conjugated secondary antibody was obtained from Thermo Fisher


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Scientific (MA, USA). The chemiluminescent signals of immunoreactive proteins visualized with an enhanced chemiluminescence (ECL) system (DoGen, Seoul, Korea) were detected with ImageQuant LAS4000 (GE Healthcare Life Sciences, PA, USA). The quantification of band intensity was performed using Image J software (National Institutes of Health, MD, USA).

Table 1. siRNA sequence pools used in the study Cell line

Target region

HeLa

HPV 18 type siRNAs

Name Sequence 5ʹ-caaccgagcacgacaggaa dTdT-3ʹ 426 5MeG (S) (19mer) Antisense (AS) 5ʹ-uuccugucgugcucgguug dTdT-3ʹ 450 4MeG (S) (21mer) 4MeU (AS)

5′-ccaacgacgcagagaaaca dTdT-3′ 5′-uguuucucugcgucguugg dTdT-3′

5'-gcaaagacaucuggacaaa dTdT-3' 3MeGU (S) 366 (19mer) Antisense (AS) 5'-uuuguccagaugucuuugc dTdT-3' CasKi

HPV 16 type siRNAs

5'-ucaagaacacguagagaaa dTdT-3' 488 4MeG (S) (19mer) Antisense (AS) 5'-uuucucuacguguucuuga dTdT-3' 497 Sense (S) (19mer) 3 MeU (AS)

HeLa/ CasKi

Control siRNA (NC)

5ʹ-gaccggucgauguaugucuug-3ʹ5ʹagacauacaucgaccggucca-3ʹ

5’-GGC UAC GUC CAG GAG CGC ACC-3’

Cell viability assay: HeLa and CaSki cells were seeded at 0.5 × 105 cells per well in a 12-well plate the day before the transfection of ND-PAMAM/E6/E7 siRNA complexes. HeLa and CaSki cells were respectively transfected with 18-type E6/E7 siRNA and 16-type E6/E7 siRNA combined with determined concentrations of PAMAM-ND (ranging from 0.01 mg/mL to 0.02 mg/mL). As a control, the maximum concentration of NC siRNA alone and NC siRNA/PAMAM-ND complexes (0.02 mg/mL) were treated for the same period. Treated cells were incubated for 72 hours at 37 °C under the ambient conditions of 5% CO2 and 100% humidity. Cell viability assaying was carried out using water soluble tetrazolium (WST) reagent (Daeil Lab Inc., Korea). WST regent (10x dilution) was added and incubated



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for 1–2 hours at 37 °C. Cell viability was determined by reading the absorbance at 460 nm, and was described as a relative percentage of the control siRNA (NC)-transfected cells; all experiments were repeated at least three times. Incucyte live cell imaging: TP53-RE-GFP reporter stable cell lines (HeLa and Caski) were transfected with a E6/E7 siRNA pool in combination with a determined concentration of PAMAM-ND (from 0.0075 mg/mL to 0.02 mg/ml) in a 24-well plate and imaged using IncuCyte HD system (Essen BioScience, MI, USA). Transfected cells were captured at one hour intervals using a 10x objective. The number of TP53-RE-GFP positive cells and total TP53-RE-GFP intensity were counted using IncuCyte ZOOM software (Essen BioScience, MI, USA). Values from all four regions of each well were averaged across the three replications, and images were extracted directly from IncuCyte ZOOM software using integrated settings. Cellular uptake: The cellular uptake of siRNA/ND-PAMAM complex was visualized using transmission electron microscopy (TEM). HeLa and CaSki cells were seeded with a cell density of 4 × 106 cells per a 100 cm2 surface area plate. After 24 hr of cell seeding, HeLa and CaSki cells were transfected, respectively, with 18 type E6/E7 siRNA and 16 type E6/E7 siRNA in combination with 0.02 mg/mL of PAMAM-ND. Hela and CaSki cells were incubated for 8 hr at 37°C in DMEM and RPMI1640 medium under of 5% CO2 and 100% humidity. After treatment, the cells were washed with PBS twice and fixed with 2% formaldehyde and 2.5% glutaraldehyde in 100 mM of sodium cacodylate buffer at pH 7.4 overnight. The cells were then post-fixed in 1% osmium tetroxide, dehydrated through a series of ethanol concentrations, and treated with 50% and 100% propylene oxide. Resin infiltration was performed with 2:1 and 1:2 mixes of propylene oxide:spur resin for 1 hr and with 100% spur resin overnight. After infiltration, the resin was changed and the sample was moved into embedding molds. The sample was polymerized at 60 °C for 24 hr. After


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embedding and sectioning, TEM observation was performed using a JEM-1010 (JEOL, Japan) with an acceleration voltage of 80 kV. Cellular uptake pathway study: HeLa and CaSki cells were seeded at 0.5 × 105 cells per well in a 12-well plate the day before the transfection of siRNA/PAMAM-ND complexes. As a control, the maximum concentration of NC siRNA alone (200 nM) and siRNA/PAMAM-ND complexes (0.02 mg/mL) were treated for the same period. Chlorpromazine hydrochloride (10 µg/ml) for the selective blockade of clathrin-dependent endocytosis and amiloride hydrochloride(100 µM) for the selective blockade of macropinocytosis were respectively added into siRNA-ND PAMAM mixture treated cells and incubated for 24 hours at 37°C. All these treatments were performed 30 minutes before the addition of siRNA PAMAM-ND complexes. Western blot assay using HRP-conjugated antibody was performed as above mentioned.

3. Result and discussion 3.1 Physical characterization of PAMAM-decorated NDs The surface of the carboxylated NDs was modified in thionyl chloride to form acyl chloride on the ND surface.

The amine group of PAMAM (3 generation) was conjugated

with the acyl chloride functional group on the ND surface, forming a positive amineterminated ND surface (Figure 1a). The morphology and characteristics of PAMAMdecorated ND were examined as shown in Figure 1. The magnified TEM images showed PAMAM decorated NDs and ND-COOH (Figure 1b). The individual core sizes of PAMAMND and ND-COOH were approximately 5 nm in spherical form. There were no significant size differences between ND-PAMAM and ND-COOH agglutinates. Although the individual core sizes of ND were about 5 nm, PAMAM-ND and ND-COOH did not present as individual core forms, rather they existed as agglutinates of irregular forms in aqueous



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dispersion due to van der Waals forces and electrostatic interaction.33 Moreover, the images of PAMAM-ND showed a thin polymeric layer on the outer ND surface, approximately 0.5– 1 nm, suggesting that PAMAM dendrimers were successfully conjugated on the ND surface. The hydrodynamic particle size of ND-COOH and PAMAM-ND measured by DLS is shown in Figure 1c. The agglutinate size of ND-COOH is significantly dependent on the environmental pH changes.34 The diameter of ND-COOH was drastically increased to the

micro-size of aggregates (2,674.1 ± 276.1 nm) at pH 2.5, while ND-COOH agglutinates showed colloidal stability with 52.6 ± 6.1 nm diameter at pH 8. After PAMAM conjugation, PAMAM-ND presented the pH-independent colloidal stability in the range 282.2 ± 2.7 nm at pH 2.5 and 485.3 ± 30.8 nm at pH 8, showing only a gradual increase in diameter with the pH change. The zeta potentials also showed the coincident properties of PAMAM-ND with 31.7 ± 3.1 mV at pH 2.9 and 15.1 ± 3.0 mV at pH 8.7, while ND-COOH drastically varied from 22.7 ± 1.8 mV at pH 2.9 to -41.4 ± 2.3 mV at pH 8.7 (Figure 1d). The zeta potentials of PAMAM-conjugated ND showed a decrease with less stiff gradient as the pH increased, compared to that of ND-COOH. The reported pKa values of the primary amines on the outer surface groups were 7.0–9.0 and the pKa values of tertiary amines in the internal cavity were 3.0–6.0 in the PAMAM dendrimer.35 The surface layer of PAMAM-conjugated ND with primary amines on the surface and tertiary amines in internal cavities showed a better buffering effect with stable nanometer-sized colloidal properties, compared to those of NDCOOH. The stable positive charges of PAMAM-decorated NDs can further bind to anionic siRNA via ion-pairing at physiological pH conditions and buffer charges.36


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Figure 1. (a) Schematic pictures of the substitution process of PAMAM dendrimer– conjugated nanodiamond (PAMAM-ND) from carboxylated nanodiamond (ND−COOH). (b) TEM images of enlarged PAMAM-ND agglutinates, and enlarged ND-COOH particles. (c) Z-average size and (d) zeta potential of PAMAM-ND and ND-COOH agglutinates in pH conditions. (e) and (f) FT-IR spectra obtained from ND−COOH, PAMAM dendrimers, and PAMAM-ND.

The FT-IR spectra of ND-COOH, PAMAM, and PAMAM-ND were obtained to confirm the conjugation between the acyl chloride of ND and amine-functionalized PAMAM dendrimer (Figure 1e and 1f). The PAMAM dendrimer showed an N-H deformation vibration peak at 1626 cm-1, a C–N stretching vibration peak at 1551 cm-1, an N-H stretching peak at 3286 cm-1, and a C-H stretching peak at 2944 cm-1.37 The peaks at 1626 cm-1 and 1551 cm-1 were respectively assigned to free N–H groups present on the periphery of the dendrimer and C–N bonds inside the core. ND-COOH showed an O-H deformation vibration peak at 1113



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cm-1, a C=O stretching peak at 1763 cm-1, a C-O stretching peak at 1629 cm-1, and an O-H stretching vibration peak at 3424 cm-1, while this was not shown for N-H stretching or bending peaks.38-39 PAMAM-ND showed both characteristics of ND-COOH and PAMAM dendrimers. The peak of PAMAM-ND at 1630 cm-1 was attributed to overlapped N-H deformation vibration and C=O stretching. The C-H stretching peak of PAMAM decorated ND was shifted from 2944 to 2968 cm-1. ND-PAMAM also showed a C=O stretching peak at 1763 cm-1. The O-H deformation vibration and stretching vibration peak of ND-COOH were shifted to 1119 cm-1 and 3421 cm-1, respectively. The C–N stretching vibrations peak and NH stretching peak of the PAMAM-ND also attributed to PAMAM dendrimer. The results of Xray photoelectron spectroscopy (XPS) for PAMAM-NDs also shows chemical conjugation of PAMAM on ND surface. ND-COCl showed tow peaks of Cl 2p3/2 and Cl 2p1/2 at 200.3 and 201.9 eV, indicating that ND-COOH was functionalized by acyl chloride group. After the graft of PAMAM dendrimer, Cl 2p peak was disappeared and N1s peak from 395 eV to 405 eV was appeared. The O 1s peaks of ND−COCl and PAMAM-ND were deconvoluted with two conventional binding energies at 531.6 eV (C=O) and 532.8 eV (C−OH). Strong peaks of C=O at 531.6 for ND-COCl presents dense functionalization by acylchloride. The reduced peaks of C=O and increased peak of C-OH for PAMAN-ND indicate that reactive acyl chloride was returned to carboxyl group after conjugation with PAMAM and ND surface. XPS spectra of C1s peaks also showed C-Cl peak in ND-COCl (286.5 eV) was successfully substituted with C-N peak in PAMAM-ND (286.0 eV). (Figure S1.) The

chemical characteristics from FT-IR spectra and XPS analysis presented that the carboxylic acid group on the ND-COOH surface was decorated with amine-functionalized PAMAM dendrimer.


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Figure 2. (a) Schematic pictures of siRNA/ PAMAM-ND complex formation via electrostatic interaction. (b) Conjugated siRNA was quantified and plotted with concentration of PAMAM-ND. (c) CD spectra of 10 µM 426 siRNA duplex with 6.75 (ND:siRNA = 1:2), 13.5 (ND:siRNA = 1:1), and 27 µg/mL (ND:siRNA = 2:1) PAMAM-ND concentrations at 20 °C. Red arrows indicate changes in the circular dichroism value as the PAMAM-ND concentration increases. (d) A schematic picture of PAMAM-ND /siRNA complexing and free siRNA. 3.2. The complex formation of PAMAM-NDs with siRNA and its structural characterization PAMAM-decorated NDs with positive charges interacted electrostatically with siRNA (Figure 2a). The quantitative amount of unbound free-siRNA on PAMAM-decorated NDs has been measured by EtBr fluorescence assay (Figure 2b). The amount of free siRNA decreased as the concentration of PAMAM-ND increased in a linear regression (R2=0.901), due to binding between siRNA and PAMAM-NDs. siRNA binding at 80% showed a 5.6 mass ratio of PAMAM-ND/siRNA complexes (PAMAM-ND 0.075 mg/mL and siRNA 0.0134 mg/mL). Supportingly, ND-COOH did not bind with siRNA due to charge repulsion.



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ND-COOH showed 96.8 ± 4.8 % free siRNA with 0.1 mg/mL and 98.7 ± 2.9 % free siRNA with 0.05 mg/mL while PAMAM-NDs showed significantly higher siRNA loading capacity of 2.9 ± 1.8 % free siRNA at 0.1 mg/mL and 37.5 ± 5.3 % at 0.05 mg/mL (Figure S2.) The secondary structural and tertiary conformational changes of siRNA upon complex formation were observed by the spectra of CD (Figure 2c). Bare siRNA duplex showed a CD spectrum with a 266 nm positive peak and 208 nm negative peak.40 At different mix ratios of siRNA and PAMAM-ND (1:1 and 1:2), the positive and negative peaks of the CD spectra were shifted from 266 nm to 267 nm and from 208 to 210 nm compared to the peaks of the bare siRNA. In contrast, the complex of PAMAM-NDs and siRNA with a 2:1 ratio showed a less intense and red-shifted positive peak at 272 nm. At a 2:1 ratio mixture of PAMAM-ND and siRNA, two negative and less intensive peaks were observed at both 208 and 211 nm, due to the 50% coexistence of free and bounded siRNA on the PAMAM-decorated ND surface. An intensive positive peak at 190 nm

is generally recognized as an indication of the right-

handed typical A-conformation of a polynucleotide helix structure.42

41

or α-helix structure in protein

A single strand of sense or antisense 426 siRNA and both simple mixture

without annealing did not show a positive peak at 190 nm, supporting that spectral change of siRNA/PAMAM-ND complexes came from interference of double helix and formation of single strand (Figure S3). The weakened intensity at 190 nm for the complex of PAMAMNDs and siRNA with a 2:1 ratio could be evidence of stable electrostatic complex formation with siRNA and PAMAM-NDs, which could disturb the shape of the helix of the siRNA duplex and make hydrogen bonds between siRNA and PAMAM dendrimer on NDs. The CD spectra of the siRNA/PAMAM-ND complexes showed structural conformational changes for bounded siRNA, induced from adsorption on the cationic solid surface via electrostatic interaction and hydrogen bonding (Figure 2d).


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Secondary structural and tertiary conformational changes of siRNA were observed for bare siRNA and complexed siRNA with PAMAM-decorated NDs as the temperature increased. The spectra of CD, UV, and DSC for bare siRNA and siRNA/PAMAM-NDs with 1:1 and 1:2 ratios were obtained from 20 °C to 90 °C (Figure 3, Table S1). The siRNA duplex showed positive peak shift from 266 nm at 20 °C to 274 nm at 90 °C and no change in the negative peak at 208 nm. The intensity of CD significantly changed over 70 °C. The ellipticity at 190 nm changed from 22.17 mdeg at 20 °C to 1.96 mdeg at 90 °C, and the ellipticity at 266 nm ranged from 8.30 mdeg at 20 °C to 1.50 mdeg at 90 °C. The intensities of the negative and positive peaks were weakened and flattened as the temperature increased, suggesting the loss of the double helix structure. The reduced UV absorbance, as heating on a structured nucleic acid, reflected the conformational disruption of base-stacking interactions.43 PAMAM-ND/siRNA (1:1 ratio) complexes showed a similar tendency with bare siRNA duplex as the temperature increased. The positive peak at 267 nm and 20 °C showed a red shift at 274 nm and 90 °C. Ellipticity was reduced from 24.43 mdeg at 20 °C to 0.92 mdeg at 90 °C (at 190 nm), and from 8.46 mdeg at 20 °C to 2.31 mdeg at 90 °C (at 267 nm), respectively. The feature of bare siRNA was dominant, rather than of the siRNA complexes, considering that 38.5% of the siRNA was bound onto the PAMAM-ND surface at the complex ratio of PAMAM-ND/siRNA (1:1). Unlike PAMAM-ND/siRNA (1:1 ratio) complexes, PAMAM-ND/siRNA (2:1 ratio) complexes showed a positive peak at 269 nm and a negative peak at 211 nm, which was not shifted during heating. PAMAM-ND/siRNA (2:1 ratio) complexes showed smaller ellipticity changes from 4.88 mdeg at 20 °C to 1.67 mdeg at 90 °C when at 190 nm. The ellipticity at 269 nm also ranged from 2.53 mdeg at 20 °C to 0.99 mdeg at 90 °C. The decreased dependence of the ellipticity on temperature over 70 °C and low ellipticity at 190 nm could be evidence that the A-conformation RNA double



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helix structure was temporarily interfered by electrostatic interaction and hydrogen bonding between the PAMAM-ND surface and siRNA.

Figure 3. CD spectra of (a) 13.5 µg/mL of PAMAM-ND, (b) 10 µM of 426 duplex siRNA, (c) 1:1 ratio PAMAM-ND /siRNA complex, and (d) a 2:1 ratio PAMAM-ND /siRNA complex as the temperature increases. The UV absorbance of (e) 13.5 µg/mL of PAMAMND, (f) 10 µM of 426 duplex siRNA, (g) 1:1 ratio PAMAM-ND /siRNA complex, and (h) a 2:1 ratio PAMAM-ND /siRNA complex as the temperature increases. (i) The DSC raw data of 135 µg/mL of PAMAM-ND, 10 µM of 426 duplex siRNA, and PAMAM-ND /siRNA complex. (j) Baseline subtracted from molar heat capacity of the PAMAM-ND /siRNA complex and siRNA duplex.

UV absorbance spectra provided complimentary information of nucleic acid as CD spectra (Figure 3b, Table S2). The UV absorbance of siRNA was originated from base


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transitions (purine and pyrimidine) at 260 nm and was caused by the phosphate backbone below 190 nm.44 The siRNA duplex showed increased optical density from 1.53 to 2.27 at 190 nm and from 0.24 and 0.28 at 260 nm upon increasing the temperature from 20 °C to 90 °C, and PAMAM-ND/siRNA (1:1 ratio) complexes showed a similar tendency of the optical density, compared to bare siRNA duplex (from 2.07 to 3.04 at 190 nm and from 0.28 to 0.33 at 260 nm). The hyperchromicity of UV absorbance at 260 nm resulted

from unwinding the

double stranded siRNA, forming single stranded RNA, and unstacking siRNA as the temperature ramped up.45 Specifically, PAMAM-ND/siRNA with 2:1 ratio complexes showed optical density changes from 1.39 to 2.21 at 190 nm and from 0.03 to 0.03 at 260 nm. ND-PAMAM/siRNA with 2:1 ratio complexes showed increased hyperchromicity at 190 nm, compared to bare siRNA and ND-PAMAM/siRNA with a 1:1 ratio. Moreover, the UV absorbance at 260 nm was weakened and did not change as the temperature ramped up. This result showed that an adequate amount of PAMAM-NDs induced adjacent neighboring between the siRNA and PAMAM surface, interfering the base stacking of the double helix. The unchanged UV absorbance at 260 nm showed the maintenance of single stranded siRNA, even as the temperature increased, and low UV absorbance (0.03) indicated that the base pair was embedded into the PAMAM polymer layer on ND. The UV absorbance of siRNA/PAMAM-ND complexes suggested intermolecular interactions between siRNA and the PAMAM layer on NDs. The sense and antisense single strand RNA was evidence that the optical density of the ND-PAMAM/siRNA complex (2:1 ratio) was dominated by single stranded RNA on the PAMAM-ND surface (Figure S3-d and e). The UV absorbance was 0.15–0.17 for sense RNA and 0.20 for the antisense RNA at 260 nm. Single-strand siRNA without the double helix structure showed hyperchromicity at 190 nm and unchanged optical density at 260 nm.



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Duplex siRNA/PAMAM-ND complex formation was evaluated with DSC analysis for PAMAM-NDs, bare siRNA, and siRNA/PAMAM-NDs, as presented in Figure 3i. Using a two-state scaled model, calorimetric enthalpy was multiplied by scaling factor for calculating total transition enthalpy (Table 2).46 The DSC spectra of siRNA and siRNA/PAMAM-ND complexes showed respective transition peaks at 78.66 ± 0.03 °C and 80.13 ± 0.03 °C, resulting from hydrogen bonding between the siRNA double helix base pairs. The enthalpy of siRNA and PAMAM-ND/siRNA complexes was 684.7 ± 11.4 kJ/mol (scaling factor 0.166 ± 0.005) and 665.6 ± 9.1 kJ/mol (scaling factor 0.071 ± 0.002). The total transition enthalpy of siRNA and siRNA/PAMAM-ND was calculated as 112.1 and 47.3 kJ/mol. The total entropies of siRNA and siRNA/PAMAM-ND were respectively 0.32 and 0.13 kJ/mol·K. The transition temperature and peak shape for PAMAM-ND/siRNA complexes were maintained without broadening or shifting the transition in the thermogram compared to that of bare siRNA, suggesting that the siRNA on the PAMAM-NDs was not thermally destabilized.47 The PAMAM-ND/siRNA complex showed significantly decreased transition enthalpy, indicating that single stranded siRNA on the PAMAM-ND surface had already been formed by intermolecular interactions and was stable, even upon heating. Thus, the thermal destabilization of double helix siRNA was reduced in the state of complex formation.

The

decreased enthalpy implies that the hydrogen bonds within the double helix base pairs were compensated with electrostatic interactions and hydrogen bonding between single stranded siRNA and the PAMAM layer on the ND surface.

Table 2. Thermodynamic properties (transition temperature, total transition enthalpy, and total entropies) of 426 duplex siRNA (10 µM) and 1:1 mass ratio PAMAM-ND /siRNA complex Sample

Tm (°C)

∆H (kJ/mol)

∆S (kJ/mol·K)

PAMAM-ND/siRNA

80.13 ± 0.03 78.66 ± 0.03

47.3 112.1

0.13

426 duplex siRNA


0.32

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Overall, the DSC results correlate with the CD and UV spectra of siRNA and siRNA/PAMAM-ND complexes. The decreased ellipticity of PAMAM-ND/siRNA complexes at a 2:1 ratio was contributed by weakened base–base interactions and interfered double helix structure formation, since siRNA was adsorbed to the PAMAM-ND surface via electrostatic interactions and hydrogen bonding (Figure 2d). The disturbance of the double helix structure on the PAMAM-ND/siRNA complexes (1:2 ratio) was observed from the transition between 70 °C and 90 °C in UV absorption. Physicochemical analysis evidenced the interaction of siRNA complexes, in that electrostatic interaction was generated by charge interactions between the negative phosphate group and the cationic -CH2-NH3+ unit present in the PAMAM-ND surface.48 3.3. Cell viability and silencing efficacy of PAMAM-NDS/E6,E7 targeting siRNA The cell viability of PAMAM-ND, siRNA pool, and a PAMAM-ND/siRNA complex were analyzed for CaSki and HeLa carcinoma cells by WST assay (Figure 4a and 4d, Table S3). The complex of siRNA and PAMAM-NDs with ratio of 1:2.78–7.41 was added with PAMAM-ND concentration of 0.0075–0.02 mg/mL and incubated for 72 hours. The bare negative control (NC) siRNA and NC siRNA/PAMAM-NDs did not show significant cytotoxicity and silencing effect for CaSki and HeLa cells after 72 hours incubation. High respective cell viabilities of 94.7 ± 3.9 % and 96.8 ± 2.8% were shown for CaSki and HeLa cells, for the existence of 0.02 mg/ml NC siRNA/PAMAM-NDs. In contrast, E6/E7 siRNA complexes with PAMAM-NDs or Dharmafect (3 µg/mL) showed concentration-dependent reduced cell viability. The cell viabilities of CaSki were reduced to 93.5 ± 3.8 % for 0.01 mg/mL of E6/E7 siRNA/PAMAM-ND complexes. In 0.015 mg/mL of E6/E7 siRNA/PAMAM-ND complexes, cell viability was significantly reduced to 88.2 ± 3.4 % (p

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