- Email: [email protected]
Lysine functionalized nanodiamonds as gene carriers - Investigation of internalization pathways and intracellular trafficking
Diamond & Related Materials 98 (2019) 107477
Contents lists available at ScienceDirect
Diamond & Related Materials journal homepage: www.elsevier.com/locate/diamond
Lysine functionalized nanodiamonds as gene carriers - Investigation of internalization pathways and intracellular trafficking
T
Saniya Alwania, QingYun Hua (Nancy)a, Safa Iftikhara, Narayan P. Appathuraib, ⁎ Deborah Michela, Chithra Karunakaranb, Ildiko Badeaa, a b
Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Canada Canadian Light Source, Saskatoon, SK, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Lysine-functionalized nanodiamonds Diamoplexes Flow cytometry Scanning transmission X-ray microscopy Cellular internalization Macropinocytosis Clathrin-mediated endocytosis Endo-lysosomal entrapment
Two major determinants for effective gene delivery are the capability of the carrier to protect genetic material during extra and intracellular trafficking, and the ability to release it at the site of action in an intact form. The cellular uptake pathway is a controlling parameter regarding the fate of nanoparticle-genetic material complexes. This study elucidates the pathways involved in the cellular uptake of pristine nanodiamonds (pND), functionalized nanodiamonds (lys-NDs) and diamoplexes (ND/genetic material complexes, namely lysNDsiRNA). Changes in membrane morphologies corresponding to a specific route of internalization were studied using transmission electron microscopy. Variations in cellular association of diamoplexes after general and targeted inhibition of endocytic pathways were quantified using flow cytometry. Electron micrographs reveal formation of unilateral membrane protrusions suggestive of macropinocytosis as one of the major uptake process for lys-NDs and lysND-siRNA diamoplexes. Formation of pits was also largely detected for lys-NDs but scarcely present for their diamoplexes, attributed to either clathrin-mediated or caveoli-mediated endocytosis. However, inhibition of caveoli-mediated endocytosis did not prevent internalization of the diamoplexes, suggesting the absence of this pathway. Due to the larger aggregate size, pNDs were mainly internalized through macropinocytosis, as indicated by unilateral pseudopods on their micrographs. The cells also showed bilateral pseudopods during internalization suggesting phagocytic uptake, possibly due to random functionalities on the surface of pNDs. Clathrin or caveoli mediated endocytosis was absent. Soft X-ray spectromicroscopy reveals strong sp3-carbon signals confirming that the internalized entities identified by electron microscopy are indeed NDs and their diamoplexes. These findings are the basis of further optimization of amino acid functionalized NDs towards increase of gene delivery efficacy through targeted internalization pathways and overcoming intracellular challenges.
1. Introduction As gene therapeutics pave their way towards precision medicine there is an increasing need for novel nanocarriers, capable of delivering the therapeutic genes to their site of action (i.e., into mammalian cells) in an intact and highly efficient form. An ideal gene carrier should have three major characteristics: (1) compatibility with the biological environment up to the level of the cellular machinery (2) the strength to protect the genetic material during biodistribution and upon cellular uptake and (3) minimal interference with the therapeutic process in action [1]. Synthetic nanocarriers ranging from organic molecules such
as lipid-based [2,3] and polymeric nanoparticles [3] to inorganic materials like gold [4], silica [5] and carbon nanoparticles [6,7] have shown promise in this regard. Of inorganic nanomaterials, nanodiamonds (NDs) are a unique member of the carbon nano-family recently studied as a biological carrier [8,9]. NDs offer an ultimate advantage of biocompatibility [10], innately reactive surface and ultra-stable diamond core [11]. Our group reported a novel approach to impart a net positive charge on the surface to modulate the conjugation with oppositely charged genetic biomolecules by functionalizing the NDs with a basic amino acid, lysine [6]. Lysine-functionalized nanodiamonds (lys-NDs) are covered by a protein
Abbreviations: pNDs, pristine nanodiamonds; lys-NDs, lysine-functionalized nanodiamonds; STXM, scanning transmission X-ray microscopy; YTZ, yttria-stabilized 0.05 mm zirconia ⁎ Corresponding author at: 107 Wiggins Road, Health Sciences Building, Room 3D01.5, Box 3D01-13, Saskatoon, SK S7N 5E5, Canada. E-mail address: [email protected] (I. Badea). https://doi.org/10.1016/j.diamond.2019.107477 Received 31 May 2019; Received in revised form 9 July 2019; Accepted 16 July 2019 Available online 18 July 2019 0925-9635/ © 2019 Elsevier B.V. All rights reserved.
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
corona upon interaction with the biological fluids, which improves their dispersion stability [9]. Contrary to other hard nanoparticle (NP) gene carriers like iron oxide [12], silica [13] and gold [14] NPs, which are less efficient in the presence of serum proteins, the corona formed on the NDs facilitates the uptake of the ND-gene conjugates [9]. Although formation of protein corona is not a condition to initiate the cellular uptake of lys-NDs, its presence enhances the internalization of lys-ND/siRNA complexes (diamoplexes) [9]. Previous analyses performed for lys-NDs have successfully laid the foundation of this novel technology by elucidating that:
Table 1 Procedures used to interrupt specific endocytic pathways of internalization of diamoplexes. Treatment–pathways affected 1 2 3 4 5
(1) lysine functionalization controls ND aggregation and provides longterm stability necessary for biological applications; (2) lys-NDs form complexes with siRNA at biologically safe weight ratios; and (3) lys-NDs internalize into cancer cells in a concentration dependent manner and distribute throughout cytosol.
Low temperature (4 °C) – General inhibition of endocytosis Reagents Concentrations (μg/ml) Sodium azide (NaN3) - General inhibition of 500 endocytosis Chlorpromazine – Clathrin-mediated endocytosis 7.1 Filipin – Caveolae-mediated endocytosis 2 Amiloride – Macropinocytosis 27
2.2. Formulation design of lysine functionalized NDs for cellular experiments Lys-NDs were synthesized in-house following the procedure previously described [6]. Briefly, pharmaceutical grade NDs were treated with strong acids allowing reoxidation of the surface in order to maximize the loading of carboxyl functional groups. Carboxylated NDs were subjected to thionylation followed by covalent conjugation of lysine amino acid via an amine terminated three-carbon chain linker. Aqueous dispersion of lys-NDs synthesized as such was prepared at 2 mg/ml concentration and YTZ grinding media was added at a ratio of 1:1. The dispersion was subjected to ultrasonication overnight and centrifugation at 5200g for 5 min to obtain a well-dispersed uniform formulation for subsequent biological experiments.
Here we aim to study in detail the mechanism of cellular internalization, an important parameter necessary to define the functional efficiency of the system. For successful gene transfection, NP-gene complexes must be able to overcome intracellular trafficking barriers and degradative machineries [15,16]. Modulation of the pathways involved in the cellular uptake has a direct impact on these obstacles, and ultimately on the fate of NP-gene complexes in the cells [16–18]. Therefore, this study focuses on understanding the processes involved in the cellular uptake of diamoplexes in mammalian cells, with an aim to further tailor the functionalization approach for maximum gene transfection.
2.3. Flow cytometry Concentrations of the inhibitor solutions were selected based on their toxicity profiles as indicated in MTT assay (Supplementary information and Fig. S1). Table 1 indicates the details of the applied pretreatments. All stock solutions were filtered using 0.22 μm syringe filter and dilutions were made in unsupplemented DMEM. HeLa cells seeded in 6-well plates at the density of 8 × 104 cells/ml and were incubated for 24 h. The cell media were replaced with inhibitor solutions and incubated for 45 min. FITC-labelled siRNA was complexed with lys-NDs dispersed in aqueous medium at a weight ratio of 50:1 by incubating the ND dispersion with siRNA for 30 min at room temperature. The final concentration of siRNA in each well was 80 pmol. The cells were incubated with diamoplexes for 6 h. After the incubation period, the cells were washed with PBS, harvested through trypsinization and centrifugation and re-suspended in 500 ml of PBS. The incubation time was selected to ensure maximum effect of pretreatments on the endocytosis of diamoplexes. Untreated cells and diamoplex-treated cells with no prior inhibitor treatments were used as negative and positive controls, respectively. Fluorescence from FITC siRNA was recorded using FL1-H band pass filter (530/30 BP) by plotting log of relative fluorescence intensity versus number of events. Healthy cell population was gated and a total of 10,000 events were recorded in the gated region per sample. The data was analyzed using BD CellQuest™ Pro software (version 6.0). Marker M1 was applied to calibrate the auto fluorescence of the cells. Positive fluorescence shift (lower to higher end) for diamoplex treated cells was compared to the untreated cells and negative fluorescence shifts (from higher to lower) after endocytic inhibitor pre-treatments were compared to the diamoplex treated cells with no prior inhibition. Higher relative fluorescence recorded in M1 region was directly correlated with greater accumulation of FITC fluorescent siRNA and subsequently diamoplexes in the cells.
2. Materials and methods 2.1. Materials Pharmaceutical-grade (ND98) carboxylic acid–functionalized NDs with an average particle size of 5 nm were purchased from Dynalene Inc. (Whitehall, PA, USA). Yttria-stabilized 0.05 mm zirconia (YTZ®) grinding media were purchased from Tosoh Corporation (Grove City, OH, USA). Lys-NDs dispersion was prepared in HyPure Molecular Biology Grade Water. HyClone® HyPure Molecular Biology Grade Water, HyClone™ 1× phosphate-buffered saline (PBS), and HyClone™ Dulbecco's Modified Eagle's Medium (DMEM)/High; glucose with Lglutamine; sodium pyruvate and Dimethyl sulfoxide (DMSO) and histology grade ethanol were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was acquired from Thermo Fisher Scientific. Human cervical cancer (HeLa) cells were obtained from American Type Culture Collection (Manassas, VA, USA). Trypsin and antibiotics were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Fluorescein isothiocyanate (FITC)-conjugated control siRNA was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Chlorpromazine, sodium azide (NaN3), filipin, amiloride, low viscosity acrylic resin (LR white)-medium grade, benzoyl peroxide blend with dicyclohexyl phthalate and osmium tetroxide 4% aqueous solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4% paraformaldehyde, 8% glutaraldehyde and Ballistic Electron Emission Microscopy (BEEM) capsules were obtained from EM Sciences (Hatfield, PA, USA). (Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent were purchased from Invitrogen, ON, Canada. Cell culture flasks and plates, centrifuge tubes, and cell strainer tubes were purchased from Falcon BD (Mississauga, ON, Canada). Formvar coated 300-mesh copper grids were provided by University of Saskatchewan Western College of Veterinary Medicine Imaging Centre.
2.4. Transmission electron microscopy HeLa cells were grown and treated with lys-NDs and lys-NDs/siRNA diamoplexes (in presence and absence of serum proteins) in similar 2
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 1. Effect of endocytic inhibitors on cellular uptake of diamoplexes. Bar graphs represent the percent fluorescence of FITC-labelled siRNA in response to various pretreatments. Decrease in FITC siRNA fluorescence as compared to the ‘No Inhibitor’ is proportional to the ability of the pre-treatment to inhibit the association of diamoplexes with the cells. Pre-treatments include - low temperature & NaN3 (general endocytosis); chlorpromazine (clathrin-mediated pathway); filipin (caveoli-mediated pathway) and amiloride (macropinocytosis). Statistical analysis: t-test (test value = 21.1 (fluorescence of cells with no inhibitor pre-treatment; 95% confidence interval; difference in fluorescence is considered significant when p-value ≤ 0.050) p-values = *0.050, **0.015, ***0.002.
3. Results
fashion as described above. Untreated cells and pNDs were used as negative and positive controls respectively. Amount of NDs in all samples was kept constant to diamoplexes at a ratio of 50:1. The cells were incubated for 4 h with all treatments in order to ensure that morphological changes of the cellular membrane resulting from endocytosis could be captured before original conformations are regained. TEM samples were prepared by a protocol described elsewhere [19]. Briefly, each sample was washed, harvested and re-suspended in PBS. Each cell pellet was transferred in a labelled BEEM capsule and was subjected centrifugation to obtain pellets of 0.5 to 1 mm size and average cell count of 1 million. Cells were then fixed at room temperature for 2 h using the solution containing 2.5% formaldehyde/ glutaraldehyde in PBS. After proper washing to remove excess fixatives from the samples, cells were subjected to second-degree fixation for 1-h using 1% osmium tetroxide aqueous solution. Pellets were then rinsed using the same buffer as the fixative diluent (i.e. PBS) and dehydrated through a graded series of ethanol concentrations (50, 70, 90 and 100%) for 10 mins each. The pellets were cured using 50:50 resin: ethanol mixture for 45 min and embedded in 100% LR white resin and heated overnight at 40 °C in the oven. Samples were sliced up to a thickness of 200 nm, plated on copper grids and viewed using TEM (Hitachi 7700 with EDS). Digital images with associated micron bars were created using the built-in TEM control software.
3.1. Effect of endocytosis inhibitors on cellular uptake of diamoplexes In our previous publication, we reported internalization of diamoplexes in mammalian cells in a concentration-dependent manner [9]. We also reported that cellular association of lys-NDs/FITC labelled siRNA diamoplexes significantly improved in presence of serum proteins [9]. In lieu of these previous findings, all nano-formulations prepared for this experiment were diluted using serum supplemented media to ascertain two major goals: (a) Maximize cellular localization of FITC labelled siRNA in order to effectively quantify differences in fluorescence resulting from inhibitor pre-treatments (b) Since there is also an evidence that serum supplementation maintains a good cellular viability for lys-ND treated cells (unpublished data), we aimed to minimize pre-treatment induced loss of healthy cells. Dilution with serum-supplemented media ensured maximum cell viability allowing us to evenly collect 10,000 events per sample. Above mentioned pre-developed method [9] to compare the relative percentage of FITC fluorescence from siRNA in different specimens. Cells without treatment with inhibitors prior to transfection, showed a highest FITC-fluorescence from siRNA (21.1%) indicating that the diamoplexes were associated with the cells (Figs. 1 and S2-A). Blocking the endocytosis non-specifically (4 °C and NaN3), resulted in decline of fluorescence intensity to 11.3% ± 1.08 and 14.3% ± 1.46, respectively (Figs. 1 and S2-B). In order to elaborate the specific endocytic routes for the uptake of these diamoplexes, we pre-treated the cells with inhibitory reagents to a specific pathway. The mean fluorescence intensity remained unchanged with chlorpromazine (22.44% ± 5.54) in comparison to the cells with no pre-treatment (21.1%) (Fig. 1). However, the cells pre-treated with chlorpromazine underwent a high degree of damage during the treatment, which ultimately affected the overall mean fluorescence. In the most viable cell population among the three samples, fluorescence reduced to 18.5% (Fig. S2-C) indicating the impact of this reagent in healthy cells. Filipin did not show any reduction in fluorescence (25.14% ± 0.53) indicating that caveolaemediated endocytosis is not a mode of internalization for diamoplexes (Figs. 1 and S2-D). Pre-treatment with amiloride reduced the FITC fluorescence intensity from siRNA to 15.98% ± 0.43 compared 21.1% corresponding to the sample that did not receive any inhibitor pretreatment (Figs. 1 and S2-E).
2.5. Scanning transmission X-ray microscopy TEM grids loaded with cell samples were pre-screened using an optical microscope to select intact cell sections and to identify the region of interest. Data was collected using scanning transmission X-ray microscope (STXM) at the soft X-ray spectromicroscopy (SM) beamline at the Canadian Light Source (Saskatoon, SK, Canada). The source point for the SM beamline is an elliptically polarizing undulator, which provides linear and circularly polarized light in the energy range of 130 to 2700 eV. A plane grating monochromator of the beamline along with a zone plate in the microscope can provide X-ray photons with spectral resolution of ~0.1 eV and spatial resolution of 25–30 nm at the carbon K-edge. The samples on TEM grids were mounted on the STXM sample holder and X-ray data were collected in the transmission mode by raster scanning the sample over the region of interest. Spectral data were collected by acquiring sequence of transmission images at the carbon Kedge from 280 to 340 eV. The sample data were then normalized (and converted into optical density-OD using Beer's law) using a carbon Kedge spectrum extracted from an empty region within the cell section. Note the empty regions that served as negative control for normalization of the sample data within these cell sections were covered by the LR white resin. The composite maps of the cells were created using the spectra extracted from different regions on the cells.
3.2. Morphological changes in the cell membrane during uptake We used TEM to map the cellular internalization and localization of different ND based treatments. Morphological changes in the cell 3
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 2. TEM images showing cellular morphologies of plasma membrane and possible uptake pathways for NDs and diamoplexes. (A) & (B) pNDs (C), (D), (E), (F) and (G) lys-NDs (H) diamoplexes (transfected in the presence of serum) (I), (J) & (K) diamoplexes (transfected in the absence of serum). Straight arrows indicate pit formation and possible clathrin or caveoli mediated endocytosis, dotted arrows indicate unilateral membrane protrusions suggestive of macropinocytosis and dashed arrow indicate bilateral membrane extension suggesting phagocytosis.
observed in significant number, suggesting combination of processes involved in the uptake (Fig. 2I, J & K). Final distributions of various ND based treatments were also assessed to gain an insight of their intracellular fate. pNDs internalized in the cells were found enclosed in large membrane-bound vesicles mainly as aggregates (Fig. 3A). The lys-NDs were distributed in the cytoplasm and existed in non-aggregated forms with uniformly sized particles (Fig. 3B). Diamoplexes internalized in the presence of serum were also well distributed in the cytoplasm (Fig. 3C). In most instances diamoplexes were entrapped in the endosomes (Fig. 3C-right inset), however, some were found with no clear boundaries (Fig. 3C-left inset) indicating that they might exist freely in the cytoplasm. Uptake of the diamoplexes administered in the absence of serum was scarce as compared to their protein coated counterparts as fewer particles were observed in the cytoplasm (Fig. 3D in comparison with Fig. 3C). Although diamoplexes (both in presence and absence of serum) travelled to close proximity to the nucleus, were majorly found entrapped in light and dark membrane-surrounded vesicles (Fig. 3E and F).
membrane such as formation of pits or pseudopods (membrane extensions) were monitored. Cells treated with pNDs presented the formation of unilateral membrane protrusions (Fig. 2A inset), suggesting macropinocytosis as a major mode of uptake. However, in some cell samples bilateral membrane extensions (Fig. 2B) were also observed. Lys-NDs, without siRNA (Fig. 2C, D & E), prompted cell membrane to form pseudopods (dotted arrows) and pits (straight arrows) indicating the presence of both macropinocytosis and clathrin mediated endocytosis. Fig. 2F shows a late stage of internalization with pit closure and initiation of intracellular vacuole formation. At high magnification, many of the pits, formed during internalization process for lys-NDs (Fig. 2E & G), showed typical features: deeply embedded and relatively uniformly shaped pits and an organized molecular coat appearing as darker edges. In the presence of serum proteins, the internalization of diamoplexes was mostly completed at the time of analysis. Plasma membrane regained its original morphology, and membrane bound clusters of diamoplexes were found distributed in the cytoplasm (Fig. 2H). In the absence of serum proteins, however, the uptake process was still underway for cells treated with diamoplexes. Both pit formation (straight arrows) and unilateral membrane extensions (dotted arrows) were 4
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 3. TEM images showing cellular distribution of NDs and diamoplexes. (A) pNDs (B) lys-NDs (C) diamoplexes (in the presence of serum) – right box shows diamoplexes in endosomes (clear membrane boundary) & left box shows diamoplexes that may exist freely in cytoplasm (no clear membrane boundary) (D) diamoplexes (in the absence of serum) in perinuclear region (E) diamoplexes (in the presence of serum) in endosomes (E1) and lysosomes (E2) (F) diamoplexes (in the absence of serum) in endosomes and lysosomes White straight arrows focus cellular distribution of treatments, black straight arrows indicate treatments entrapped in endosomes and black dotted arrows indicate treatments entrapped in lysosomes.
indicates the presence of NDs. The large dip in the absorption spectrum at ~302 eV was previously observed and can be attributed to a second absolute gap in the diamond band structure [22]. The unique diamond features (broadband at 290–302 eV and the dip at 302 eV) were not observed from any regions within the control sample (Figs. 4H and S3). The features at 305 eV and 325 eV were reported earlier as being characteristic to diamond nanocrystals [21], however the exact origins are not clear. The peaks at 288.2 and 289.3 eV (green line marked as “Protein” in Fig. 4G) can be attributed to the C1s → 1π* C]O of carbonyl bond of the amide group from the protein and C1s → 1π* C]N of DNA, respectively [23]. The peak at 288.2 also appears in red line marked as “Lys-NDs” which corresponds to the protein corona formed around lysine ND particles.
3.3. Confirmation of identities of internalized nanodiamonds Since we could not confirm the identity of NDs by TEM, STXM was performed to capture the sp3 carbon signals from NDs. The optical micrograph of the TEM grid (Fig. 4A), TEM image of the analyzed cell (Fig. 4B) and X-ray images (Fig. 4C–E) illustrate that the same section of the cell was analyzed. The X-ray image at 288.2 eV shows protein details within the cell (Fig. 4D) and the image at 292.5 eV highlights two regions of absorption by NDs (Fig. 4E). Portions F–H of Fig. 4 entail spectromicroscopic data collected in the cellular region with the NDs inclusions. The X-ray image (Fig. 4F) confirms the presence of lys-NDs inside the cell. Spectroscopy data collected from the three marked regions in Fig. 4F and presented in Fig. 4G demonstrates that NDs signals and cell compositions are distinct: the lys-NDs spectrum shows peaks at energies 285.2, 288.2, 289.5, 292, 305, 325 eV and a prominent dip at 302 eV in agreement with the literature [9,20]. The pre-edge peak at 285.2 eV is attributed to C1s → π* transition, which is a characteristic feature of sp2 carbon such as graphite or amorphous silicon [20]. The presence of a peak at 289.5 eV is due to the core excitation resonance and a broad feature between 290 and 302 eV arises from σ* states [21]. Observation of these two features in the lys-ND spectrum unequivocally
4. Discussion Physicochemical parameters of lysine-NDs and diamoplexes are one of the major determinants for cellular uptake. Extensive study of these parameters performed previously became foundational basis to design nano-formulations for assays presented in this publication. Table 2 summarizes the size and the zeta potentials for all nano-formulations 5
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 4. TEM and STXM images corresponding to the same cell confirming the identity of internalized entities. A) optical micrograph of the cells showing the region of interest (ROI); B) TEM image of the cell at 5K magnification showing lys-NDs in the boxed region; C) STXM image of the same cell analyzed at 280.0 eV showing the whole cell including lys-NDs in boxed region; D) STXM image at 288.2 eV showing only protein absorption; E) STXM image at 292.5 eV highlighting two regions of strong absorption by lys-NDs in the boxed region; F) STXM color composite map of boxed cell region; G) X-ray absorption spectra extracted from three areas marked in different colors in figure F: no carbonaceous background (blue) protein (green) and ND (red); and H) overlay of X-ray absorption spectra of lys-ND treated cell (red) and untreated cells (green) showing specific diamond features in the treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
complexes: clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis [16–18]. We found typical features of pits during the uptake of lys-NDs corresponding to clathrin mediated endocytosis, which can engulf particles up to 100–105 nm [24]. Endosomal entrapment in clathrin-mediated endocytosis occurs due to a sudden pH drop upon detachment of encapsulated vesicles from the cell
utilized in above experiments. Previous analyses confirmed that diamoplexes of lys-NDs carried functional siRNA into the cells at significantly higher level compared to non-functionalized carboxylated NDs or naked siRNA delivery [9]. In this study, we determined that all three receptor-mediated endocytic processes are involved in the cellular uptake of lys-NDs/siRNA nanoTable 2 Particle size and surface charge for lys-NDs based nano-formulations. Nano-formulations
Average particle size (nm)
Zeta potential (mV)
Ref
Lys-ND Lys-NDs coated with a protein corona (lys-NDs/FBS) Diamoplexes of lys-NDs/siRNA at 50:1 mass ratio
51 ± 16 264 ± 8 272 ± 6
+26.3 ± 0.3 −12.4 ± 0.1 +30 ± 2
[6,9] [9] [6]
Aqueous dispersion of lys-NDs was used for preparation of all nano-formulations. FBS = fetal bovine serum. 6
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
diamoplexes observed here. In the presence of filipin, a caveolae-mediated endocytosis inhibitor, the siRNA fluorescence from diamoplexes remained unchanged suggesting absence of this pathway as a prominent mode for uptake. Caveolae-mediated endocytosis is evidenced to be charge specific, favoring negatively charged gene-complexes [31]. As the overall zeta potential of the diamoplexes at ratio 50:1 is positive (+30 mV) [6] the absence of this internalization pathway is justifiable. Unlike lys-NDs and associated diamoplexes which showed a controlled uptake mechanism, pNDs showed signs of internalization through phagocytosis. They may initiate a non-specific random phagocytic engulfment due to their high variability in aggregate sizes and chemical conformation of their surface. Although HeLa cells are generally considered non-phagocytic in nature, there is evidence of the presence of non-specific endocytosis, mainly energy-dependent phagocytosis in early stages of internalizing hard NPs [32,33]. Nanocarriers should be ideally designed to bypass the phagocytic uptake in mammalian cells, especially during their transport in the blood stream. While pNDs do show this behavior, the lys-ND diamoplexes did not exhibit cellular internalization by phagocytosis, indicating that the absence of aggregation and positive zeta potential are favorable properties for mitigating phagocytosis. An interesting finding was that internalization of diamoplexes was higher in the presence of serum proteins, indicating that diamoplex uptake is significantly influenced by the protein corona [9]. The above observations depicting intracellular distribution of diamoplexes, as an anomaly is more abundant and faster uptake in the presence of serum, reestablishes our previous findings that protein coating facilitates the uptake of diamoplexes [9]. Although diamoplexes are internalized by the cells, most of them ended up in endosomes risking their degradation in the cytoplasm (Fig. 3E & F). Darker vesicles, as evident in Fig. 3E2, are marked by accumulation of degradative enzymes representing late stage lysosomal entrapment [34,35]. Therefore, regardless of the pathway involved in the uptake, diamoplexes need to be equipped with a machinery that allows them to escape from the endosome before reaching the stage of lysosomal degradation to exist freely in the cytosol. Functionalization of the NDs with pH-responsive residues may facilitate the escape of diamoplexes from the endosomes before acidification, and may improve the bioactivity of the genetic material. Moving forward, biodistribution assays will provide more detailed insight about in vivo behavior of diamoplexes. This knowledge can then be employed to tailor the final formulation by: (a) adjusting the overall ND functionalization strategy as such, that it allows complexation of siRNA at optimum mass ratios (b) controlling the thickness of protein adsorption layer formed on final diamoplexes. Newer techniques like STXM employed here will provide better understanding of sub-cellular distribution of diamoplexes in live cells. Moreover, other techniques like correlative light electron microscopy employed more recently in the field [36–38] may also be used to study fluorescent labelled diamoplexes in cellular models.
membrane [16,17]. Soon after detachment, the clathrin-coated vesicles lose their clathrin coating and undergo dynamic fusion in the cytoplasm forming early endosomes with a pH-drop to 6.1–6.8. Some cargo of the vesicle is recycled back to the cell surface through recycling endosomes. The remainder vesicles face a further acidifying environment in late endosomes (pH up to 4.8), triggering lysosomal fusion and ultimately classic degradation of the genetic cargo [16,17]. Previous investigations of non-covalently functionalized ND-gene complexes show clathrinmediated endocytosis as a strongly possible mode of uptake [18]. In our studies, uptake of diamoplexes was not significantly blocked in the presence of chlorpromazine, a reagent which inhibits the formation of pits by depleting the assembly of clathrin on the plasma membrane [25]. However, TEM images of lys-NDs and diamoplexes in the absence of serum proteins illustrate pit formation indicating a possible involvement of this pathway in the uptake of covalently functionalized lys-ND and their diamoplexes. Moreover, during flow cytometry analysis we observed a high degree of variation in fluorescence values recorded between samples pre-treated with chlorpromazine. This was possibly due to cellular toxicity associated with the reagent at tested concentrations and treatment time. In the few viable cells, chlorpromazine was able to knock down the fluorescence by 3%, further confirming that pits observed during TEM analysis correspond to clathrinmediated uptake process. Upon clathrin-mediated endocytosis, the presence of nano-complexes in endosomes ultimately triggers enzymatic degradation of the genetic cargo; therefore, it is imperative to equip the ND-based gene carrier with an ability to escape from the endosomes before this stage of acidification. Another prominent route in the uptake of NDs and diamoplexes was macropinocytosis, which is typically involved in uptake of larger sized particles [24]. This is an actin-dependent process, where the plasma membrane envelopes the complexes via unilateral membrane extension forming macropinosomes [25]. In the presence of amiloride, which interrupts the Na (+)/H (+) exchange at plasma membrane [26], the reduction in the uptake of diamoplexes was statistically significant indicating macropinocytosis as another major contributing pathway. Quantitative data indicates that macropinocytosis might be more prevalent than any other mode of uptake (Fig. 1). Although the cargo in macropinosomes may also end up in lysosomes for degradation [27], macropinocytosis may still be a preferred target as a primary mode of diamoplex uptake, especially in cancer applications. Cancer cells exploit macropinocytosis to replenish nutrients for abnormal proliferation and engulfing cell surface receptors for the purpose of down-regulating them to combat natural defense mechanisms [28]. Keeping in mind this established link between macropinocytosis and cancer, this process can be targeted to ensure maximum loading of diamoplexes carrying the therapeutic genes into these cells. Since, macropinocytosis promotes formation of larger vesicles ranging in size from 200 nm to 5 μm [29], upsizing the diamoplexes within this size range may bias towards this pathway for uptake. This can be done either by: a) Selecting the right NDs to siRNA mass ratio, which protects the cargo during travel and releases it efficiently in the target cells, or b) Forming a defined protein corona surrounding diamoplexes, through adsorbing a series of selective proteins in the designing phase.
5. Conclusion In this study, we demonstrated that the covalently functionalized lys-NDs and their diamoplexes internalized in mammalian cancer cells through formation of pits and unilateral membrane protrusions, illustrating clathrin-mediated endocytosis and macropinocytosis as mechanisms involved. Unchanged fluorescence and hence siRNA delivery after filipin treatment and the overall surface charge of diamoplexes being highly positive, there is theoretical absence of caveolae-mediated endocytosis as a mode of internalization. Moreover, fewer diamoplexes found in the cytoplasm in the absence of serum proteins further confirms our findings that protein corona enhances the cellular uptake of diamoplexes. Identification of the pathways will inform future design and engineering of diamoplexes. Specific composition of surface
However, maximum allowable size of the diamoplexes needs to be conservative towards the lower end of 200 nm, as larger size may present other challenges during in vivo delivery. These barriers might be: opsonization by phagocytes during circulation or accumulation in liver and spleen. Moreover, in order to utilize enhanced permeability and retention effect for tumor accumulation, < 200 nm size is preferred [30]. The ratio of lys-NDs to siRNA being 50:1 in above experiments, the average size of the formed diamoplexes was around 270 nm [6]. This also explains the predominance of macropinocytic uptake of 7
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
functional groups, size and overall surface charges to improve uptake and delivery of the genetic cargo will be pursued.
[7] S. Dizaj, S. Jafari, A. Khosroushahi, A sight on the current nanoparticle-based gene delivery vectors, Nanoscale Res. Lett. 9 (1) (2014) 252, https://doi.org/10.1186/ 1556-276X-9-252. [8] Zhang, X.; Chen, M.; Lam, R.; Xu, X.; Osawa; Ho, D., Polymer-functionalized nanodiamond platforms as vehicles for gene delivery. ACS Nano 2009, 3 (9), 2609–2616. DOI:https://doi.org/10.1021/nn900865g. [9] S. Alwani, R. Kaur, D. Michel, M. Chitanda, R. Verrall, C. Karunakaran, I. Badea, Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application, Int. J. Nanomedicine 11 (2016) 687–702, https://doi.org/10.2147/IJN.S92218. [10] A. Schrand, L. Dai, J. Schlager, S. Hussain, E. Osawa, Differential biocompatibility of carbon nanotubes and nanodiamonds, Diam. Relat. Mater. 16 (12) (2007) 2118–2123, https://doi.org/10.1016/j.diamond.2007.07.020. [11] S. Alwani, I. Badea, Nanodiamonds in biomedicine, in: K.D. Sattler (Ed.), Carbon Nanomaterials Sourcebook: Graphene, Fullerenes, Nanotubes, and Nanodiamonds, vol. I, 2016 9781482252682. [12] L. Guo, Z. Feng, L. Cai, Z. Xi, X. Mou, T. Wang, N. He, Effects of a protein-corona on the cellular uptake of ferroferric oxide nanoparticles, J. Nanosci. Nanotechnol. 16 (7) (2016) 7125–7128, https://doi.org/10.1166/jnn.2016.11361. [13] A. Lesniak, F. Fenaroli, M. Monopoli, C. Åberg, K. Dawson, A. Salvati, Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells, ACS Nano 6 (7) (2012) 5845–5857, https://doi.org/10.1021/nn300223w. [14] X. Cheng, T. Xin, A. Wu, J. Li, J. Tian, Y. Chong, Z. Chai, Y. Zhao, C. Chen, C. Ge, Protein corona influences cellular uptake of gold nanoparticles by phagocytic and nonphagocytic cells in a size-dependent manner, ACS Appl. Mater. Interfaces 7 (37) (2015) 20568–20575, https://doi.org/10.1021/acsami.5b04290. [15] J. Wang, Z. Lu, M. Wientjes, J. Au, Delivery of siRNA therapeutics: barriers and carriers, AAPS J. 12 (4) (2010) 492–503, https://doi.org/10.1208/s12248-0109210-4. [16] I. Khalil, K. Kogure, H. Akita, H. Harashima, Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery, Pharmacol. Rev. 58 (1) (2006) 32–45, https://doi.org/10.1124/pr.58.1.8. [17] A. El-Sayed, H. Harashima, Endocytosis of gene delivery vectors: from clathrindependent to lipid raft-mediated endocytosis, Mol. Ther. 21 (6) (2013) 1118–1130, https://doi.org/10.1038/mt.2013.54. [18] A. Alhaddad, C. Durieu, G. Dantelle, E. Cam, C. Malvy, F. Treussart, J.-R. Bertrand, Influence of the internalization pathway on the efficacy of siRNA delivery by cationic fluorescent nanodiamonds in the Ewing sarcoma cell model, PLoS One 7 (12) (2012) e52207, , https://doi.org/10.1371/journal.pone.0052207. [19] A. Schrand, J. Schlager, Dai; D & Hussain, S., Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy, Nat. Protoc. 5 (4) (2010) 744–757, https://doi.org/10.1038/nprot.2010.2. [20] A. Sumant, P. Gilbert, D. Grierson, A. Konicek, M. Abrecht, J. Butler, T. Feygelson, S. Rotter, R. Carpick, Surface composition, bonding, and morphology in the nucleation and growth of ultra-thin, high quality nanocrystalline diamond films, Diam. Relat. Mater. 16 (4–7) (2007) 718–724, https://doi.org/10.1016/j.diamond. 2006.12.011. [21] Y. Chang, H. Hsieh, W. Pong, M. Tsai, F. Chien, P. Tseng, L. Chen, T. Wang, K. Chen, D. Bhusari, J. Yang, S. Lin, Quantum confinement effect in diamond nanocrystals studied by X-ray-absorption spectroscopy, Phys. Rev. Lett. 82 (1999) 5377, https:// doi.org/10.1103/PhysRevLett.82.5377. [22] J. Morar, F. Himpsel, G. Hollinger, G. Hughes, J. Jordan, Observation of a C-1s core exciton in diamond, Phys. Rev. Lett. 54 (1985) 1960, https://doi.org/10.1103/ PhysRevLett.54.1960. [23] Z. Yangquanwei, S. Neethirajan, C. Karunakaran, Cytogenetic analysis of quinoa chromosomes using nanoscale imaging and spectroscopy techniques, Nanoscale Res. Lett. 8 (1) (2013) 463, https://doi.org/10.1186/1556-276X-8-463. [24] L. Kou, J. Sun, Y. Zhai, Z. He, The endocytosis and intracellular fate of nanomedicines: implication for rational design, Asian J. Pharm. Sci. 8 (1) (2013) 1–10, https://doi.org/10.1016/j.ajps.2013.07.001. [25] D. Dutta, J. Donaldson, Search for inhibitors of endocytosis - intended specificity and unintended consequences, Cell Logist. 2 (4) (2012) 203–208, https://doi.org/ 10.4161/cl.23967. [26] M. Koivusalo, C. Welch, H. Hayashi, C. Scott, M. Kim, T. Alexander, N. Touret, K. Hahn, S. Grinstein, Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling, J. Cell Biol. 188 (4) (2010) 547–563, https://doi.org/10.1083/jcb.200908086. [27] G. Sahay, W. Querbes, A. C, A. Eltoukhy, S. Sarkar, C. Zurenko, E. Karagiannis, K. Love, D. Chen, R. Zoncu, Y. Buganim, A. Schroeder, R. Langer, D. Anderson, Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling, Nat. Biotechnol. 31 (7) (2013) 653–658, https://doi.org/10.1038/nbt.2614. [28] K. Ha, S. Bidlingmaier, B. Liu, Macropinocytosis exploitation by cancers and cancer therapeutics, Front. Physiol. 7 (2016) 381, https://doi.org/10.3389/fphys.2016. 00381. [29] N. Oh, J. Park, Endocytosis and exocytosis of nanoparticles in mammalian cells, Int. J. Nanomedicine 9 (1) (2014) 51–63, https://doi.org/10.2147/IJN.S26592. [30] J. Perry, K. Reuter, J. Luft, C. Pecot, W. Zamboni, J. DeSimone, Mediating passive tumor accumulation through particle size, tumor type, and location, Nano Lett. 17 (5) (2017) 2879–2886, https://doi.org/10.1021/acs.nanolett.7b00021. [31] L. Billiet, J. Gomez, M. Berchel, P. Jaffrès, T. Le Gall, T. Montier, E. Bertrand, H. Cheradame, P. Guégan, M. Mével, B. Pitard, T. Benvegnu, P. Lehn, C. Pichon, P. Midoux, Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line, Biomaterials 33 (10) (2012) 2980–2990, https://doi.org/10.1016/j.biomaterials.2011.12.027. [32] S. Gratton, P. Ropp, P. Pohlhaus, J. Luft, V. Madden, M. Napier, J. DeSimone, The effect of particle design on cellular internalization pathways, Proc. Natl. Acad. Sci.
Funding Natural Sciences and Engineering Research Council of Canada (NSERC) has supported this work (grant number: 2015-03689). Author contribution statements Saniya Alwani and Ildiko Badea developed the theory. Saniya Alwani designed and carried out all experimental work. Saniya Alwani prepared and edited the manuscript in entirety. Nancy Hua and Safa Iftikhar assisted in conducting cellular assays using endocytosis inhibitors. Narayan P. Appathurai, and Chithra Karunakaran contributed in conducting and interpreting of scanning transmission X-ray microscopy experiments at the Canadian Light Source. Deborah Michel contributed in conducting flow cytometry analysis. Declaration of Competing Interest The authors report no conflicts of interest in this work. Acknowledgments The authors acknowledge the Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan for funding this project. Saniya Alwani would also like to thank the College of Pharmacy and Nutrition for financial support of the experiments. The authors thank Tosoh Corporation, USA, for the kind donation of the YTZ grinding media used in these studies. The TEM analysis was conducted at Western College of Veterinary Medicine Image Center at the University of Saskatchewan, a Canadian Foundation for Innovationfunded facility. The authors would also like to thank Larhonda Sobchishin for sample preparation, training on microscope and optimizing results. The scanning transmission X-ray microscopy data described in this paper were collected at the Soft X-ray Spectromicroscopy beamline of the Canadian Light Source, which is supported by National Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. We thank Dr. Jian Wang for his contribution in scanning transmission X-ray microscopy data collection and processing. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.diamond.2019.107477. References [1] M. Davis, Non-viral gene delivery systems, Curr. Opin. Biotechnol. 13 (2) (2002) 128–131, https://doi.org/10.1016/S0958-1669(02)00294-X. [2] B. Martin, M. Sainlos, A. Aissaoui, N. Oudrhiri, M. Hauchecorne, J. Vigneron, J. Lehn, P. Lehn, The design of cationic lipids for gene delivery, Curr. Pharm. Des. 11 (3) (2005) 375–394, https://doi.org/10.2174/1381612053382133. [3] H. Yin, R. Kanasty, A. Eltoukhy, A. Vegas, J. Dorkin, D. Anderson, Non-viral vectors for gene-based therapy, Nat. Rev. Genet. 15 (8) (2014) 541–555, https://doi.org/ 10.1038/nrg3763. [4] R. Bahadur, B. Thapa, N. Bhattarai, Gold nanoparticle-based gene delivery: promises and challenges, Nanotechnol. Rev. 3 (3) (2014), https://doi.org/10.1515/ ntrev-2013-0026. [5] S. Hartono, N. Phuoc, M. Yu, Z. Jia, M. Monteiro, S. Qiao, C. Yu, Functionalized large pore mesoporous silica nanoparticles for gene delivery featuring controlled release and co-delivery, J. Mater. Chem. B 2 (6) (2014) 718–726, https://doi.org/ 10.1039/C3TB21015D. [6] R. Kaur, J. Chitanda, D. Michel, J. Maley, F. Borondics, P. Yang, R. Verrall, I. Badea, Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies, Int. J. Nanomedicine 7 (2012) 3851–3866, https://doi.org/10.2147/IJN.S32877.
8
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
[33]
[34]
[35]
[36]
P. Hänninen, STED-TEM correlative microscopy leveraging nanodiamonds as intracellular dual-contrast markers, Small 14 (5) (2018) 1701807, , https://doi.org/ 10.1002/smll.201701807. [37] F. Hsieh, Y. Chen, Y. Huang, H. Lee, C. Lin, H. Chang, Correlative light-electron microscopy of lipid-encapsulated fluorescent nanodiamonds for nanometric localization of cell surface antigens, Anal. Chem. 90 (3) (2018) 1566–1571, https://doi. org/10.1021/acs.analchem.7b04549. [38] S. Han, M. Raabe, L. Hodgson, J. Mantell, P. Verkade, T. Lasser, K. Landfester, T. Weil, I. Lieberwirth, High-contrast imaging of nanodiamonds in cells by energy filtered and correlative light-electron microscopy: toward a quantitative nanoparticle-cell analysis, Nano Lett. 19 (3) (2019) 2178–2185, https://doi.org/10. 1021/acs.nanolett.9b00752.
U. S. A. 105 (33) (2008) 11613–11618, https://doi.org/10.1073/pnas. 0801763105. J. Khan, B. Pillai, T. Das, Y. Singh, S. Maiti, Molecular effects of uptake of gold nanoparticles in HeLa cells, Chembiochem 8 (11) (2007) 1237–1240, https://doi. org/10.1002/cbic.200700165. B. Liu, S.Y. Lo, C.C. Liu, C.L. Chyan, Y.W. Huang, R. Aronstam, H.J. Lee, Endocytic trafficking of nanoparticles delivered by cell-penetrating peptides comprised of nona-arginine and a penetration accelerating sequence, PLoS One 8 (6) (2013) e67100, , https://doi.org/10.1371/journal.pone.0067100. A. Kitamura, S. Mastumoto, I. Asahina, Growth inhibition of HeLa cell by internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Tokyo, Cancer Cell Int. 9 (2009) 30, https://doi.org/10.1186/1475-2867-9-30. N. Prabhakar, M. Peurla, S. Koho, T. Deguchi, T. Näreoja, H. Chang, J. Rosenholm,
9
Contents lists available at ScienceDirect
Diamond & Related Materials journal homepage: www.elsevier.com/locate/diamond
Lysine functionalized nanodiamonds as gene carriers - Investigation of internalization pathways and intracellular trafficking
T
Saniya Alwania, QingYun Hua (Nancy)a, Safa Iftikhara, Narayan P. Appathuraib, ⁎ Deborah Michela, Chithra Karunakaranb, Ildiko Badeaa, a b
Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Canada Canadian Light Source, Saskatoon, SK, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Lysine-functionalized nanodiamonds Diamoplexes Flow cytometry Scanning transmission X-ray microscopy Cellular internalization Macropinocytosis Clathrin-mediated endocytosis Endo-lysosomal entrapment
Two major determinants for effective gene delivery are the capability of the carrier to protect genetic material during extra and intracellular trafficking, and the ability to release it at the site of action in an intact form. The cellular uptake pathway is a controlling parameter regarding the fate of nanoparticle-genetic material complexes. This study elucidates the pathways involved in the cellular uptake of pristine nanodiamonds (pND), functionalized nanodiamonds (lys-NDs) and diamoplexes (ND/genetic material complexes, namely lysNDsiRNA). Changes in membrane morphologies corresponding to a specific route of internalization were studied using transmission electron microscopy. Variations in cellular association of diamoplexes after general and targeted inhibition of endocytic pathways were quantified using flow cytometry. Electron micrographs reveal formation of unilateral membrane protrusions suggestive of macropinocytosis as one of the major uptake process for lys-NDs and lysND-siRNA diamoplexes. Formation of pits was also largely detected for lys-NDs but scarcely present for their diamoplexes, attributed to either clathrin-mediated or caveoli-mediated endocytosis. However, inhibition of caveoli-mediated endocytosis did not prevent internalization of the diamoplexes, suggesting the absence of this pathway. Due to the larger aggregate size, pNDs were mainly internalized through macropinocytosis, as indicated by unilateral pseudopods on their micrographs. The cells also showed bilateral pseudopods during internalization suggesting phagocytic uptake, possibly due to random functionalities on the surface of pNDs. Clathrin or caveoli mediated endocytosis was absent. Soft X-ray spectromicroscopy reveals strong sp3-carbon signals confirming that the internalized entities identified by electron microscopy are indeed NDs and their diamoplexes. These findings are the basis of further optimization of amino acid functionalized NDs towards increase of gene delivery efficacy through targeted internalization pathways and overcoming intracellular challenges.
1. Introduction As gene therapeutics pave their way towards precision medicine there is an increasing need for novel nanocarriers, capable of delivering the therapeutic genes to their site of action (i.e., into mammalian cells) in an intact and highly efficient form. An ideal gene carrier should have three major characteristics: (1) compatibility with the biological environment up to the level of the cellular machinery (2) the strength to protect the genetic material during biodistribution and upon cellular uptake and (3) minimal interference with the therapeutic process in action [1]. Synthetic nanocarriers ranging from organic molecules such
as lipid-based [2,3] and polymeric nanoparticles [3] to inorganic materials like gold [4], silica [5] and carbon nanoparticles [6,7] have shown promise in this regard. Of inorganic nanomaterials, nanodiamonds (NDs) are a unique member of the carbon nano-family recently studied as a biological carrier [8,9]. NDs offer an ultimate advantage of biocompatibility [10], innately reactive surface and ultra-stable diamond core [11]. Our group reported a novel approach to impart a net positive charge on the surface to modulate the conjugation with oppositely charged genetic biomolecules by functionalizing the NDs with a basic amino acid, lysine [6]. Lysine-functionalized nanodiamonds (lys-NDs) are covered by a protein
Abbreviations: pNDs, pristine nanodiamonds; lys-NDs, lysine-functionalized nanodiamonds; STXM, scanning transmission X-ray microscopy; YTZ, yttria-stabilized 0.05 mm zirconia ⁎ Corresponding author at: 107 Wiggins Road, Health Sciences Building, Room 3D01.5, Box 3D01-13, Saskatoon, SK S7N 5E5, Canada. E-mail address: [email protected] (I. Badea). https://doi.org/10.1016/j.diamond.2019.107477 Received 31 May 2019; Received in revised form 9 July 2019; Accepted 16 July 2019 Available online 18 July 2019 0925-9635/ © 2019 Elsevier B.V. All rights reserved.
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
corona upon interaction with the biological fluids, which improves their dispersion stability [9]. Contrary to other hard nanoparticle (NP) gene carriers like iron oxide [12], silica [13] and gold [14] NPs, which are less efficient in the presence of serum proteins, the corona formed on the NDs facilitates the uptake of the ND-gene conjugates [9]. Although formation of protein corona is not a condition to initiate the cellular uptake of lys-NDs, its presence enhances the internalization of lys-ND/siRNA complexes (diamoplexes) [9]. Previous analyses performed for lys-NDs have successfully laid the foundation of this novel technology by elucidating that:
Table 1 Procedures used to interrupt specific endocytic pathways of internalization of diamoplexes. Treatment–pathways affected 1 2 3 4 5
(1) lysine functionalization controls ND aggregation and provides longterm stability necessary for biological applications; (2) lys-NDs form complexes with siRNA at biologically safe weight ratios; and (3) lys-NDs internalize into cancer cells in a concentration dependent manner and distribute throughout cytosol.
Low temperature (4 °C) – General inhibition of endocytosis Reagents Concentrations (μg/ml) Sodium azide (NaN3) - General inhibition of 500 endocytosis Chlorpromazine – Clathrin-mediated endocytosis 7.1 Filipin – Caveolae-mediated endocytosis 2 Amiloride – Macropinocytosis 27
2.2. Formulation design of lysine functionalized NDs for cellular experiments Lys-NDs were synthesized in-house following the procedure previously described [6]. Briefly, pharmaceutical grade NDs were treated with strong acids allowing reoxidation of the surface in order to maximize the loading of carboxyl functional groups. Carboxylated NDs were subjected to thionylation followed by covalent conjugation of lysine amino acid via an amine terminated three-carbon chain linker. Aqueous dispersion of lys-NDs synthesized as such was prepared at 2 mg/ml concentration and YTZ grinding media was added at a ratio of 1:1. The dispersion was subjected to ultrasonication overnight and centrifugation at 5200g for 5 min to obtain a well-dispersed uniform formulation for subsequent biological experiments.
Here we aim to study in detail the mechanism of cellular internalization, an important parameter necessary to define the functional efficiency of the system. For successful gene transfection, NP-gene complexes must be able to overcome intracellular trafficking barriers and degradative machineries [15,16]. Modulation of the pathways involved in the cellular uptake has a direct impact on these obstacles, and ultimately on the fate of NP-gene complexes in the cells [16–18]. Therefore, this study focuses on understanding the processes involved in the cellular uptake of diamoplexes in mammalian cells, with an aim to further tailor the functionalization approach for maximum gene transfection.
2.3. Flow cytometry Concentrations of the inhibitor solutions were selected based on their toxicity profiles as indicated in MTT assay (Supplementary information and Fig. S1). Table 1 indicates the details of the applied pretreatments. All stock solutions were filtered using 0.22 μm syringe filter and dilutions were made in unsupplemented DMEM. HeLa cells seeded in 6-well plates at the density of 8 × 104 cells/ml and were incubated for 24 h. The cell media were replaced with inhibitor solutions and incubated for 45 min. FITC-labelled siRNA was complexed with lys-NDs dispersed in aqueous medium at a weight ratio of 50:1 by incubating the ND dispersion with siRNA for 30 min at room temperature. The final concentration of siRNA in each well was 80 pmol. The cells were incubated with diamoplexes for 6 h. After the incubation period, the cells were washed with PBS, harvested through trypsinization and centrifugation and re-suspended in 500 ml of PBS. The incubation time was selected to ensure maximum effect of pretreatments on the endocytosis of diamoplexes. Untreated cells and diamoplex-treated cells with no prior inhibitor treatments were used as negative and positive controls, respectively. Fluorescence from FITC siRNA was recorded using FL1-H band pass filter (530/30 BP) by plotting log of relative fluorescence intensity versus number of events. Healthy cell population was gated and a total of 10,000 events were recorded in the gated region per sample. The data was analyzed using BD CellQuest™ Pro software (version 6.0). Marker M1 was applied to calibrate the auto fluorescence of the cells. Positive fluorescence shift (lower to higher end) for diamoplex treated cells was compared to the untreated cells and negative fluorescence shifts (from higher to lower) after endocytic inhibitor pre-treatments were compared to the diamoplex treated cells with no prior inhibition. Higher relative fluorescence recorded in M1 region was directly correlated with greater accumulation of FITC fluorescent siRNA and subsequently diamoplexes in the cells.
2. Materials and methods 2.1. Materials Pharmaceutical-grade (ND98) carboxylic acid–functionalized NDs with an average particle size of 5 nm were purchased from Dynalene Inc. (Whitehall, PA, USA). Yttria-stabilized 0.05 mm zirconia (YTZ®) grinding media were purchased from Tosoh Corporation (Grove City, OH, USA). Lys-NDs dispersion was prepared in HyPure Molecular Biology Grade Water. HyClone® HyPure Molecular Biology Grade Water, HyClone™ 1× phosphate-buffered saline (PBS), and HyClone™ Dulbecco's Modified Eagle's Medium (DMEM)/High; glucose with Lglutamine; sodium pyruvate and Dimethyl sulfoxide (DMSO) and histology grade ethanol were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was acquired from Thermo Fisher Scientific. Human cervical cancer (HeLa) cells were obtained from American Type Culture Collection (Manassas, VA, USA). Trypsin and antibiotics were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Fluorescein isothiocyanate (FITC)-conjugated control siRNA was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Chlorpromazine, sodium azide (NaN3), filipin, amiloride, low viscosity acrylic resin (LR white)-medium grade, benzoyl peroxide blend with dicyclohexyl phthalate and osmium tetroxide 4% aqueous solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4% paraformaldehyde, 8% glutaraldehyde and Ballistic Electron Emission Microscopy (BEEM) capsules were obtained from EM Sciences (Hatfield, PA, USA). (Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent were purchased from Invitrogen, ON, Canada. Cell culture flasks and plates, centrifuge tubes, and cell strainer tubes were purchased from Falcon BD (Mississauga, ON, Canada). Formvar coated 300-mesh copper grids were provided by University of Saskatchewan Western College of Veterinary Medicine Imaging Centre.
2.4. Transmission electron microscopy HeLa cells were grown and treated with lys-NDs and lys-NDs/siRNA diamoplexes (in presence and absence of serum proteins) in similar 2
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 1. Effect of endocytic inhibitors on cellular uptake of diamoplexes. Bar graphs represent the percent fluorescence of FITC-labelled siRNA in response to various pretreatments. Decrease in FITC siRNA fluorescence as compared to the ‘No Inhibitor’ is proportional to the ability of the pre-treatment to inhibit the association of diamoplexes with the cells. Pre-treatments include - low temperature & NaN3 (general endocytosis); chlorpromazine (clathrin-mediated pathway); filipin (caveoli-mediated pathway) and amiloride (macropinocytosis). Statistical analysis: t-test (test value = 21.1 (fluorescence of cells with no inhibitor pre-treatment; 95% confidence interval; difference in fluorescence is considered significant when p-value ≤ 0.050) p-values = *0.050, **0.015, ***0.002.
3. Results
fashion as described above. Untreated cells and pNDs were used as negative and positive controls respectively. Amount of NDs in all samples was kept constant to diamoplexes at a ratio of 50:1. The cells were incubated for 4 h with all treatments in order to ensure that morphological changes of the cellular membrane resulting from endocytosis could be captured before original conformations are regained. TEM samples were prepared by a protocol described elsewhere [19]. Briefly, each sample was washed, harvested and re-suspended in PBS. Each cell pellet was transferred in a labelled BEEM capsule and was subjected centrifugation to obtain pellets of 0.5 to 1 mm size and average cell count of 1 million. Cells were then fixed at room temperature for 2 h using the solution containing 2.5% formaldehyde/ glutaraldehyde in PBS. After proper washing to remove excess fixatives from the samples, cells were subjected to second-degree fixation for 1-h using 1% osmium tetroxide aqueous solution. Pellets were then rinsed using the same buffer as the fixative diluent (i.e. PBS) and dehydrated through a graded series of ethanol concentrations (50, 70, 90 and 100%) for 10 mins each. The pellets were cured using 50:50 resin: ethanol mixture for 45 min and embedded in 100% LR white resin and heated overnight at 40 °C in the oven. Samples were sliced up to a thickness of 200 nm, plated on copper grids and viewed using TEM (Hitachi 7700 with EDS). Digital images with associated micron bars were created using the built-in TEM control software.
3.1. Effect of endocytosis inhibitors on cellular uptake of diamoplexes In our previous publication, we reported internalization of diamoplexes in mammalian cells in a concentration-dependent manner [9]. We also reported that cellular association of lys-NDs/FITC labelled siRNA diamoplexes significantly improved in presence of serum proteins [9]. In lieu of these previous findings, all nano-formulations prepared for this experiment were diluted using serum supplemented media to ascertain two major goals: (a) Maximize cellular localization of FITC labelled siRNA in order to effectively quantify differences in fluorescence resulting from inhibitor pre-treatments (b) Since there is also an evidence that serum supplementation maintains a good cellular viability for lys-ND treated cells (unpublished data), we aimed to minimize pre-treatment induced loss of healthy cells. Dilution with serum-supplemented media ensured maximum cell viability allowing us to evenly collect 10,000 events per sample. Above mentioned pre-developed method [9] to compare the relative percentage of FITC fluorescence from siRNA in different specimens. Cells without treatment with inhibitors prior to transfection, showed a highest FITC-fluorescence from siRNA (21.1%) indicating that the diamoplexes were associated with the cells (Figs. 1 and S2-A). Blocking the endocytosis non-specifically (4 °C and NaN3), resulted in decline of fluorescence intensity to 11.3% ± 1.08 and 14.3% ± 1.46, respectively (Figs. 1 and S2-B). In order to elaborate the specific endocytic routes for the uptake of these diamoplexes, we pre-treated the cells with inhibitory reagents to a specific pathway. The mean fluorescence intensity remained unchanged with chlorpromazine (22.44% ± 5.54) in comparison to the cells with no pre-treatment (21.1%) (Fig. 1). However, the cells pre-treated with chlorpromazine underwent a high degree of damage during the treatment, which ultimately affected the overall mean fluorescence. In the most viable cell population among the three samples, fluorescence reduced to 18.5% (Fig. S2-C) indicating the impact of this reagent in healthy cells. Filipin did not show any reduction in fluorescence (25.14% ± 0.53) indicating that caveolaemediated endocytosis is not a mode of internalization for diamoplexes (Figs. 1 and S2-D). Pre-treatment with amiloride reduced the FITC fluorescence intensity from siRNA to 15.98% ± 0.43 compared 21.1% corresponding to the sample that did not receive any inhibitor pretreatment (Figs. 1 and S2-E).
2.5. Scanning transmission X-ray microscopy TEM grids loaded with cell samples were pre-screened using an optical microscope to select intact cell sections and to identify the region of interest. Data was collected using scanning transmission X-ray microscope (STXM) at the soft X-ray spectromicroscopy (SM) beamline at the Canadian Light Source (Saskatoon, SK, Canada). The source point for the SM beamline is an elliptically polarizing undulator, which provides linear and circularly polarized light in the energy range of 130 to 2700 eV. A plane grating monochromator of the beamline along with a zone plate in the microscope can provide X-ray photons with spectral resolution of ~0.1 eV and spatial resolution of 25–30 nm at the carbon K-edge. The samples on TEM grids were mounted on the STXM sample holder and X-ray data were collected in the transmission mode by raster scanning the sample over the region of interest. Spectral data were collected by acquiring sequence of transmission images at the carbon Kedge from 280 to 340 eV. The sample data were then normalized (and converted into optical density-OD using Beer's law) using a carbon Kedge spectrum extracted from an empty region within the cell section. Note the empty regions that served as negative control for normalization of the sample data within these cell sections were covered by the LR white resin. The composite maps of the cells were created using the spectra extracted from different regions on the cells.
3.2. Morphological changes in the cell membrane during uptake We used TEM to map the cellular internalization and localization of different ND based treatments. Morphological changes in the cell 3
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 2. TEM images showing cellular morphologies of plasma membrane and possible uptake pathways for NDs and diamoplexes. (A) & (B) pNDs (C), (D), (E), (F) and (G) lys-NDs (H) diamoplexes (transfected in the presence of serum) (I), (J) & (K) diamoplexes (transfected in the absence of serum). Straight arrows indicate pit formation and possible clathrin or caveoli mediated endocytosis, dotted arrows indicate unilateral membrane protrusions suggestive of macropinocytosis and dashed arrow indicate bilateral membrane extension suggesting phagocytosis.
observed in significant number, suggesting combination of processes involved in the uptake (Fig. 2I, J & K). Final distributions of various ND based treatments were also assessed to gain an insight of their intracellular fate. pNDs internalized in the cells were found enclosed in large membrane-bound vesicles mainly as aggregates (Fig. 3A). The lys-NDs were distributed in the cytoplasm and existed in non-aggregated forms with uniformly sized particles (Fig. 3B). Diamoplexes internalized in the presence of serum were also well distributed in the cytoplasm (Fig. 3C). In most instances diamoplexes were entrapped in the endosomes (Fig. 3C-right inset), however, some were found with no clear boundaries (Fig. 3C-left inset) indicating that they might exist freely in the cytoplasm. Uptake of the diamoplexes administered in the absence of serum was scarce as compared to their protein coated counterparts as fewer particles were observed in the cytoplasm (Fig. 3D in comparison with Fig. 3C). Although diamoplexes (both in presence and absence of serum) travelled to close proximity to the nucleus, were majorly found entrapped in light and dark membrane-surrounded vesicles (Fig. 3E and F).
membrane such as formation of pits or pseudopods (membrane extensions) were monitored. Cells treated with pNDs presented the formation of unilateral membrane protrusions (Fig. 2A inset), suggesting macropinocytosis as a major mode of uptake. However, in some cell samples bilateral membrane extensions (Fig. 2B) were also observed. Lys-NDs, without siRNA (Fig. 2C, D & E), prompted cell membrane to form pseudopods (dotted arrows) and pits (straight arrows) indicating the presence of both macropinocytosis and clathrin mediated endocytosis. Fig. 2F shows a late stage of internalization with pit closure and initiation of intracellular vacuole formation. At high magnification, many of the pits, formed during internalization process for lys-NDs (Fig. 2E & G), showed typical features: deeply embedded and relatively uniformly shaped pits and an organized molecular coat appearing as darker edges. In the presence of serum proteins, the internalization of diamoplexes was mostly completed at the time of analysis. Plasma membrane regained its original morphology, and membrane bound clusters of diamoplexes were found distributed in the cytoplasm (Fig. 2H). In the absence of serum proteins, however, the uptake process was still underway for cells treated with diamoplexes. Both pit formation (straight arrows) and unilateral membrane extensions (dotted arrows) were 4
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 3. TEM images showing cellular distribution of NDs and diamoplexes. (A) pNDs (B) lys-NDs (C) diamoplexes (in the presence of serum) – right box shows diamoplexes in endosomes (clear membrane boundary) & left box shows diamoplexes that may exist freely in cytoplasm (no clear membrane boundary) (D) diamoplexes (in the absence of serum) in perinuclear region (E) diamoplexes (in the presence of serum) in endosomes (E1) and lysosomes (E2) (F) diamoplexes (in the absence of serum) in endosomes and lysosomes White straight arrows focus cellular distribution of treatments, black straight arrows indicate treatments entrapped in endosomes and black dotted arrows indicate treatments entrapped in lysosomes.
indicates the presence of NDs. The large dip in the absorption spectrum at ~302 eV was previously observed and can be attributed to a second absolute gap in the diamond band structure [22]. The unique diamond features (broadband at 290–302 eV and the dip at 302 eV) were not observed from any regions within the control sample (Figs. 4H and S3). The features at 305 eV and 325 eV were reported earlier as being characteristic to diamond nanocrystals [21], however the exact origins are not clear. The peaks at 288.2 and 289.3 eV (green line marked as “Protein” in Fig. 4G) can be attributed to the C1s → 1π* C]O of carbonyl bond of the amide group from the protein and C1s → 1π* C]N of DNA, respectively [23]. The peak at 288.2 also appears in red line marked as “Lys-NDs” which corresponds to the protein corona formed around lysine ND particles.
3.3. Confirmation of identities of internalized nanodiamonds Since we could not confirm the identity of NDs by TEM, STXM was performed to capture the sp3 carbon signals from NDs. The optical micrograph of the TEM grid (Fig. 4A), TEM image of the analyzed cell (Fig. 4B) and X-ray images (Fig. 4C–E) illustrate that the same section of the cell was analyzed. The X-ray image at 288.2 eV shows protein details within the cell (Fig. 4D) and the image at 292.5 eV highlights two regions of absorption by NDs (Fig. 4E). Portions F–H of Fig. 4 entail spectromicroscopic data collected in the cellular region with the NDs inclusions. The X-ray image (Fig. 4F) confirms the presence of lys-NDs inside the cell. Spectroscopy data collected from the three marked regions in Fig. 4F and presented in Fig. 4G demonstrates that NDs signals and cell compositions are distinct: the lys-NDs spectrum shows peaks at energies 285.2, 288.2, 289.5, 292, 305, 325 eV and a prominent dip at 302 eV in agreement with the literature [9,20]. The pre-edge peak at 285.2 eV is attributed to C1s → π* transition, which is a characteristic feature of sp2 carbon such as graphite or amorphous silicon [20]. The presence of a peak at 289.5 eV is due to the core excitation resonance and a broad feature between 290 and 302 eV arises from σ* states [21]. Observation of these two features in the lys-ND spectrum unequivocally
4. Discussion Physicochemical parameters of lysine-NDs and diamoplexes are one of the major determinants for cellular uptake. Extensive study of these parameters performed previously became foundational basis to design nano-formulations for assays presented in this publication. Table 2 summarizes the size and the zeta potentials for all nano-formulations 5
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
Fig. 4. TEM and STXM images corresponding to the same cell confirming the identity of internalized entities. A) optical micrograph of the cells showing the region of interest (ROI); B) TEM image of the cell at 5K magnification showing lys-NDs in the boxed region; C) STXM image of the same cell analyzed at 280.0 eV showing the whole cell including lys-NDs in boxed region; D) STXM image at 288.2 eV showing only protein absorption; E) STXM image at 292.5 eV highlighting two regions of strong absorption by lys-NDs in the boxed region; F) STXM color composite map of boxed cell region; G) X-ray absorption spectra extracted from three areas marked in different colors in figure F: no carbonaceous background (blue) protein (green) and ND (red); and H) overlay of X-ray absorption spectra of lys-ND treated cell (red) and untreated cells (green) showing specific diamond features in the treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
complexes: clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis [16–18]. We found typical features of pits during the uptake of lys-NDs corresponding to clathrin mediated endocytosis, which can engulf particles up to 100–105 nm [24]. Endosomal entrapment in clathrin-mediated endocytosis occurs due to a sudden pH drop upon detachment of encapsulated vesicles from the cell
utilized in above experiments. Previous analyses confirmed that diamoplexes of lys-NDs carried functional siRNA into the cells at significantly higher level compared to non-functionalized carboxylated NDs or naked siRNA delivery [9]. In this study, we determined that all three receptor-mediated endocytic processes are involved in the cellular uptake of lys-NDs/siRNA nanoTable 2 Particle size and surface charge for lys-NDs based nano-formulations. Nano-formulations
Average particle size (nm)
Zeta potential (mV)
Ref
Lys-ND Lys-NDs coated with a protein corona (lys-NDs/FBS) Diamoplexes of lys-NDs/siRNA at 50:1 mass ratio
51 ± 16 264 ± 8 272 ± 6
+26.3 ± 0.3 −12.4 ± 0.1 +30 ± 2
[6,9] [9] [6]
Aqueous dispersion of lys-NDs was used for preparation of all nano-formulations. FBS = fetal bovine serum. 6
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
diamoplexes observed here. In the presence of filipin, a caveolae-mediated endocytosis inhibitor, the siRNA fluorescence from diamoplexes remained unchanged suggesting absence of this pathway as a prominent mode for uptake. Caveolae-mediated endocytosis is evidenced to be charge specific, favoring negatively charged gene-complexes [31]. As the overall zeta potential of the diamoplexes at ratio 50:1 is positive (+30 mV) [6] the absence of this internalization pathway is justifiable. Unlike lys-NDs and associated diamoplexes which showed a controlled uptake mechanism, pNDs showed signs of internalization through phagocytosis. They may initiate a non-specific random phagocytic engulfment due to their high variability in aggregate sizes and chemical conformation of their surface. Although HeLa cells are generally considered non-phagocytic in nature, there is evidence of the presence of non-specific endocytosis, mainly energy-dependent phagocytosis in early stages of internalizing hard NPs [32,33]. Nanocarriers should be ideally designed to bypass the phagocytic uptake in mammalian cells, especially during their transport in the blood stream. While pNDs do show this behavior, the lys-ND diamoplexes did not exhibit cellular internalization by phagocytosis, indicating that the absence of aggregation and positive zeta potential are favorable properties for mitigating phagocytosis. An interesting finding was that internalization of diamoplexes was higher in the presence of serum proteins, indicating that diamoplex uptake is significantly influenced by the protein corona [9]. The above observations depicting intracellular distribution of diamoplexes, as an anomaly is more abundant and faster uptake in the presence of serum, reestablishes our previous findings that protein coating facilitates the uptake of diamoplexes [9]. Although diamoplexes are internalized by the cells, most of them ended up in endosomes risking their degradation in the cytoplasm (Fig. 3E & F). Darker vesicles, as evident in Fig. 3E2, are marked by accumulation of degradative enzymes representing late stage lysosomal entrapment [34,35]. Therefore, regardless of the pathway involved in the uptake, diamoplexes need to be equipped with a machinery that allows them to escape from the endosome before reaching the stage of lysosomal degradation to exist freely in the cytosol. Functionalization of the NDs with pH-responsive residues may facilitate the escape of diamoplexes from the endosomes before acidification, and may improve the bioactivity of the genetic material. Moving forward, biodistribution assays will provide more detailed insight about in vivo behavior of diamoplexes. This knowledge can then be employed to tailor the final formulation by: (a) adjusting the overall ND functionalization strategy as such, that it allows complexation of siRNA at optimum mass ratios (b) controlling the thickness of protein adsorption layer formed on final diamoplexes. Newer techniques like STXM employed here will provide better understanding of sub-cellular distribution of diamoplexes in live cells. Moreover, other techniques like correlative light electron microscopy employed more recently in the field [36–38] may also be used to study fluorescent labelled diamoplexes in cellular models.
membrane [16,17]. Soon after detachment, the clathrin-coated vesicles lose their clathrin coating and undergo dynamic fusion in the cytoplasm forming early endosomes with a pH-drop to 6.1–6.8. Some cargo of the vesicle is recycled back to the cell surface through recycling endosomes. The remainder vesicles face a further acidifying environment in late endosomes (pH up to 4.8), triggering lysosomal fusion and ultimately classic degradation of the genetic cargo [16,17]. Previous investigations of non-covalently functionalized ND-gene complexes show clathrinmediated endocytosis as a strongly possible mode of uptake [18]. In our studies, uptake of diamoplexes was not significantly blocked in the presence of chlorpromazine, a reagent which inhibits the formation of pits by depleting the assembly of clathrin on the plasma membrane [25]. However, TEM images of lys-NDs and diamoplexes in the absence of serum proteins illustrate pit formation indicating a possible involvement of this pathway in the uptake of covalently functionalized lys-ND and their diamoplexes. Moreover, during flow cytometry analysis we observed a high degree of variation in fluorescence values recorded between samples pre-treated with chlorpromazine. This was possibly due to cellular toxicity associated with the reagent at tested concentrations and treatment time. In the few viable cells, chlorpromazine was able to knock down the fluorescence by 3%, further confirming that pits observed during TEM analysis correspond to clathrinmediated uptake process. Upon clathrin-mediated endocytosis, the presence of nano-complexes in endosomes ultimately triggers enzymatic degradation of the genetic cargo; therefore, it is imperative to equip the ND-based gene carrier with an ability to escape from the endosomes before this stage of acidification. Another prominent route in the uptake of NDs and diamoplexes was macropinocytosis, which is typically involved in uptake of larger sized particles [24]. This is an actin-dependent process, where the plasma membrane envelopes the complexes via unilateral membrane extension forming macropinosomes [25]. In the presence of amiloride, which interrupts the Na (+)/H (+) exchange at plasma membrane [26], the reduction in the uptake of diamoplexes was statistically significant indicating macropinocytosis as another major contributing pathway. Quantitative data indicates that macropinocytosis might be more prevalent than any other mode of uptake (Fig. 1). Although the cargo in macropinosomes may also end up in lysosomes for degradation [27], macropinocytosis may still be a preferred target as a primary mode of diamoplex uptake, especially in cancer applications. Cancer cells exploit macropinocytosis to replenish nutrients for abnormal proliferation and engulfing cell surface receptors for the purpose of down-regulating them to combat natural defense mechanisms [28]. Keeping in mind this established link between macropinocytosis and cancer, this process can be targeted to ensure maximum loading of diamoplexes carrying the therapeutic genes into these cells. Since, macropinocytosis promotes formation of larger vesicles ranging in size from 200 nm to 5 μm [29], upsizing the diamoplexes within this size range may bias towards this pathway for uptake. This can be done either by: a) Selecting the right NDs to siRNA mass ratio, which protects the cargo during travel and releases it efficiently in the target cells, or b) Forming a defined protein corona surrounding diamoplexes, through adsorbing a series of selective proteins in the designing phase.
5. Conclusion In this study, we demonstrated that the covalently functionalized lys-NDs and their diamoplexes internalized in mammalian cancer cells through formation of pits and unilateral membrane protrusions, illustrating clathrin-mediated endocytosis and macropinocytosis as mechanisms involved. Unchanged fluorescence and hence siRNA delivery after filipin treatment and the overall surface charge of diamoplexes being highly positive, there is theoretical absence of caveolae-mediated endocytosis as a mode of internalization. Moreover, fewer diamoplexes found in the cytoplasm in the absence of serum proteins further confirms our findings that protein corona enhances the cellular uptake of diamoplexes. Identification of the pathways will inform future design and engineering of diamoplexes. Specific composition of surface
However, maximum allowable size of the diamoplexes needs to be conservative towards the lower end of 200 nm, as larger size may present other challenges during in vivo delivery. These barriers might be: opsonization by phagocytes during circulation or accumulation in liver and spleen. Moreover, in order to utilize enhanced permeability and retention effect for tumor accumulation, < 200 nm size is preferred [30]. The ratio of lys-NDs to siRNA being 50:1 in above experiments, the average size of the formed diamoplexes was around 270 nm [6]. This also explains the predominance of macropinocytic uptake of 7
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
functional groups, size and overall surface charges to improve uptake and delivery of the genetic cargo will be pursued.
[7] S. Dizaj, S. Jafari, A. Khosroushahi, A sight on the current nanoparticle-based gene delivery vectors, Nanoscale Res. Lett. 9 (1) (2014) 252, https://doi.org/10.1186/ 1556-276X-9-252. [8] Zhang, X.; Chen, M.; Lam, R.; Xu, X.; Osawa; Ho, D., Polymer-functionalized nanodiamond platforms as vehicles for gene delivery. ACS Nano 2009, 3 (9), 2609–2616. DOI:https://doi.org/10.1021/nn900865g. [9] S. Alwani, R. Kaur, D. Michel, M. Chitanda, R. Verrall, C. Karunakaran, I. Badea, Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application, Int. J. Nanomedicine 11 (2016) 687–702, https://doi.org/10.2147/IJN.S92218. [10] A. Schrand, L. Dai, J. Schlager, S. Hussain, E. Osawa, Differential biocompatibility of carbon nanotubes and nanodiamonds, Diam. Relat. Mater. 16 (12) (2007) 2118–2123, https://doi.org/10.1016/j.diamond.2007.07.020. [11] S. Alwani, I. Badea, Nanodiamonds in biomedicine, in: K.D. Sattler (Ed.), Carbon Nanomaterials Sourcebook: Graphene, Fullerenes, Nanotubes, and Nanodiamonds, vol. I, 2016 9781482252682. [12] L. Guo, Z. Feng, L. Cai, Z. Xi, X. Mou, T. Wang, N. He, Effects of a protein-corona on the cellular uptake of ferroferric oxide nanoparticles, J. Nanosci. Nanotechnol. 16 (7) (2016) 7125–7128, https://doi.org/10.1166/jnn.2016.11361. [13] A. Lesniak, F. Fenaroli, M. Monopoli, C. Åberg, K. Dawson, A. Salvati, Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells, ACS Nano 6 (7) (2012) 5845–5857, https://doi.org/10.1021/nn300223w. [14] X. Cheng, T. Xin, A. Wu, J. Li, J. Tian, Y. Chong, Z. Chai, Y. Zhao, C. Chen, C. Ge, Protein corona influences cellular uptake of gold nanoparticles by phagocytic and nonphagocytic cells in a size-dependent manner, ACS Appl. Mater. Interfaces 7 (37) (2015) 20568–20575, https://doi.org/10.1021/acsami.5b04290. [15] J. Wang, Z. Lu, M. Wientjes, J. Au, Delivery of siRNA therapeutics: barriers and carriers, AAPS J. 12 (4) (2010) 492–503, https://doi.org/10.1208/s12248-0109210-4. [16] I. Khalil, K. Kogure, H. Akita, H. Harashima, Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery, Pharmacol. Rev. 58 (1) (2006) 32–45, https://doi.org/10.1124/pr.58.1.8. [17] A. El-Sayed, H. Harashima, Endocytosis of gene delivery vectors: from clathrindependent to lipid raft-mediated endocytosis, Mol. Ther. 21 (6) (2013) 1118–1130, https://doi.org/10.1038/mt.2013.54. [18] A. Alhaddad, C. Durieu, G. Dantelle, E. Cam, C. Malvy, F. Treussart, J.-R. Bertrand, Influence of the internalization pathway on the efficacy of siRNA delivery by cationic fluorescent nanodiamonds in the Ewing sarcoma cell model, PLoS One 7 (12) (2012) e52207, , https://doi.org/10.1371/journal.pone.0052207. [19] A. Schrand, J. Schlager, Dai; D & Hussain, S., Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy, Nat. Protoc. 5 (4) (2010) 744–757, https://doi.org/10.1038/nprot.2010.2. [20] A. Sumant, P. Gilbert, D. Grierson, A. Konicek, M. Abrecht, J. Butler, T. Feygelson, S. Rotter, R. Carpick, Surface composition, bonding, and morphology in the nucleation and growth of ultra-thin, high quality nanocrystalline diamond films, Diam. Relat. Mater. 16 (4–7) (2007) 718–724, https://doi.org/10.1016/j.diamond. 2006.12.011. [21] Y. Chang, H. Hsieh, W. Pong, M. Tsai, F. Chien, P. Tseng, L. Chen, T. Wang, K. Chen, D. Bhusari, J. Yang, S. Lin, Quantum confinement effect in diamond nanocrystals studied by X-ray-absorption spectroscopy, Phys. Rev. Lett. 82 (1999) 5377, https:// doi.org/10.1103/PhysRevLett.82.5377. [22] J. Morar, F. Himpsel, G. Hollinger, G. Hughes, J. Jordan, Observation of a C-1s core exciton in diamond, Phys. Rev. Lett. 54 (1985) 1960, https://doi.org/10.1103/ PhysRevLett.54.1960. [23] Z. Yangquanwei, S. Neethirajan, C. Karunakaran, Cytogenetic analysis of quinoa chromosomes using nanoscale imaging and spectroscopy techniques, Nanoscale Res. Lett. 8 (1) (2013) 463, https://doi.org/10.1186/1556-276X-8-463. [24] L. Kou, J. Sun, Y. Zhai, Z. He, The endocytosis and intracellular fate of nanomedicines: implication for rational design, Asian J. Pharm. Sci. 8 (1) (2013) 1–10, https://doi.org/10.1016/j.ajps.2013.07.001. [25] D. Dutta, J. Donaldson, Search for inhibitors of endocytosis - intended specificity and unintended consequences, Cell Logist. 2 (4) (2012) 203–208, https://doi.org/ 10.4161/cl.23967. [26] M. Koivusalo, C. Welch, H. Hayashi, C. Scott, M. Kim, T. Alexander, N. Touret, K. Hahn, S. Grinstein, Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling, J. Cell Biol. 188 (4) (2010) 547–563, https://doi.org/10.1083/jcb.200908086. [27] G. Sahay, W. Querbes, A. C, A. Eltoukhy, S. Sarkar, C. Zurenko, E. Karagiannis, K. Love, D. Chen, R. Zoncu, Y. Buganim, A. Schroeder, R. Langer, D. Anderson, Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling, Nat. Biotechnol. 31 (7) (2013) 653–658, https://doi.org/10.1038/nbt.2614. [28] K. Ha, S. Bidlingmaier, B. Liu, Macropinocytosis exploitation by cancers and cancer therapeutics, Front. Physiol. 7 (2016) 381, https://doi.org/10.3389/fphys.2016. 00381. [29] N. Oh, J. Park, Endocytosis and exocytosis of nanoparticles in mammalian cells, Int. J. Nanomedicine 9 (1) (2014) 51–63, https://doi.org/10.2147/IJN.S26592. [30] J. Perry, K. Reuter, J. Luft, C. Pecot, W. Zamboni, J. DeSimone, Mediating passive tumor accumulation through particle size, tumor type, and location, Nano Lett. 17 (5) (2017) 2879–2886, https://doi.org/10.1021/acs.nanolett.7b00021. [31] L. Billiet, J. Gomez, M. Berchel, P. Jaffrès, T. Le Gall, T. Montier, E. Bertrand, H. Cheradame, P. Guégan, M. Mével, B. Pitard, T. Benvegnu, P. Lehn, C. Pichon, P. Midoux, Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line, Biomaterials 33 (10) (2012) 2980–2990, https://doi.org/10.1016/j.biomaterials.2011.12.027. [32] S. Gratton, P. Ropp, P. Pohlhaus, J. Luft, V. Madden, M. Napier, J. DeSimone, The effect of particle design on cellular internalization pathways, Proc. Natl. Acad. Sci.
Funding Natural Sciences and Engineering Research Council of Canada (NSERC) has supported this work (grant number: 2015-03689). Author contribution statements Saniya Alwani and Ildiko Badea developed the theory. Saniya Alwani designed and carried out all experimental work. Saniya Alwani prepared and edited the manuscript in entirety. Nancy Hua and Safa Iftikhar assisted in conducting cellular assays using endocytosis inhibitors. Narayan P. Appathurai, and Chithra Karunakaran contributed in conducting and interpreting of scanning transmission X-ray microscopy experiments at the Canadian Light Source. Deborah Michel contributed in conducting flow cytometry analysis. Declaration of Competing Interest The authors report no conflicts of interest in this work. Acknowledgments The authors acknowledge the Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan for funding this project. Saniya Alwani would also like to thank the College of Pharmacy and Nutrition for financial support of the experiments. The authors thank Tosoh Corporation, USA, for the kind donation of the YTZ grinding media used in these studies. The TEM analysis was conducted at Western College of Veterinary Medicine Image Center at the University of Saskatchewan, a Canadian Foundation for Innovationfunded facility. The authors would also like to thank Larhonda Sobchishin for sample preparation, training on microscope and optimizing results. The scanning transmission X-ray microscopy data described in this paper were collected at the Soft X-ray Spectromicroscopy beamline of the Canadian Light Source, which is supported by National Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. We thank Dr. Jian Wang for his contribution in scanning transmission X-ray microscopy data collection and processing. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.diamond.2019.107477. References [1] M. Davis, Non-viral gene delivery systems, Curr. Opin. Biotechnol. 13 (2) (2002) 128–131, https://doi.org/10.1016/S0958-1669(02)00294-X. [2] B. Martin, M. Sainlos, A. Aissaoui, N. Oudrhiri, M. Hauchecorne, J. Vigneron, J. Lehn, P. Lehn, The design of cationic lipids for gene delivery, Curr. Pharm. Des. 11 (3) (2005) 375–394, https://doi.org/10.2174/1381612053382133. [3] H. Yin, R. Kanasty, A. Eltoukhy, A. Vegas, J. Dorkin, D. Anderson, Non-viral vectors for gene-based therapy, Nat. Rev. Genet. 15 (8) (2014) 541–555, https://doi.org/ 10.1038/nrg3763. [4] R. Bahadur, B. Thapa, N. Bhattarai, Gold nanoparticle-based gene delivery: promises and challenges, Nanotechnol. Rev. 3 (3) (2014), https://doi.org/10.1515/ ntrev-2013-0026. [5] S. Hartono, N. Phuoc, M. Yu, Z. Jia, M. Monteiro, S. Qiao, C. Yu, Functionalized large pore mesoporous silica nanoparticles for gene delivery featuring controlled release and co-delivery, J. Mater. Chem. B 2 (6) (2014) 718–726, https://doi.org/ 10.1039/C3TB21015D. [6] R. Kaur, J. Chitanda, D. Michel, J. Maley, F. Borondics, P. Yang, R. Verrall, I. Badea, Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies, Int. J. Nanomedicine 7 (2012) 3851–3866, https://doi.org/10.2147/IJN.S32877.
8
Diamond & Related Materials 98 (2019) 107477
S. Alwani, et al.
[33]
[34]
[35]
[36]
P. Hänninen, STED-TEM correlative microscopy leveraging nanodiamonds as intracellular dual-contrast markers, Small 14 (5) (2018) 1701807, , https://doi.org/ 10.1002/smll.201701807. [37] F. Hsieh, Y. Chen, Y. Huang, H. Lee, C. Lin, H. Chang, Correlative light-electron microscopy of lipid-encapsulated fluorescent nanodiamonds for nanometric localization of cell surface antigens, Anal. Chem. 90 (3) (2018) 1566–1571, https://doi. org/10.1021/acs.analchem.7b04549. [38] S. Han, M. Raabe, L. Hodgson, J. Mantell, P. Verkade, T. Lasser, K. Landfester, T. Weil, I. Lieberwirth, High-contrast imaging of nanodiamonds in cells by energy filtered and correlative light-electron microscopy: toward a quantitative nanoparticle-cell analysis, Nano Lett. 19 (3) (2019) 2178–2185, https://doi.org/10. 1021/acs.nanolett.9b00752.
U. S. A. 105 (33) (2008) 11613–11618, https://doi.org/10.1073/pnas. 0801763105. J. Khan, B. Pillai, T. Das, Y. Singh, S. Maiti, Molecular effects of uptake of gold nanoparticles in HeLa cells, Chembiochem 8 (11) (2007) 1237–1240, https://doi. org/10.1002/cbic.200700165. B. Liu, S.Y. Lo, C.C. Liu, C.L. Chyan, Y.W. Huang, R. Aronstam, H.J. Lee, Endocytic trafficking of nanoparticles delivered by cell-penetrating peptides comprised of nona-arginine and a penetration accelerating sequence, PLoS One 8 (6) (2013) e67100, , https://doi.org/10.1371/journal.pone.0067100. A. Kitamura, S. Mastumoto, I. Asahina, Growth inhibition of HeLa cell by internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Tokyo, Cancer Cell Int. 9 (2009) 30, https://doi.org/10.1186/1475-2867-9-30. N. Prabhakar, M. Peurla, S. Koho, T. Deguchi, T. Näreoja, H. Chang, J. Rosenholm,
9