Decoding Live-Cell Interactions with Multi-Nanoparticle Systems

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Biological and Medical Applications of Materials and Interfaces

Decoding Live-Cell Interactions with Multi-Nanoparticle Systems: Differential Implications for Uptake, Trafficking and Gene Regulation Le Liang, Zhenhui Liu, and Ishan Barman ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11315 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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ACS Applied Materials & Interfaces

Decoding Live-Cell Interactions with MultiNanoparticle Systems: Differential Implications for Uptake, Trafficking and Gene Regulation Le Liang†, Zhenhui Liu†, Ishan Barman* †, ‡, §

†Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA, ‡Department of Oncology and §The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA

KEYWORDS: Cell multi-nanoparticle interactions, Nanoparticle endocytosis, Intracellular transport, Gene regulation, Surface modification

ABSTRACT: Surface modification with oligonucleotides renders gold nanoparticles to endocytose through very different pathways as compared to unmodified ones. Such oligonucleotide modified gold nanoparticles (OGNs) have been exploited as effective nanocarriers for gene regulation

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therapies. Notably, in an effort to reduce overall dosage and provide safer transition to the clinic, cooperative systems comprised of two or more discrete nanomaterials have been recently proposed as an alternative to intrinsically multifunctional nanoparticles. Yet, our understanding of such systems designed to synergistically cooperate in their diagnostic or therapeutic functions remain acutely limited. Specifically, cellular interactions and uptake of OGNs are poorly understood when the cell simultaneously interacts with other types of nanoparticles. Here, we investigated the impact of simultaneous uptake of similar-sized iron oxide nanoparticles (IOPs) on the endocytosis and gene regulation function of OGNs, analogs of which have been proposed for sensitization, targeting and treatment of tumors. We discovered that both the OGN uptake amount and, remarkably, the gene regulation function remained stable when exposed to a very wide range of extracellular concentrations of IOPs. Additionally, the colocalization analysis showed a proportion of OGNs were colocalized with IOPs inside cells, which hints at the presence of similar trafficking pathways for OGNs and IOPs following endocytosis. Taken together, our observations indicate that while the OGN endocytosis is highly independent of the IOP endocytosis, it shares transport pathways inside cells – but does so without affecting gene regulation behavior. These results provide key insights into concomitant interactions of cells with


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diverse nanoparticles, and offers a basis for the future design and optimization of cooperative nanomaterials for diverse theranostic applications.

1. INTRODUCTION

Personalized medicine relies on the incorporation of appropriate molecular species to target specific cells — more specifically, molecularly targeted therapy. The need to visualize the performance of molecularly targeted therapies, in turn, has motivated the development of the field of nanoparticle-based imaging.

Nanoparticles provide an attractive agent to address the

molecular imaging requirement, as they possess the ability to amplify contrast signal by incorporating large numbers of reporting elements, unique physicochemical properties,1 the ability to modulate pharmacokinetics through surface chemistry ,2-4 and to combine multiple functions in a single scaffold.5 Building on this idea, the integration of imaging and therapeutic capabilities into single nanoparticle systems has also been realized, permitting confirmation of drug delivery to tumor sites6-8 as well as image-guided selective tumor ablation.9

While the integration of multiple functions into a single nanostructure appears attractive, it can diminish the efficacy of the individual functions due to space and surface-chemistry limitations in



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these tiny platforms. For instance, magnetic nanoparticles and drug molecules have been coencapsulated in liposomes to simultaneously achieve multiple functions,10 however, this often results in a reduced loading capacity and stability with respect to a single-component liposome. Alternatively, recent reports have suggested separation of functions into two or more nanoparticle formulations to simplify this drawback.11-14 Such research is aimed at engineering combinations of diverse nanoparticles that can cooperate in their diagnostic or therapeutic functions. Designing such combinations, however, remains challenging owing to a rudimentary knowledge of simultaneous cellular interactions with multiple, distinct types of nanoparticles. An urgent need in developing imaging and therapeutic strategies, therefore, is the cataloguing of differences in nanoparticle endocytosis and intracellular transport as well as their effect on gene regulation behavior when two or more nanoparticle formulations concomitantly interact with the cell.

Prior work focusing on endocytosis of single nanoparticle formulations have uncovered the different factors, notably the physico-chemical properties,15-18 that govern the process. The endocytosis process is usually energy-dependent and receptor-mediated, as has been carefully elucidated by recent studies.16, 19, 20 The surface properties of the nanoparticles can be adjusted by a variety of modifications in order to target specific endocytic receptors and boost uptake rate, and has been widely exploited for smart nanocarrier design.21,


22

Following endocytosis, the

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nanoparticles undergo intracellular transport and localization that can be broadly divided into the following steps: formation of early endosome, fusion of endosome and transport to lysosome, which is the general destination except for cases with special targeting ligand modification or lysosome escaping design.23, 24

To catalogue and probe these processes when the cells interact with combination of nanoparticles, we selected oligonucleotide-modified gold nanoparticles (OGN) and streptavidincoated iron oxide nanoparticles (IOP) as our model system. Our choice of the model system was governed by the versatility and demonstrated utility of these individual nanomaterials in the literature,8,

25

and, crucially, by use of their close analogs (gold nanorods and iron-oxide

nanoworms) in designing cooperative nanomaterial system.11 Gold nanoparticles are bioinert, can be easily synthesized and functionalized, and have proven to be effective nanocarriers in gene delivery systems,7,

26

Since antisense oligonucleotide therapies are powerful gene-therapy

candidates for clinical treatments of cancer and HIV/AIDS amongst other disorders,27-31 significant attention has been focused on developing antisense OGN. OGNs display prominent advantages over traditional liposome carriers, including greater knockdown of gene expression, higher endocytic efficiency, stronger binding affinity for targets, nuclease resistance, and lower cytotoxicity.25, 32 On the other hand, IOPs have been harnessed in the clinical setting as magnetic



resonance (MR) contrast probes, for instance, in the imaging of lesions in the reticuloendothelial system (RES) organs, such as the liver and lymph nodes.33, 34 In particular, IOPs decorated with streptavidin have shown the ability to home to diseased tissues through interactions with multiple tissue-specific receptors.8

In this article, we specifically focus on understanding the (differences in) endocytosis, trafficking and downstream impact of the OGNs while the cells are simultaneously exposed to the streptavidin-coated IOPs (Fig. 1A). Here, the antisense oligonucleotides for the surface modification of gold nanoparticles were designed to suppress the expression of green fluorescent protein (GFP). For visualization and quantification purposes, OGNs were labeled with cyanine-3 (Cy3) dye through oligonucleotide hybridization, and IOPs were labeled with Alexa 488 through streptavidin-biotin conjugation (Fig. 1B). This design allowed us to shed light on the relatively unexplored and poorly understood phenomena of simultaneous cellular interactions with diverse nanoparticles, which resides at the heart of emergent multi-nanoparticle systems that are being developed for theranostic purposes.


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Figure 1. A) Schematic depicting the process used to synthesize the Cy3-labeled OGNs and Alexa 488 labeled IOPs as well as the subsequent uptake and trafficking of the nanoparticles co-incubated with the mammalian cell. Here, gold nanoparticles were first conjugated with thiol-modified oligonucleotides, then hybridized with complementary Cy3 labeled oligonucleotides. Streptavidin modified iron oxide nanoparticles bind with biotin labeled Alexa 488. B) Representative duo-color confocal image showing the distribution of OGNs (red) and IOPs (green) inside a HeLa cell following 2hr of incubation. Yellow signal depicts the colocalization of OGNs and IOPs. Scale bar 10µm.

2. RESULTS AND DISCUSSION 2.1.

Decoding cellular interactions with streptavidin-coated IOPs



Commonly used 15nm diameter IOPs, which were coated with 5nm streptavidin, were employed as one of the nanomaterials in the model system. IOP concentrations ranging from 0.5µg/ml to 2µg/ml have been regularly employed for in vitro cellular studies without significant cytotoxicity.35 As can be evidenced by comparing Fig. 2A (control, i.e. where no nanoparticles are used) with Fig. 2B-D, fluorescent imaging of NIH 3T3 fibroblast cells display substantial changes in cell morphology and size (Fig. S1) when the cells were exposed to a high concentration of IOPs. We also separately tested the cytotoxicity of such streptavidin-modified IOPs in the 3T3 cells as well as HeLa cells at 1µg/ml, 5µg/ml and 25µg/ml IOPs concentration after an incubation period of 2 hours. Overall, MTT assays revealed an increasing adverse impact of the nanoparticles with increasing concentration. Both kinds of cells suffer from significant cytotoxicity at an IOP concentration of 25µg/ml, although HeLa cells display higher tolerance to the IOPs (Fig. 2F). Additionally, we quantified the endocytosis of Alexa 488 labeled IOPs (Fig. S2) in the HeLa cells using flow cytometry (Fig. 2E). The fluorescence activated cell sorting (FACS) results revealed that the amount of nanoparticle uptake in the Hela cells is nearly linear at the lower end of IOP concentration range – but this behavior changes markedly when the cells are exposed to higher IOP concentrations. Such saturation curves are commonly observed for receptor-mediated endocytosis, where the particles (at high concentration) compete for receptor sites on the cell


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surface,36 while the initial uptake rate is proportional to the active surface area.37 Crucially, the plateauing behavior of IOP endocytosis at the higher concentrations indicates that if other nanoparticles were to share the same set of pathways (as the IOPs) it would create a competitive (rather than a cooperative) landscape likely resulting in lower-than-expected uptake rates for both sets of nanoparticles.

Figure 2. A-D) Fluorescent microscopy images of 3T3 cells with GFP expression following a 2-hr incubation period with IOPs, at different concentrations of 0, 1, 5 and 25µg/ml. Scale bar: 10µm. E) Amount of Alexa 488-labeled IOPs internalized by the Hela cells was quantified using FACS, with



measurements being performed in triplicate. Average and standard deviation values of the IOP intensity recorded over ca. 10,000 cells is provided along with the nonlinear curve-fit (Hill). F) MTT analysis (average and standard deviation (N =5)) of HeLa and 3T3 cell viability when incubated with different concentrations of IOPs. Significant differences in viability were noted when the cell is incubated with 25µg/ml IOPs and when no nanoparticles are used (control) (p-value < 0.05).

2.2.

Probing the simultaneous endocytosis of OGNs and IOPs

Since the endocytosis rate is strongly related to the nanoparticle size,36, 38 we sought to minimize the size difference between IOPs and OGNs to reduce the impact of such dimensional variations on our observations. Here, we used gold nanoparticles with 15nm diameter, i.e. the same as that of the aforementioned IOPs. It is worth noting that the antisense oligonucleotide has 27 bases, corresponding to a 9nm-long double helix strand. Considering that the single strand is usually shorter than the full length of the double strand, the size of the modified nanoparticle was estimated to be ~20nm.39 Cy3 labeled oligonucleotides were hybridized with the antisense oligonucleotide for visualization and quantification purposes with confocal imaging and FACS, respectively (Fig. S3). We mixed the OGNs (at a concentration of 1 µg/ml) with IOPs of different concentrations in the cell culture medium, and performed confocal imaging and FACS after 2hour incubation with the HeLa cells.


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The fluorescent images (Fig. 3A-D) shows the endocytosis of OGNs while cells were also incubated with IOPs at the previous set of studied concentrations, i.e. 0, 1, 5 and 25 µg/ml, respectively. These images do not exhibit any obvious differences in terms of the amount of OGNs taken up by the HeLa cells. The flow cytometry measurements (Fig. 3E) further reinforce the confocal imaging observations. For the FACS measurements, ca. 10,000 cells were measured for each of the samples. While there is an evident shift in the fluorescence levels from the control sample, no differences between any of the other samples could be discerned thereby indicating the similarity in level of uptake of the OGNs. Together, the confocal imaging and FACS measurements confirm that the endocytosis of IOPs do not have an impact on the number of OGNs internalized by the cell.

In combination with the previous result of the IOPs uptake as a function of its concentration, one can reasonably infer that there is little competition between these two types of nanoparticles owing to the utilization of distinct endocytic pathways by them. We attribute this primarily to the DNA surface modification of the gold nanoparticles as opposed to the streptavidin coating of the IOPs. Previous research suggests that the pathway of endocytosis of spherical nucleic acid nanoparticle conjugates is mediated by lipid-rafts, in particular caveolae.17,

40

Mirkin and co-

workers have noted the importance of scavenger reporters (specifically, SR-A) in specific



recognition of and strong binding to the dense oligonucleotide shell of such nanoparticle conjugates.17 Following such binding, oligonucleotide-modified gold nanoparticles were postulated to enter cells via the caveolae-mediated pathway, owing to the close proximity of the nanoparticle conjugate, SR-A, and the lipid-raft microdomains. In contrast, unmodified gold nanoparticles do not exhibit the same predilection for caveolae-mediated pathway.41 Indeed, cells that were pretreated with chlorpromazine, which inhibits the formation of clathrin-coated vesicles, were observed to have significantly reduced uptake of 15nm gold nanoparticles.

Figure 3. A-D) Representative confocal images showing the uptake of OGNs in HeLa cells when both OGNs and IOPs (at 0, 1, 5 and 25µg/ml) are co-incubated with the cells. Red signals


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emanate from the Cy3 labeling of the OGNs. Scale bar: 10µm. E) Flow cytometry results showing the endocytosis of Cy3-labeled OGNs as the IOP concentration was increased from 0 to 25 µg/ml. 10,000 cells were counted for each sample.

2.3.

Putative interference of IOPs in the gene regulation function of OGNs

While spherical nucleic acids have been proposed as therapeutic payloads via gene regulation, little is known at this time about how other nanoparticles may interfere in this key function during simultaneous entry and uptake. To address this unmet need, we examined if and how the gene regulation activity of OGNs is affected by the concomitant uptake of the IOPs. Previous work has demonstrated the efficacy of DNA-gold nanoparticle for antisense gene regulation, where the unique ensemble properties of the conjugate confer several important advantages in the context of intracellular target recognition and binding.25 Consistent with the demonstrated results, the use of the antisense oligonucleotide for the OGNs in our experiments induces downregulation of GFP level in the 3T3 fibroblasts.

Clearly, the confocal images (Fig. 4A-B) obtained following a 24-hour incubation period show that the OGNs induce significantly lower GFP intensity in the fibroblasts. In sharp contrast, the control case where nonsense oligonucleotides were employed exhibit similar fluorescence levels



to the nontreated cells. FACS measurements verified that the GFP expression was reduced by

ca. 10% when the 3T3 cells were incubated with 1 µg/ml of OGNs (Fig. 4F). Subsequently, we performed an independent set of measurements where the IOPs and OGNs were simultaneously added to the cell culture medium. Remarkably, the level of GFP downregulation, as seen from the confocal images, do not show any perceptible difference – irrespective of whether the IOP concentrations was 1, 5 or 25 µg/ml (Fig. 4C-E). This observation was also confirmed by FACS, as shown in Fig. 4F. While GFP downregulation of OGNs alone is well-known and has been demonstrated by previous research studies (as OGNs efficiently scavenge intracellular DNA or RNA25), our results provide the first indication that such gene regulation activity may remain unperturbed even when the cell is exposed to other nanoparticles. We note, though, that this observation may not be generalizable to all other nanoparticles, and further investigation is required to understand the specific parameters of the types of nanoparticles that preclude interference in gene regulation.


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Figure 4. A-E) Representative confocal images displaying the lack of interference of IOPs in the gene regulation activity of the OGNs. Scale bar: 10µm. F) Flow cytometry results based on triplicate measurements confirmed that the action of OGNs remains consistent, despite the different concentrations of IOPs used during the incubation process (p-value650 nm.

4.7. Flow cytometry

Cells were incubated with nanoparticles for the time, as detailed in the Results section. Before measurements, the cell growth medium was removed, and the cells were washed three times with 1×PBS. Next, trypsin was added to each sample and the samples were incubated for 2 min at 37 ºC. Then, DMEM was added and the resulting cell suspensions were transferred to tubes.



10000 cells were analyzed using a FACS canto flow cytometer (BD Biosciences, USA). Consistent gating based on cell size and granularity (forward and side scatter) was applied to select the fluorescence signals of counted cells.

ASSOCIATED CONTENT

Supporting Information

The following files are available free of charge.

The average area of NIH 3T3 cell at different IOPs concentration; Gel-electrophoresis measurements performed on unmodified IOPs and Alexa 488 labelled IOPs; Gel-electrophoresis measurements performed on Cy3-labeled OGN, unlabeled OGN, and unmodified gold nanoparticles; Duo-color confocal images recorded after the HeLa cells were co-incubated with 1 µg/ml of OGNs and different IOP concentrations; Flow cytometry results of cellular uptake of OGNs when the cells are co-incubated with both oligonucleotide-modified and unmodified gold nanoparticles (PDF)


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AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected] (Ishan Barman)

Notes

There are no conflicts of interest to declare

ACKNOWLEDGEMENTS

We acknowledge the support of the National Institute of Biomedical Imaging and Bioengineering (2-P41-EB015871-31), National Institute of General Medical Sciences (DP2GM128198), National Cancer Institute (R01 CA213492) and the JHU Catalyst Award.

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