In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.
Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.
Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. These vehicles can be loaded with drugs, proteins, and nucleic acids designed to impact cellular responses and achieve a desired effect in target tissues. Many types of nanocarriers have been explored for therapeutic and diagnostic benefit including lipids, polymers, silicon, and magnetic materials. These systems are attractive due to their potential for localized drug delivery, increased therapeutic concentration in target tissues, and reduction of systemic toxicity.
Solid lipid nanoparticles (SLNs) are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle in recent years. SLNs can be readily formulated for multiple applications including bio-sensing 1, cosmetics 2, and therapeutic delivery 3-7. Their utility stems from the fact that they are comprised entirely of resorbable, nontoxic lipids, resulting in enhanced biocompatibility. During synthesis, lipophilic drugs can be incorporated into SLN vehicles, thereby increasing drug solubility and suitability for parenteral administration. SLN vehicles also help to stabilize encapsulated therapeutics, reducing their degradation and clearance, and maximizing therapeutic action. These vehicles are particularly well suited for long acting, controlled-release preparations due to their stability at body temperature 3,4,8,9. Importantly, encapsulation of drugs in lipid nanoparticles alters the intrinsic pharmacokinetic profiles of the drug molecules. This provides a potential advantage by allowing the controlled release of drugs with a narrow therapeutic index. The release rate of SLN-incorporated therapeutics can be tuned based on the lipid degradation rate or the drug diffusion rate in the lipid matrix.
SLNs are often engineered to accumulate in specific target tissues. For example, their size (typically greater than 10 nm) potentiates retention in the circulation, where the leaky vasculature of tumor tissue facilitates deposition. In addition, the route of particle administration has been shown to alter biodistribution with the potential to target specific physiological structures such as lymph nodes 10,11. Upon deposition in target tissues, achieving appropriate cellular interactions and eventual internalization of nanoparticles is challenging due to the ability of cell membranes to selectively control the flow of ions and molecules into and out of the cell 12. To facilitate cellular uptake, it is possible to modify nanocarriers with specific ligands including peptides, small molecules, and monoclonal antibodies13,14. Several mechanisms including both passive penetration and active transport of nanoparticles across the cell membrane have been previously described 3,12,15. In general, it has been demonstrated that cell-nanoparticle interactions are influenced by the physicochemical properties of the nanoparticles including size, shape, surface charge and surface chemistry, in addition to cell-specific parameters such as cell type or cell cycle phase 12.
A previous investigation demonstrated the synthesis of sub-10 nm SLNs for topical 16 and biomarker detection applications 1 using the phase inversion temperature (PIT) method 17. This is a gentle synthesis method where 2 the composition remains constant while the temperature is gradually changed. Continuous stirring of the heated solution, as it cools to RT results in a nanoemulsion. This process results in the synthesis of SLNs with smaller particle size1 than that previously reported using various methods for the synthesis of lipid nanoparticles17-22. The resulting size scale, less than 20 nm, provides an advantage for intracellular targeting applications due to increased surface area and the potential for enhanced cellular interactions.
A schematic of SLNs, designed to deliver a fluorescent dye or therapeutic, is shown in Figure 1. The SLNs consist of a lipid interior (e.g., linear alkane) allowing the incorporation of lipophilic compounds (e.g., dyes or therapeutics) and a surfactant exterior (e.g., linear nonionic surfactant) surrounded by water. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. Primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time in order to characterize interactions with respect to toxicity and particle uptake. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels. Fluorescence microscopy and flow cytometry were two methods employed to examine particle uptake in vitro.
Figure 1. Schematic of SLN showing the major constituents. Please click here to view a larger version of this figure.
1. Processing of SLNs
2. SLNs Interaction with Fibroblasts and Dendritic Cells
The PIT method was used to synthesize the SLNs and the phase inversion temperature was determined utilizing a water bath. The samples were slowly heated and gently agitated until the solution appeared clear. The phase inversion temperature for the SLNs made using heneicosane lipid is 45 °C. Table 1 summarizes the particle size, polydispersity, melting point and latent heat of melting for the SLNs. The SLNs synthesized using the processing conditions described above resulted in control SLNs and NiR-loaded SLNs with an average particle diameter of 18.59 and 16.87 nm and with a polydispersity of 5.83 and 4.47, respectively (Figure 3). The stability of the SLNs dispersion was monitored by the measurement of the particle size over 6 days at two different storage temperatures (4 and 23 °C). The particle size did not change over 6 days (data not shown).
The thermal behavior of the SLNs was investigated using differential scanning calorimetry. The synthesized SLNs have a melting point of 38.0 (± 0.4) and 36.8 (± 0.2) °C for the control SLNs and NiR-loaded SLNs, respectively. In contrast, the melting point for the bulk lipid is 40.0 °C, indicating the suppression of the melting point of the SLNs relative to the bulk lipid. As expected, the confinement to small dimensions and high surface area-to-volume ratio depress the nanoparticle melting point 1. Moreover, the latent heat of melting for the control SLNs and NiR-loaded SLNs is 5.1 (± 0.2), and 4.0 (± 0.1) J/g, respectively. These results indicate that the level of crystallinity of the SLNs is affected by the payload, where the NiR-loaded SLNs showed a lower crystallinity in comparison with the control SLNs.
Figure 3. Particle size distribution of typical nanoemulsions. Please click here to view a larger version of this figure.
Sample | Particle Size (nm) | Polydispersity | Melting Point (°C) | Latent Heat of Melting (J/g) |
SLNs (Control) | 18.59 | 5.83 | 38.0 ± 0.4 | 5.1 ± 0.2 |
NiR-SLNs | 16.87 | 4.47 | 36.8 ± 0.2 | 4.0 ± 0.1 |
Table 1. Summary of the physical properties and thermal behavior of the control SLNs and NiR-loaded SLNs including particle size, polydispersity, melting point and latent heat of melting.
In order to explore the interactions of particles and cellular models, the dose limitations of the SLN as directly applied to primary cells in culture, were first established. Using an MTT assay, the dose-response effect on cellular metabolism was measured, where increasing particle concentration resulted in a decrease in cellular viability (with respect to the untreated control cells). There was no observed difference in toxicity between cells exposed to SLN alone versus NiR-loaded SLN at these pre-determined doses. In parallel, cells were visually examined for adherence to the tissue culture polystyrene and images were taken to demonstrate both representative cellular morphology and the uptake of fluorescent particles over time (Figure 5). Using a dose equal to 5 μg/ml lipid (approximately 80% cellular viability) particle uptake was observed by 2 hr post exposure.
Figure 4. Viability of primary human dermal fibroblasts after 24 hr incubation with nanoparticles. Viability was measured using an MTT assay, and data represent percent viable cells with respect to untreated controls. Concentrations represent weight/volume lipid. Data reflect at least three independent experiments, performed in triplicate. Error bars indicate the standard error of the mean. Please click here to view a larger version of this figure.
Figure 5. Primary human dermal fibroblasts after (A) 2 and (B) 24 hr incubation with NiR-loaded nanoparticles. Nanoparticle concentration is equal to 5 µg/ml lipid. Images are representative of at least three independent experiments, performed in duplicate. Please click here to view a larger version of this figure.
Based on the results of the dose limitation studies, the correlation between dose of SLNs and level of incorporation in murine BMDC was investigated. These cells are widely considered the best in vitro model of multiple DC subsets existing in vivo and allow for basic research level investigations of both cellular and molecular effects of intended manipulations. BMDC were left untreated or exposed O/N to either 0.5 or 5.0 μg/ml lipid of the NiR-loaded SLNs (similarly to human fibroblast, use of 50 μg/ml lipid resulted in less than 10% viable cells) and the level of nanoparticle incorporation determined by measuring the intensity of NiR fluorescence in BMDCs via flow cytometry. A direct correlation between the concentration of SLNs used and the amount of fluorescence assessed in BMDCs was observed (Figure 6A). An analysis of the kinetic of NiR-loaded SLNs incorporation revealed a very rapid uptake by BMDC. The SLN fluorescent signal was already evident after 1 hr of exposure, while plateauing at around 5h of exposure (Figure 6B).
Figure 6. SLN incorporation by dendritic cells correlates with concentration of exposure. (A) Murine bone marrow-derived dendritic cells (BMDC) were exposed O/N to NiR-SLNs, at the concentration of lipid indicated, and the level of incorporation assessed via flow cytometry. Histogram overlays are all gated on the CD11c+ population. (B) Kinetic of SLN incorporation by BMDC. Cells were exposed to NiR-SLNs (5 μg/ml) for the indicated time and the level of fluorescence (on gated CD11c+ cells) was assessed by flow cytometry. Please click here to view a larger version of this figure.
In this study, the synthesis of SLNs and their applicability for intracellular targeting applications were explored. These biocompatible nanoparticles have shown promise as delivery vehicles for multiple applications including drug delivery, gene silencing, and vaccine technologies 25-30. Ultra-small SLNs were synthesized using a facile process, and their interactions with primary skin cells and primary immune cells were explored. SLNs were designed to include encapsulation of a fluorescent dye (NiR), which served as a model therapeutic cargo.
The PIT method was employed to synthesize SLN and the resulting particle properties (particle size, polydispersity, melting point, and latent heat of melting) were evaluated using dynamic light scattering and differential scanning calorimetry. Particle size and polydispersity analysis revealed the synthesis of ultra-small SLNs with narrow polydispersity, ideal for intracellular targeting applications. As previously reported, particle size can play an important role on cellular interactions 12. The size of nanoparticles has been shown to have a dramatic effect, influencing uptake efficiency, internalization pathway selection, intracellular localization, and cytotoxicity 12. Some of the overall trends with respect to cell-nanoparticle interactions documented in the literature include: critical size can vary with cell type and surface properties of the nanoparticles, and small nanoparticles have higher probability to be internalized by passive up-take than large ones 12. Thermal analysis of the particles in this study revealed a different level of crystallinity (i.e., latent heat of melting) between the control SLNs and those loaded with NiR dye. The crystallinity of the SLNs decreased due to the addition of the fluorescent dye, which acts as an impurity. The crystallinity of the therapeutic delivery system has been shown to be an important factor that affects the delivery and dose 31. A decrease in crystallinity of the SLN vehicle can influence parameters such as therapeutic loading capacity and drug release kinetics 31. The PIT method is a more gentle synthesis technique in comparison with other methods, with the limitation that only lipophilic drugs can be incorporated into the particles. Despite this limitation, SLNs synthesized using this method are highly biocompatible, making then suitable delivery platform for many applications in biomedical research.
The effects of SLN interaction on human dermal fibroblast viability were analyzed using an MTT assay. A dose-dependent decrease in viability was observed with increasing concentrations of SLN. During SLN synthesis, the lipid particles are stabilized with a surfactant layer. As the particle size decreases, surface area increases, as does the potential for cell surface interactions and cellular exposure to the surfactant layer, which may damage cell membranes. These experiments allowed the determination of the dosing concentration at which cellular uptake of particles could be observed while avoiding a significant decrease in cell viability due to membrane disruption. MTT analysis is a quantitative technique that can be broadly applied to understanding cellular health. In parallel, cells were examined using fluorescence microscopy in order to visualize the fluorescence of the cargo fluorescent dye, NiR. Using this technique, particle uptake was observed after only 2 hr of incubation with human fibroblasts. Fluorescence microscopy provides qualitative information related to cellular morphology and characteristics. Care must be taken to avoid excessive background staining and imaging of artifacts. In addition, as mouse models are generally the first choice for in depth cellular and molecular investigations, we analyzed the interaction between SLNs and murine cells. In particular, a great interest lies in the use of nanoparticles for the selective and controlled release of drugs that would modify the behavior of the immune system. Consequently, flow cytometry was employed to measure the uptake of particles by mouse dendritic cells. These data confirmed that phagocytic cells like dendritic cells can tolerate a similar range of SLNs concentration (with reduced viability observed at 50 µg/ml lipid as with human dermal fibroblasts) and the level of incorporation directly correlates with the concentration of SLN exposure. Moreover, DC revealed a very rapid incorporation of SLNs, suggesting that this population might represent a valuable target via SLN-mediated drug delivery.
This study includes the description of a method for the synthesis of ultra-small populations of biocompatible nanoparticles, as well as several in vitro methods by which to assess their cellular interactions. In future studies, additional assays may be employed to further characterize the particle-cell interactions and ultimately guide the successful development of therapeutic nanocarriers. These could include studies of drug release kinetics, analysis of cellular tropism, measurement of pro-inflammatory cytokine levels, and analysis of cellular transcriptomic changes over time.
The authors have nothing to disclose.
Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory’s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.
Nile Red (NiR) | Sigma | 19123 | BioReagent, suitable for fluorescence, ≥98.0% |
Heneicosane | Aldrich | 286052 | 98% |
Brij O10 | Sigma | P6136 | Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether |
Water | Sigma | W3500 | Sterile-filtered, BioReagent, suitable for cell culture |
Syringe Filter 0.2 µm Supor Membrane Low Protein Binding | Life Sciences | PN4612 | Non-Pyrogenic |
Nanotrac Ultra | Microtrac | serial number U1985IS | Instrument |
Differential Scanning Calorimeter | Mettlet-Toledo | —- | Instument |
Primary human fibroblasts | Life Technologies | C-004-5C | Neonatal (HDFn) |
Medium 106 | Life Technologies | M-106-500 | A sterile, liquid medium for the culture of human dermal fibroblasts. |
Low Serum Growth Supplement Kit (LSGS Kit) | Life Technologies | S-003-K | All the components of complete LSGS |
MTT Cell Proliferation Assay Kit | Trevigen | 4890-025-K | Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT) |
Safire2 microplate reader | Tecan | —- | Instrument |
Phosphate buffered saline | Sigma | P5493 | For molecular biology |
Recombinant murine GM-CSF | R&D Systems | 415 | >97%, by SDS-PAGE under reducing conditions and visualized by silver stain. |
Recombinant murine IL-4 | R&D Systems | 404 | >97%, by SDS-PAGE under reducing conditions and visualized by silver stain. |