Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Processing of SLNs

1. Synthesis of SLNs
   Note: Use the PIT method ^1,17,20 to prepare the sub-20 nm SLNs ¹.
   1. Use aseptic technique, bioreagents or cell culture grade reagents, and sterile materials (i.e., pipettes, spatulas, vials, etc.).
   2. Use a biosafety cabinet for the synthesis of SLNs (Figure 2).
   3. Combine 0.6 mg of fluorescent dye (Nile Red (NiR), NiR concentration is approximately 0.3 mg/ml) and 0.10 g of Heneicosane (Lipid, 5 wt/wt% lipid concentration) into a vial (15 ml), co-melt at 90 °C, and stir.
   4. Add 0.11 g polyoxyethylene (10) oleyl ether (Surf....

Results

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.......

Discussion

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 se.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	Peprotech	315-03	>97%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Recombinant murine IL-4	Peprotech	214-14	>97%, by SDS-PAGE under reducing conditions and visualized by silver stain.