Nanocarriers Made of Proteins: Intracellular Visualization of a Smart Biodegradable Drug Delivery System

Abstract

be observed by electron microscopy (EM). Correlative light and electron microscopy (CLEM) combine the strengths of both techniques and contribute to a precise localization of the nanocarriers in the cell. Using fluorescent nanodiamonds, for example, can serve as a fluorescent marker [11,12] These markers have the advantage of being biocompatible and can be easily modified to achieve a certain surface functionalization. [13] CLEM preparation ideally features a fluorophore with a stable inherent fluorescence suitable for confocal laser scanning microscopy (cLSM) which, in combination, has a contrasting component suitable for transmission electron microscopy (TEM). Another approach is using inorganic, fluorescent nanoparticles. Quantum dots are the commonly used fluorescent markers for intracellular tracking, but they are not as bright as NPLs for both one photon and two photon excitation. [19] Moreover, due to their small size and spherical shape it can be challenging to identify quantum dots-but also the larger, rectangular NPLs-in a cellular environment by TEM. [14,15] This might involve additional elemental analysis (e.g., energy dispersive x-ray spectroscopy [EDS] or electron energy loss spectroscopy [EELS]) to confirm the identity of the quantum dots to ultimately locate the nanocarrier. [16][17][18] Here, we develop a model system consisting of an organic nanocapsule (NC) labeled with a fluorescent labeling system. As fluorescent marker we utilize large (20-50 nm), rectangular CdSe-CdZnS nanoplatelets (NPLs). We encapsulated these NPLs into biocompatible NCs by a polyaddition reaction at the droplet interface in an inverse miniemulsion. We used NCs made of bovine serum albumin (BSA), crosslinked at the interface with toluene diisocyanate (TDI), forming a dense polymeric shell. [6,7] The NPLs were added to the aqueous dispersed phase during the miniemulsion process, leading to the encapsulation of the NPLs into the NCs. The key to success of the NPL markers hinged on the protective coating on the NPLs. [19] This coating makes NPLs both easy to disperse in aqueous medium and protects the NPLs' surface from major damage during the encapsulation process, thus leading to high fluorescence after encapsulation. The potential toxicity of CdSe nanoparticles is not an issue at this stage. The fluorescent marker can either be easily replaced or even omitted completely.To follow the intracellular pathway of the protein NCs, RAW264.7 macrophages were incubated with these NCs followed by different techniques like flow cytometry, cLSM, and This work analyzes the intracellular fate of protein-based nanocarriers along their endolysosomal pathway by means of correlative light and electron microscopy methods. To unambiguously identify the nanocarriers and their degradation remnants in the cellular environment, they are labeled with fluorescent, inorganic nanoplatelets. This allows tracking the nanocarriers on their intracellular pathway by means of electron microscopy imaging. From the present data, it is possible to identify d...

A super-resolution and transmission electron microscopy correlative approach to study intracellular trafficking of nanoparticles

“…33,34 Despite the potential of combining two very powerful techniques, only a handful of articles demonstrate the applicability of CLEM on intracellular trafficking of nanomaterials. 35,36 Whilst these studies clearly demonstrate the power of CLEM in understanding NP intracellular metabolism, there is still a resolution gap between the FM and EM techniques used, preventing the assignment of single particles to specific compartments.…”

Biobased Agents for Single‐Particle Detection with Optoacoustics

“…[34] This allows to precisely tailor the properties of capsules, including degradation speed, shell permeability, and size, for different applications. [40][41][42][43][44] We present here an adapted inverse emulsion approach to demonstrate contrast-enhanced OA imaging in vivo with nanocarriers and the first proof-of-concept single-particle tracking with bio-based, ICG-loaded microcarriers smaller than red blood cells. We selected casein as shell material, which is the main protein component of milk and cheese.…”