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Multitasking Nanoparticles for Intracellular Bioimaging and Biosensing

Periodic Reporting for period 2 - NanoIntra (Multitasking Nanoparticles for Intracellular Bioimaging and Biosensing)

Periodo di rendicontazione: 2019-05-01 al 2020-04-30

The development of specifically designed nanomaterials for advanced medical applications such as theranostics has become a major technical challenge with great potential societal and economic outcomes. Nanomedicine and related techniques hold great promises for improved diagnostic and treatment of widely spread diseases such as cancer.
This project aims at developing novel multitasking nanoparticles as advanced tools for highly localized and ultrasensitive intracellular imaging and biosensing. These nanoprobes may provide new insights in the spatial and temporal processes at play in live cells. Such functional objects are achieved through the design of complex architectures involving semiconductor quantum dots as bright and stable luminescent material. These nanocrystals are combined with plasmonic nanoparticles, which offer new strategies for their photothermally induced intracellular delivery. The targeting and imaging of specific subcellular structures is then achieved thanks to proper surface functionalization of the nanoparticles. In addition, the plasmonic nanocrystals also allow these nanoparticles to be used as Surface Enhanced Raman Scattering (SERS) biosensors.
Besides their application in cell labeling and biosensing, these functional nanoparticles have tremendous potential applications in the fields of theranostics, nanomedicine, nanobiophotonics, and NanoIntra constitutes a first step toward the development of a full lab-on-a-particle. Through the synthesis and characterization of novel materials and their application to biotechnology, NanoIntra is a multidisciplinary research and training project at the interface between physics, chemistry and biology.

Conclusions of the action:
NanoIntra saw the development of new nanoparticles with complex architecture and advanced properties. A new surface chemistry was in particular developed for efficient endocytosis of inorganic nanoparticles. NanoIntra also resulted in the discovery of new low dimensional structures including nano-cages, rings and 2D superstructures. Finally, an innovative approach for the integration of these materials with 3D printing techniques was demonstrated, enabling the versatile fabrication of devices with applications in extracellular biosensing and bioassays.
The first two years of the project were conducted in the group of Prof. Wiesner at Cornell University in the United States. This period was dedicated to the synthesis and development of these new materials with complex architectures. In this regards, new methods have been established for the synthesis of luminescent quantum dots with improved optical properties and their encapsulation in silica nanoparticles as a biocompatible material (Fig. 1). In addition, this silica matrix provided a versatile platform for the subsequent surface functionalization with zwitterionic groups. During this period, it was demonstrated that such functionalization allowed endosomal uptake of the nanoparticles by live cells (Fig. 1), while ensuring good colloidal stability. New methods have also been investigated for the synthesis of ultrasmall and water soluble plasmonic gold and/or silver nanoparticles. These metal nanoparticles can thereafter bind to silica coated quantum dots thanks to prior surface modification with amine or thiol groups.
In parallel, new nanoparticles structures have also been discovered through the self-assembly of mesoporous silica. This approach resulted in particular in the formation of ultrasmall particles with dodecahedral cage and ring morphologies (Fig. 2). Given the versatility of silica surface chemistry, and the ability to distinguish the inside and outside of the cages and rings, these materials might prove themselves particularly useful as cargo containers for smart drug delivery strategies.
The final year of NanoIntra was carried out in the group of Prof. Zeger Hens at Ghent University, Belgium. Following the discovery of dodecahedral silica cages in the first part of the project, a new 2D materials (Fig. 3) was conceived through the confined growth of these cages at an interface between two immiscible liquids. Varying the synthesis conditions offered a fine control over the thickness and structure of these 2D superstructures, revealing emergence of structural order in mesoporous materials.
Finally a new approach was developed for the integration of silica cages with 3D printing by digital light processing. To this end, the nanostructure were functionalized with photoresponsive ligands, enabling the direct printing of mesoporous parts without any post-treatment required. In addition the versatile silica chemistry studied in NanoIntra, allowed for the positioning of functionalities within the printed parts. This approach can be implemented for selective binding mechanisms in bioassays for instance.

Overview of the results and dissemination:
The results obtained within NanoIntra were disseminated through publication in peer-reviewed articles in scientific journals:
- The Impact of core/shell sizes on the optical gain characteristics of CdSe/CdS quantum dots, S. Bisschop, P. Geiregat, T. Aubert, Z. Hens, ACS Nano 2018, 12 (9), 9011-9021.
- Self-assembly of highly symmetrical, ultrasmall inorganic cages directed by surfactant micelles, K. Ma, Y. Gong, T. Aubert, M.Z. Turker, T. Kao, P.C. Doerschuk, U. Wiesner, Nature 2018, 558 (7711), 577-580.
- Fluorescent silica nanoparticles with well-separated intensity distributions from batch reactions, T. Kao, F. Kohle, K. Ma, T. Aubert, A. Andrievsky, U. Wiesner, Nano Lett. 2018, 18 (2), 1305-1310.
- Efficient Endocytosis of Inorganic Nanoparticles with Zwitterionic Surface Functionalization, E. Drijvers, J. Liu, A. Harizaj, U. Wiesner, K. Braeckmans, Z. Hens, T. Aubert, ACS Appl. Mater. Interfaces 2019, 11 (42), 38475-38482.
- Two‐Dimensional Superstructures of Silica Cages, T. Aubert, K. Ma, K. W. Tan, U. Wiesner, Adv. Mater. 2020, 32, 1908362.
As an important finding of this project, the surface functionalization of the nanoparticles with zwitterionic group was optimized in order to balance colloidal stability and efficient endosomal uptake. This provides a good alternative to traditional functionalization with polyethylene glycol which typical hinder the uptake of nanoparticles by live cells. This approach can thereafter be extended to other inorganic nanoparticles with a wide range of properties and compositions, thereby enabling a number of intracellular applications based on nanomaterials.
Nevertheless, a variety of new diagnostic and therapeutic probes with drugs hidden inside nanoparticles with cage-like structures can also be envisaged, hence offering interesting complementarities to the multitasking nanoparticles investigated in this project, with important potential impact for the field of nanomedicine. The subsequent 2D assembly of these cages resulted in the discovery of a hitherto unknown 2D material with high potential for SERS sensing, as well as for catalysis and separation applications.
In the light of the current COVID-19 pandemic, 3D printing techniques have been on the front stage for their potential to address numerous needs in such context. In this regard, the latest results obtained in NanoIntra, such as 3D printed mesoporous materials for biosensing and bioassay applications, may play an essential role in the future development of these critical technologies.
Fig. 2. Cryogenic transmission electron microscopy of silica nano-cages and rings.
Fig. 3. Transmission electron microscopy image of a silica cage 2D superstructure.
Fig. 1. Functional silica coated quantum dots and their uptake in live cells.