Mechanism-Driven Technology Development for Solving the Intracellular Delivery Problem of Hard-To-Transfect Cells.

The so-called "hard-to-transfect cells" are well-known to present great challenges to intracellular delivery, but detailed understandings of the delivery behaviors are lacking. Recently, we discovered that vesicle trapping is a likely bottleneck of delivery into a type of hard-to-transfect cells, namely, bone-marrow-derived mesenchymal stem cells (BMSCs). Driven by this insight, herein, we screened various vesicle trapping-reducing methods on BMSCs. Most of these methods failed in BMSCs, although they worked well in HeLa cells. In stark contrast, coating nanoparticles with a specific form of poly(disulfide) (called PDS1) nearly completely circumvented vesicle trapping in BMSCs, by direct cell membrane penetration mediated by thiol-disulfide exchange. Further, in BMSCs, PDS1-coated nanoparticles dramatically enhanced the transfection efficiency of plasmids of fluorescent proteins and substantially improved osteoblastic differentiation. In addition, mechanistic studies suggested that higher cholesterol content in plasma membranes of BMSCs might be a molecular-level reason for the greater difficulty of vesicle escape in BMSCs.

Probing the Intracellular Delivery of Nanoparticles into Hard-to-Transfect Cells.

Hard-to-transfect cells are cells that are known to present special difficulties in intracellular delivery of exogenous entities. However, the special transport behaviors underlying the special delivery problem in these cells have so far not been examined carefully. Here, we combine single-particle motion analysis, cell biology studies, and mathematical modeling to investigate nanoparticle transport in bone marrow-derived mesenchymal stem cells (BMSCs), a technologically important type of hard-to-transfect cells. Tat peptide-conjugated quantum dots (QDs-Tat) were used as the model nanoparticles. Two different yet complementary single-particle methods, namely, pair-correlation function and single-particle tracking, were conducted on the same cell samples and on the same viewing stage of a confocal microscope. Our results reveal significant differences in each individual step of transport of QDs-Tat in BMSCs vs a commonly used model cell line, HeLa cells. Single-particle motion analysis demonstrates that vesicle escape and cytoplasmic diffusion are dramatically more difficult in BMSCs than in HeLa cells. Cell biology studies show that BMSCs use different biological pathways for the cellular uptake, vesicular transport, and exocytosis of QDs-Tat than HeLa cells. A reaction-diffusion-advection model is employed to mathematically integrate the individual steps of cellular transport and can be used to predict and design nanoparticle delivery in BMSCs. This work provides dissective, quantitative, and mechanistic understandings of nanoparticle transport in BMSCs. The investigative methods described in this work can help to guide the tailored design of nanoparticle-based delivery in specific types and subtypes of hard-to-transfect cells.

Spontaneous and instant formation of highly stable protein-nanoparticle supraparticle co-assemblies driven by hydrophobic interaction.

Recently, supraparticle protein-nanoparticle co-assemblies (or 'supraparticle co-assemblies' for short) have attracted considerable interest due to their fundamental and technological value. However, it remains challenging to form supraparticle co-assemblies with high stability. Here, we show that using hydrophobic interaction, instead of the previously used electrostatic and van der Waals interactions, as the primary driving force can lead to instant formation of exceptionally stable supraparticle co-assemblies with minimal external energy input. Our formation method of supraparticle co-assemblies simply involves mixing globular proteins (e.g., bovine serum albumin) with hydrophobic nanoparticles (e.g., hydrophobic magnetic nanoparticles and hydrophobic quantum dots) without significant energy input (e.g., sonication or stirring). Upon mixing of hydrophobic nanoparticles and proteins, the formation of supraparticle co-assemblies only takes <1 minute. Further incubation of the mixture for several hours results in a gradual increase of the size uniformity of supraparticle co-assemblies. The formed supraparticle co-assemblies have been colloidally stable for 6 months and counting, and can withstand harsh environments such as basic and acidic pH, high temperature, high dilution, and serum. Co-encapsulation of different sizes/types of nanoparticles is found to be feasible and the co-encapsulation number ratio of different nanoparticles is well-controlled by the feeding ratio. Proof-of-concept studies show the potential of the supraparticle co-assemblies for biological imaging, delivery, and modulation. The combination of very rapid formation, minimal energy consumption, highly stable products, and inexpensive raw materials of this hydrophobic interaction-driven process meets many of the main goals of 'ideal' nano-manufacturing. Thus, this process could serve as the foundation of ideal manufacturing of supraparticle co-assemblies.

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

Highly sensitive "turn-on" fluorescent chemical sensor for trace analysis of Cr3+ using electro-synthesized poly(N-(9-fluorenylmethoxycarbonyl)-l-histidine).

Trivalent chromium (Cr3+) can cause severely environment pollution, declining quality of edible agro-products in plants and animals, and human diseases. Poly(N-(9-fluorenylmethoxycarbonyl)-l-histidine) (PFLH) synthesized by the direct electro-polymerization of its corresponding commercially available monomer in both boron trifluoride diethyl etherate and dichloromethane mixed system. The "turn-on" type fluorescent sensor based on PFLH displayed high sensitivity and selectivity for Cr3+ detecting. The structure of PFLH was rationally proved by 1H NMR spectra, FT-IR spectra, quantum chemical calculations, and its optical properties were characterized. The electro-synthesized PFLH exhibited a "turn-on" fluorescent response towards Cr3+, which was employed as a sensing platform for the "turn-on" fluorescent analysis of Cr3+ in a wide linear range from 5.1nM to 25µM with a low limit of detection as low as 1.7nM. The possible mechanism of fluorescent "turn-on" sensor based on PFLH for Cr3+ was proposed. The sensor displayed high sensitivity, good selectivity, satisfactory practicability, suggesting that PFLH has potential fluorescent application for "turn-on" sensing Cr3+ in agricultural environments and edible agro-products of plants and animals.

Highly selective "turn-on" fluorescent sensing of fluoride ion based on a conjugated polymer thin film-Fe3+ complex.

We designed a new fluorescent conjugated polymer thin film sensor via direct electropolymerization of the corresponding electroactive monomer M onto the surface of ITO electrode, and the thin film-Fe3+ complex was used for the highly-selective detection of fluoride ion (F-) in water environmental samples. The as-obtained thin film could effectively detect Fe3+ as a selective turn-off fluorescent sensor, and exhibited outstanding reversibility. This film in the presence of Fe3+ showed a highly selective turn-on response toward F- over other anions with a 5-fold enhancement in the fluorescence intensity. F- with a relatively wide concentration range from 10 µM to 3 mM could be determined in a rather simple and sensitive manner with a detection limit of 6.78 µM (0.128 ppm). Analytical applicability of the film-Fe3+ complex for determining the levels of F- in environmental water samples has been successfully demonstrated by fluorescent analysis with satisfactory results. This strategy will provide a new approach for the facile design of new molecular sensing devices and practical application in environments.