Protein-nanoparticle co-assembly supraparticles for drug delivery: Ultrahigh drug loading and colloidal stability, and instant and complete lysosomal drug release.

Two frequent problems hindering clinical translation of nanomedicine are low drug loading and low colloidal stability. Previous efforts to achieve ultrahigh drug loading (>30 %) introduce new hurdles, including lower colloidal stability and others, for clinical translation. Herein, we report a new class of drug nano-carriers based on our recent finding in protein-nanoparticle co-assembly supraparticle (PNCAS), with both ultrahigh drug loading (58 % for doxorubicin, i.e., DOX) and ultrahigh colloidal stability (no significant change in hydrodynamic size after one year). We further show that our PNCAS-based drug nano-carrier possesses a built-in environment-responsive drug release feature: once in lysosomes, the loaded drug molecules are released instantly (<1 min) and completely (∼100 %). Our PNCAS-based drug delivery system is spontaneously formed by simple mixing of hydrophobic nanoparticles, albumin and drugs. Several issues related to industrial production are studied. The ultrahigh drug loading and stability of DOX-loaded PNCAS enabled the delivery of an exceptionally high dose of DOX into a mouse model of breast cancer, yielding high efficacy and no observed toxicity. With further developments, our PNCAS-based delivery systems could serve as a platform technology to meet the multiple requirements of clinical translation of nanomedicines.

Evaluation of Cd2+ stress on Synechocystis sp. PCC6803 based on single-cell elemental accumulation and algal toxicological response.

With the development of single cell analysis techniques, the concept of precision toxicology has been proposed in recent years. Due to the heterogeneity of cells, we need to perform toxicological assessments on individual cells. Microalgae, one kind of important primary producers, play as a major pathway by which heavy metals enter the food chain and thus accumulate/transfer to higher trophic levels. Herein, the biosorption of Cd (Ex-Cd) and bioaccumulation of Cd (In-Cd) for Synechocystis sp. PCC 6803 were investigated by online 3D droplet microfluidic device combined with inductively coupled plasma mass spectrometry detection. Meanwhile, the algal toxicological responses of the algae cell to Cd2+ exposure under different concentration (50, 100, and 150 µg L - 1) and time (15 min, 24, 48 and 96 h) were studied. Combining single-cell analysis with toxicological indicators, the toxicity mechanism of Cd2+to algal was discussed. The single cell analysis results revealed heterogeneity in cellular uptake of Cd2+. The proportion of Cd-containing cells and Cd content in single algal cells all reached the maximum at 24 h. The uptake of Cd2+ occurred within 15 min under all tested exposure concentrations and a large part of Cd2+ were adsorbed on the algal cells surface. The Pearson correlation analysis showed that cell density, chlorophyll a and carotenoids were significantly negatively correlated with Cd accumulation, whereas ROS level and SOD activity were significantly positively correlated with Cd accumulation. It suggested that Cd2+accumulated intracellular would show toxic effects on the algal cells and oxidative stress is the main mechanism of Cd toxicity to algal cells. This work promotes our understanding of the toxicological responses of microalgae under Cd stress at single cells level.

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.

Improving crossing of multiple bio-delivery barriers by a novel bio-interface design based on hydrophobic nanoparticle surfaces.

Biological delivery remains a major challenge in biotechnology, partly because it is often not enough to overcome a single delivery barrier. It is highly desirable, yet rarely available, to design delivery carriers with both simple structures and the ability to cross multiple delivery barriers with high efficiency. Herein, we describe a distinct design (dubbed 'SDot') of delivery carriers with a single structural feature that can enhance the crossing of multiple delivery barriers. The bio-interface (the interface with a biological environment) of an SDot nanoparticle is highly hydrophobic, thus enhancing its interactions with lipid membranes, which are the primary components of many bio-delivery barriers. We used quantum dots (QDs) as the model core material of SDots and conjugated them with a RGD peptide. Thus-formed SDots-RGD demonstrated greatly improved abilities of cellular uptake and transcytosis in a brain tumor cell line, U87MG, compared with the conventional nanoparticle counterpart with a hydrophilic bio-interface (wQDs-RGD). Further, after loading a microtubule-binding anticancer drug, paclitaxel (PTX), onto the nanoparticle surface of SDots-RGD, the resulting drug formulation PTX@SDots-RGD displayed excellent ability of intracellular targeting to microtubules in U87MG cells. In a small animal cancer model, PTX@SDots-RGD exhibited significantly higher ability to slow down brain tumor growth than that of PTX@wQDs-RGD and free PTX. Taken together, these experimental results indicated the significant potential of SDots-RGD for bio-delivery, although the possible long-term toxicity of QDs used as the core material needs to be addressed in future work by replacing QDs with clinically approved materials.

Investigation of toxic effect of mercury on Microcystis aeruginosa: Correlation between intracellular mercury content at single cells level and algae physiological responses.

Single-cell studies can help to understand individual differences and obtain atypical cellular characteristics in view of cellular heterogeneity. Herein, the accumulation of mercury (Hg) in single algae cells was studied by droplet chip-time resolved inductively coupled plasma mass spectrometry analytical system, and the relation of Hg accumulation to the physiological responses of algae cell was explored. When low concentrations of Hg2+ (5-20 µg/L) were used in the exposure experiment, the content of Hg in single cells increased in first 2 h, then decreased with further increase of exposure time to 96 h, probably due to the growth dilution effect of the algae. When exposed to 30 µg/L Hg2+, the uptake of Hg by individual cells increased over time, which was associated with increased cell membrane permeability. The exposure to Hg2+ (5-30 µg/L) inhibited the growth of algae in a concentration-dependent manner and serious growth inhibition occurred under the exposure concentration of 30 µg/L. While the exposure concentration was lower than 20 µg/L, algal cells exhibited a recover tendency due to the self-protection mechanism of algal cells. Bivariate results showed that intracellular Hg accumulation was significantly negatively correlated with cells growth in terms of OD680, photosynthetic pigments, Fv/Fm and PIabs. On the contrast, reactive oxygen species content, superoxide dismutase activity, and cell membrane permeability were significantly positively correlated with the accumulation of intracellular Hg. These results are helpful to further understand the toxic effect of Hg on algae.

Examining the Cellular Transport Pathway of Fusogenic Quantum Dots Conjugated With Tat Peptide.

Understanding the underlying transport mechanism of biological delivery is important for developing delivery technologies for pharmaceuticals, imaging agents, and nanomaterials. Recently reported by our group, SDots are a novel class of nanoparticle delivery systems with distinct biointerface features and excellent fusogenic capabilities (i.e., strong ability to interact with the hydrophobic portions of biomembranes). In this study, we investigate the cellular transport mechanism of SDots conjugated with Tat peptide (SDots-Tat) by live-cell spinning-disk confocal microscopy combined with molecular biology methods. Mechanistic studies were conducted on the following stages of cellular transport of SDots-Tat in HeLa cells: cellular entry, endosomal escape, nucleus entry, and intranuclear transport. A key finding is that, after escaping endosomes, SDots-Tat enter the cell nucleus via an importin ß-independent pathway, bypassing the usual nucleus entry mechanism used by Tat. This finding implies a new approach to overcome the nucleus membrane barrier for designing biological delivery technologies.

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.

Remote neurostimulation with physical fields at cellular level enabled by nanomaterials: Toward medical applications.

Most neurological diseases have no cure today; innovations in neurotechnology are in urgent need. Nanomaterial-based remote neurostimulation with physical fields (NNSPs) is an emerging class of neurotechnologies that has generated tremendous interest in recent years. This perspective focuses on the clinical translation of this new class of neurotechnologies, an issue that so far has not received enough attention. We outline the major barriers in their clinical translation. We highlight our recent efforts to tackle these translational barriers, with a focus on the biological delivery problem. In particular, for the first time, we have shown that it is feasible to use noninvasive brain delivery to generate significant physiological responses in living animals by NNSP. However, much more work is needed to overcome the translational barriers.

Encapsulating insoluble antifungal drugs into oleic acid-modified silica mesocomposites with enhanced fungicidal activity.

Recently, with increasing medical practices, including organ transplantation and tumor chemotherapy, fungal infections, particularly the occurrence of drug-resistant fungal strains, remain a severe problem for the public health, which cause worse complications in the immunocompromised patients. The search for efficacious yet safe antifungal agents is in high demand in precision medicine. However, fungicides are often poorly water soluble for oral absorption, which is difficult for pharmaceutical efficacy evaluation. In this study, lipophilic oleic acid (OA)-grafted mesoporous silica (SBA-15) was facilely modified by cetyltrimethylammonium bromide (CTAB), which acts as an efficient antifungal drug matrix of itraconazole (ITZ). Characterized by physicochemical methods, the rod-like SBA-15-OA-CTAB/ITZ composite with retained mesostructural regularity shows that the loading amount of ITZ in the mesopore is ∼18%, contributing to the enhanced antifungal activity against Aspergillus fumigatus (A. fumigatus) and Candida albicans (C. albicans). The antimicrobial mechanism study suggests that the reactive oxygen species (ROS) were formed when fungal cells were incubated with the formulated ITZ, while there was no ROS formation in the presence of pure ITZ, which may result from the quaternary ammonium moieties of CTAB in the nanocomposites. Due to the potential toxicity of CTAB on mammalian cells, the as-synthesized mesoporous SBA-15-OA-CTAB/ITZ provides an alternative molecular design for the formulation improvement of a lipophilic antifungal drug applicable for external uses such as topical therapy.

Biomolecular detection, tracking, and manipulation using a magnetic nanoparticle-quantum dot platform.

Fluorescent and magnetic materials play a significant role in biosensor technology, enabling sensitive quantification and separations with applications in diagnostics, purification, quality control, and therapeutics. Here, we present a magneto-fluorescent biosensor/separations platform consisting of quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) that are separately encapsulated in amphiphilic block co-polymer micelles conjugated to DNA or protein (i.e., single-stranded (ss) DNA derived from the mRNA of the tumor suppressor protein p53 or avidin protein). Analytes were detected via an aggregation sandwich assay upon binding of at least 1 QD and 1 SPION-containing micelle to result in a fluorescent/magnetic composite. Multiplexed isolation of protein and DNA biomolecules was demonstrated by using QDs of varying emission wavelength; QD fluorescence intensity could be correlated with analyte concentration. Sequential or parallel biomolecule separation was achieved by adding appropriately functionalized SPION-containing micelles and applying user-controlled magnetic fields via patterned magnetic disks and wires. QD fluorescence was used to continuously visualize analyte separation during this process. This QD/SPION platform is simple to use, demonstrates ∼10-16 M sensitivity in analyte detection (comparable to competing QD biosensors based on energy transfer) with specificity against 1 and 2 basepair mismatches in DNA detection, molecular separations capability in solutions of ∼10-10 M, and permits simultaneous or parallel, multiplexed separation of protein and DNA. Thus, this versatile platform enables self-assembly-based rapid, sensitive, and specific detection and separation of biomolecules, simultaneously and with real-time visualization. This technology demonstrates potential for nanoscale assembly, biosensing, and bioseparations.

Producing protein-nanoparticle co-assembly supraparticles by the interfacial instability process.

Originally discovered in fundamental research of nanomaterial-biomolecule interactions, protein-nanoparticle co-assembly supraparticles (PNCAS) have become an emerging class of nanomaterials with various biological applications. We apply the interfacial instability process, which was originally reported for forming nanoparticles-encapsulated polymeric micelles, to produce PNCAS. By doing so hydrophobic nanoparticles, which are often the product formed from the upstream nanoparticle synthesis step, can be directly used as the raw materials of the production process of PNCAS. On the other hand, we take advantage of the structural features of protein molecules, in comparison with amphiphilic block copolymers, to mitigate two common problems encountered in the original interfacial instability-mediated nanoparticle encapsulation process, namely (1) poor encapsulation number control and (2) inconvenience and high cost to vary the assembly size. Additionally, we achieve semi-continuous and scalable production of PNCAS by combining the electrospray process and the interfacial instability process. We also conduct proof-of-concept studies of biological applications of the PNCAS products.

Enhancing the effects of transcranial magnetic stimulation with intravenously injected magnetic nanoparticles.

Transcranial magnetic stimulation (TMS) is a non-invasive and clinically approved method for treating neurological disorders. However, the relatively weak intracranial electric current induced by TMS is an obvious inferiority which can only produce limited treatment effects in clinical application. The present study aimed to investigate the possibility of enhancing the effects of TMS with intravenously administrated magnetic nanoparticles. To facilitate crossing of the blood-brain barrier (BBB), the superparamagnetic iron oxide nanoparticles (SPIONs) were coated with carboxylated chitosan and poly(ethylene glycol). To aid the nanoparticles in crossing the BBB and targeting the predesigned brain regions, an external permanent magnet was attached to the foreheads of the rats before the intravenous administration of SPIONs. The electrophysiological tests showed that the maximum MEP amplitude recorded in an individual rat was significantly higher in the SPIONs + magnet group than in the saline group (5.78 ± 2.54 vs. 1.80 ± 1.55 mV, P = 0.015). In the M1 region, biochemical tests detected that the number density of c-fos positive cells in the SPIONs + magnet group was 3.44 fold that of the saline group. These results suggest that intravenously injected SPIONs can enhance the effects of TMS in treating neurological disorders.

Direct and Noninvasive Penetration of Bare Hydrophobic Quantum Dots through Live Cell Membranes.

Semiconductor quantum dots (QDs) possess outstanding optical properties as fluorescent probes, but their applications in live cell intracellular imaging are hindered by various cellular transport barriers. Inspired by membrane proteins inserting their nanometer-scale hydrophobic surface into biomembranes, the present work aims to investigate the possibility that bare hydrophobic QDs could penetrate through live cell membranes without disrupting the membrane integrity. We utilize live cell spinning disk confocal microscopy to image and track the cellular transport process of bare hydrophobic QDs in the presence of a small percentage of three different organic cosolvents, namely, tetrahydrofuran (THF), chloroform, and hexane. A major finding is that, under certain cosolvent conditions, bare hydrophobic QDs can indeed penetrate through biomembranes in a noninvasive manner. Results of this work offer us guidance to design a new class of nanobioprobes based on combining hydrophobic nanoscale surface and cosolvent, and they provide key new pieces to the emerging complex and sophisticated picture of nanostructure-biosystem interactions.

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.

Intracellular targeted delivery of quantum dots with extraordinary performance enabled by a novel nanomaterial design.

Quantum dots (QDs) have emerged as a major class of fluorescent probes with unique optical properties, but applying QDs for imaging specific intracellular entities in live cells has been hindered by the poor performance of targeted intracellular delivery of QDs due to various cellular transport barriers. We describe a novel QD nanoprobe design, which is termed a cosolvent-bare hydrophobic QD-biomolecule (cS-bQD-BM, or 'SDot' for short), combining a cosolvent, a bare hydrophobic nanoparticle surface, ultrasmall size and biomolecular function. SDots show extraordinary intracellular targeting performance with the nucleus as the model target, including near-perfect specificity, excellent efficiency and reproducibility, high-throughput ability, minimal toxicity, and ease of operation, as well as superb optical properties and colloidal stability. We introduce integrated single-particle tracking and pair-correlation function analysis of a spinning-disk confocal microscope platform (iSPT-pCF-SDCM) to study SDot's cellular transport. Endocytosed SDots can undergo a highly potent and noninvasive process of vesicle escape, yielding complete vesicle escape with no serious vesicle disruption. We exploit SDots' unprecedented ability to overcome cellular transport barriers to enhance drug and macromolecule delivery.

A potent, minimally invasive and simple strategy of enhancing intracellular targeted delivery of Tat peptide-conjugated quantum dots: organic solvent-based permeation enhancer.

Targeted delivery of nanomaterials to specific intracellular locations is essential for the development of many nanomaterials-based biological applications. Thus far the targeting performance has been limited due to various intracellular transport barriers, especially intracellular vesicle trapping. Here we report the application of permeation enhancers based on organic solvents in small percentage to enhance the intracellular targeted delivery of nanomaterials. Previously permeation enhancers based on organic solvents and ionic liquids have been used in overcoming biological transport barriers at tissue, organ, and cellular levels, but this strategy has so far rarely been examined for its potential in facilitating transport of nanometer-scale entities across intracellular barriers, particularly intracellular vesicle trapping. Using the cell nucleus as a model intracellular target and Tat peptide-conjugated quantum dots (QDs-Tat) as a model nanomaterial-based probe, we demonstrate that a small percentage (e.g. 1%) of organic solvent greatly enhances nucleus targeting specificity as well as increasing endocytosis-based cellular uptake of QDs. We combine vesicle colocalization (DiO dye staining), vesicle integrity (calcein dye release), and single-particle studies (pair-correlation function microscopy) to investigate the process of organic solvent-enhanced vesicle escape of QDs-Tat. The organic solvent based vesicle escape-enhancing approach is found to be not only very effective but minimally invasive, resulting in high vesicle escape efficiency with no significant disruption to the membrane integrity of either intracellular vesicles or cells. This approach drastically outperforms the commonly used vesicle escape-enhancing agent (i.e., chloroquine, whose enhancement effect is based on disrupting vesicle integrity) in both potency and minimal invasiveness. Finally, we apply organic solvent-based targeting enhancement to improve the intracellular delivery of the anticancer drug doxorubicin (DOX).

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.

Examining the Roles of Emulsion Droplet Size and Surfactant in the Interfacial Instability-Based Fabrication Process of Micellar Nanocrystals.

The interfacial instability process is an emerging general method to fabricate nanocrystal-encapsulated micelles (also called micellar nanocrystals) for biological detection, imaging, and therapy. The present work utilized fluorescent semiconductor nanocrystals (quantum dots or QDs) as the model nanocrystals to investigate the interfacial instability-based fabrication process of nanocrystal-encapsulated micelles. Our experimental results suggest intricate and intertwined roles of the emulsion droplet size and the surfactant poly (vinyl alcohol) (PVA) used in the fabrication process of QD-encapsulated poly (styrene-b-ethylene glycol) (PS-PEG) micelles. When no PVA is used, no emulsion droplet and thus no micelle is successfully formed; Emulsion droplets with large sizes (~25 µm) result in two types of QD-encapsulated micelles, one of which is colloidally stable QD-encapsulated PS-PEG micelles while the other of which is colloidally unstable QD-encapsulated PVA micelles; In contrast, emulsion droplets with small sizes (~3 µm or smaller) result in only colloidally stable QD-encapsulated PS-PEG micelles. The results obtained in this work not only help to optimize the quality of nanocrystal-encapsulated micelles prepared by the interfacial instability method for biological applications but also offer helpful new knowledge on the interfacial instability process in particular and self-assembly in general.