Poly‐L‐Arginine Grafted Silica Mesoporous Nanoparticles for Enhanced Cellular Uptake and their Application in DNA Delivery and Controlled Drug Release

Mesoporous silica nanoparticles (MSNs), that are capable of delivering gene and drugs to organisms in an effective and selective way have attracted much attention lately for its potential in the treatment of cancer. However, the successful application of MSNs for delivery of plasmid DNA or drugs requires surface modification of the silica with positively charged functional groups so that it binds to the negatively charged nucleic acids and also helps it penetrate through the cell membrane. We report for the first time the synthesis of a hybrid MSN where the cell penetrating cationic polypeptide poly‐L‐arginine synthesized by NCA polymerization is grafted onto the external surface of MSN using click chemistry. These poly‐L‐arginine grafted MSNs show low cytotoxity (85% cell viability at 100 μg/mL MSN concentration) and high cellular uptake by both HeLa and A549 (>90%). The poly‐L‐arginine grafted MSNs were used effectively to deliver mCherry DNA plasmid into cells leading to expression of the protein mCherry inside the cells (transfection efficiency 60%). In contrast, poly‐L‐arginine grafted non‐porous silica nanoparticles were unable to express the protein mCherry inside the cells although their uptake into the cells was as efficient as with poly‐L‐arginine grafted MSNs. We also show preliminary results to demonstrate that these hybrid MSNs can be used as a delivery vehicle for the anticancer drug Doxorubicin towards cancerous cells HeLa and A549. The biocompatibility of poly‐L‐arginine and its cell penetrating ability are expected to make these MSN conjugates very useful carriers for the delivery of genes and drugs into cancer cells.     

Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique

The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs' G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized.     

Facile Functionalization of Gold Nanoparticles with PLGA Polymer Brushes and Efficient Encapsulation into PLGA Nanoparticles: Toward Spatially Precise Bioimaging of Polymeric Nanoparticles

Nanocarriers prepared from poly(lactide‐co‐glycolide) (PLGA) have broad biomedical applications. Understanding their cellular uptake and distribution requires appropriate visualization in complex biological compartments with high spatial resolution, which cannot be offered by traditional imaging techniques based on fluorescent or radioactive probes. Herein, the encapsulation of gold nanoparticles (GNPs) into PLGA nanoparticles is proposed, which should allow precise spatial visualization in cells using electron microscopy. Available protocols for encapsulating GNPs into polymeric matrices are limited and associated with colloidal instability and low encapsulation efficiency. In this report, the following are described: 1) a facile protocol to functionalize GNPs with PLGA polymer followed by 2) encapsulation of the prepared PLGA‐capped GNPs into PLGA nanocarriers with 100% encapsulation efficiency. The remarkable encapsulation of PLGA‐GNPs into PLGA matrix obeys the general rule in chemistry “like dissolves like” as evident from poor encapsulation of GNPs capped with other polymers. Moreover, it is shown that how the encapsulated gold nanoparticles serve as nanoprobes to visualize PLGA polymeric hosts inside cancer cells at the spatial resolution of the electron microscope. The described methods should be applicable to a wide range of inorganic nanoprobes and provide a new method of labeling pharmaceutical polymeric nanocarriers to understand their biological fate at high spatial resolution.     

Synthesis,Characterization, Properties,and Novel Applications of Fluorescent Nanodiamonds

In the last few years, fluorescent nanodiamonds (FNDs)  have been developed significantly as a new member in the nanocarbon family. The surface of FNDs is embedded with some crystallographic defects containing color centres which surmount the properties of other fluorochromes including up conversion and down conversion nanoparticles, quantum dots, nano tubes, fullerenes, organic dyes, silica etc. Some of the intriguing properties like inevitable photostability, inherent bio-compatibility, outstanding optical and robust mechanical properties, excellent magnetic field, and electric field sensing potentiality make FNDs appealing to some benevolent applications in numerous fields like bio-imaging, delivering drugs, fighting cancer, spin electronics, imaging of magnetic structure at nanoscale and as promising nanometric temperature sensor. The structure of FNDs has certain point defects on the surface among which negatively charged nitrogen vacancy centre (NV^?) is the most investigated color centre. The production of NV^? fluorescence nanodiamonds is the most challenging task as substitution of carbon atoms is required to create vacancies by causing irradiation from an electron beam which is followed by high temperature annealing. Thus, this review points out the relative advantages of FNDs containing negatively charged nitrogen vacancy centres produced from HPHT method or CVD method with those nanodiamonds produced through detonation process or pulsed laser ablation (PLA) method. The steps involved in the fabrication of FNDs are described along with the major challenges and struggles underwent during the process in this review. This review also summarizes the recent developments made in the functionalization and applications predominantly made in the field of biological science and it is understood that depending on the defect color centres they can exhibit different emitted wavelengths ranging from UV–visible to near infrared with broad or narrow bandwidths. This review also highlights some of the fluorescent NDs that emit stable and strong red or green photoluminescence from the defect centers of NV^? which are implanted in the crystal lattice. This critical and extensive review will be useful for the further progress in this futuristic field of FNDs.

     

Intracellular Breakable and Ultrasound‐Responsive Hybrid Microsized Containers for Selective Drug Release into Cancerous Cells

This work reports an efficient and straightforward strategy to fabricate hybrid microsized containers with reduction‐sensitive and ultrasound‐responsive properties. The ultrasound and reductive sensitivity are visualized using scanning electron microscopy, with the results showing structural decomposition upon ultrasound irradiation and in the presence of reducing agent. The ultrasound‐responsive functionalities of hybrid carriers can be used as external trigger for rapid controlled release, while prolonged drug release can be achieved in the presence of reducing agent. To evaluate the potential for targeted drug delivery, hybrid microsized containers are loaded with the anticancer drug doxorubicin (Dox). Such hybrid capsules can undergo structural intracellular degradation after cellular uptake by human cervical cancer cell line (HeLa), resulting in Dox release into cancer cells. In contrast, there is no Dox release when hybrid capsules are incubated with human mesenchymal stem cells (MSCs) as an example of normal human cells. The cell viability results indicate that Dox‐loaded capsules effectively killed HeLa cells, while they have lower cytotoxicity against MSCs as an example of healthy cells. Thus, the newly developed intracellular‐ and ultrasound‐responsive microcarriers obtained via sol‐gel method and layer‐by‐layer technique provide a high therapeutic efficacy for cancer, while minimizing adverse side effect.     

Early Membrane Responses to Magnetic Particles are Predictors of Particle Uptake in Neural Stem Cells

Magnetic particles (MPs) offer several advantages for neural cell therapy, but limited particle uptake by neural cells is a barrier to translation. It is recently proved that tailoring particle physicochemical properties (by enhancing their iron content) dramatically improves uptake in neural stem cells (NSCs)—a major transplant population. High‐throughput screening of particles with varying physicochemical properties can therefore aid in identifying particles with optimal uptake features, but research is hampered by the lack of simple methodologies for studying neural cell membrane responses to nanoparticle platforms. A high‐resolution–high throughput method has been used to study early membrane responses of primary rodent NSCs to particles of variant magnetite loading, to attempt to correlate these responses with known particle internalization profiles. Membrane imaging is enhanced through sequential staining with osmium (O) and thiocarbohydrazide (T), a method termed OTOTO, combined with field‐emission scanning electron microscopy (FESEM). A five‐point classification system was used to systematically evaluate early MP‐induced membrane responses to particles possessing distinct physicochemical properties. Significantly different profiles of membrane activation were noted that correlate with particle uptake profiles. It is suggested that our method can serve as a valuable predictor of particle internalization in neural cells for diverse particle platforms.     

Cytotoxicity and Cellular Uptake of Amorphous Silica Nanoparticles in Human Cancer Cells

The cytotoxic effects of silica nanoparticles (SNPs) on different human cancer cells, as well as the uptake kinetics and pathways of SNPs have been studied here. SNPs with the diameter of ≈20 nm induced a dose‐dependent cytotoxicity in both gastric cancer cells (MGC80–3) and cervical adenocarcinoma epithelial cells (HeLa), but MGC80–3 cells were more susceptible to the cytotoxic effect induced by SNPs. Changes in the nuclear morphology and flow cytometric analysis with annexin V/PI double staining show that SNPs induced a higher degree of apoptosis in MGC80–3 cells. Accordingly, more remarkable reactive oxygen species (ROS) burst is detected in SNP‐treated MGC80–3 cells. Using fluorescein isothiocyanate (FITC)‐labeled SNPs and flow cytometry, it is found that the uptake of SNPs is more efficient in MGC80–3 than in HeLa cells. SNPs are internalized into both cancer cells through energy‐dependent pathway. Inhibitor studies with dynasore and methyl‐β‐cyclodextrin show that these cancer cells took up 20 nm SNPs mainly through the caveolin‐mediated endocytosis, while in HeLa cells SNPs internalization was also via dynamin‐dependent clathrin‐mediated pathway. These findings indicate that SNPs cause differential cytotoxic effects in different human cancer cells, which might be related to the uptake process and efficiency toward these cancer cells.     

Fluorescence labeling and interaction of atherogenic lipoproteins with cultured cells

We prepared lipoprotein (a) and LDL covalently labeled with either BODIPY or rhodamine. A dual wavelength method was used for the microscopic observation of both lipoproteins during their interaction with HepG2 cells. Since a large proportion of Lp(a) colocalized with LDL on the cell surface and inside the cells, it was concluded that Lp(a) uptake into cells is mediated by LDL via internalization of LDL.     

Large Scale Synthesis of Highly Stable Fluorescent Carbon Dots Using Silica Spheres as Carriers for Targeted Bioimaging of Cancer Cells

In this paper, fluorescent carbon dots (CDs) loaded on silica (SiO₂) spheres are synthesized by the one‐pot hydrothermal route, and then folic acids (FA) are covalently conjugated on the surface of SiO₂ spheres. The formed SiO₂@CDs‐FA composites can target specific tissues, e.g., cancer. The key of this method is the employment of (3‐aminopropyl)trimethoxysilane as bridge joint, which not only serves as surface passivation agents allowing the large scale synthesis of CDs with high quantum yield, but also enables SiO₂@CDs composites further covalent conjugation of FA. The resultant SiO₂@CDs composites have many advantages such as easy separation and purification, highly stable, well water‐soluble, and biocompatible. Moreover, the SiO₂@CDs‐FA could be used as fluorescent probes for biological imaging in vitro. The uptake of the SiO₂@CDs‐FA into HeLa cells is receptor‐mediated endocytosis, which is confirmed by a comparative study using FR‐negative 293T cells. Findings from this study suggest that the SiO₂@CDs‐FA composites could be used as a platform for cancer diagnosis studies in various biological systems.     

Polymeric‐Micelle‐Based Nanomedicine for siRNA Delivery

Small interfering RNAs (siRNAs) are a rapidly emerging class of innovative nucleic acid medicines for the treatment of diseases such as cancer. However, significant hurdles hamper their clinical application, including poor cellular uptake, instability under physiological conditions, off‐target effects, and possible immunogenicity. The development of suitable delivery systems that protect and efficiently transport siRNA to targeted cells has been pursued. Nanoparticle‐based vectors have been widely investigated as potential candidates for effective siRNA delivery. Among the different nanoparticles, polymeric micelles, which are self‐assembled nanoparticles composed of amphiphilic materials with a core‐shell structure, have attracted great attention in recent years. Polymeric micelles in the range of several tens to hundreds of nanometers can be prepared, regulated, and modified relatively easily. The outer hydrophilic segments can prolong the in vivo lifetime of siRNA to achieve effective accumulation in tumors and can also be modified with cationic charges that interact electrostatically with siRNA and be introduced with different moieties to target specific cells. The inner cores can improve the stability of micelles and serve as payloads for hydrophobic drugs. Here, the barriers impeding siRNA delivery, the different polymeric micelles of siRNA developed to date, their gene silencing or therapeutic activity, and advanced applications for the co‐delivery of drugs and siRNA by these delivery systems are reviewed.     

Facile Synthesis of Mg2+‐Doped Carbon Dots as Novel Biomaterial Inducing Cell Osteoblastic Differentiation

Carbon dots (CDs), as an emerging fluorescent nanomaterial with low toxicity, has been widely applied in various bio‐related fields. However, investigations on their capabilities in guiding osteogenic differentiation are rarely seen, which has great significance in osteoporosis therapy and bone regeneration. Herein, for the first time, a new kind of Mg²⁺‐doped CDs is facilely synthesized through a one‐step hydrothermal method from metal gluconate salts. The CDs can serve as nanocarrier of Mg²⁺ ions entering into cells, and the bioessential metal ions subsequently stimulate osteoblastic differentiation by improving alkaline phosphatase (ALP) activity and upregulation related mRNA expression. Noteworthy, the raw material has almost negligible performance on osteoblastic differentiation compared to Mg‐CDs, which is due to the ultrasmall sizes of CDs and the efficient uptake by cells. Moreover, benefitting from the fluorescence properties, Mg‐CDs can also be applied as cell labeling agents. This work proposes a new strategy to synthesize multifunctional metal ion‐doped CDs, which might had great potential in serving as promising nanodrugs for bone loss therapy.     

Surface enhanced Raman scattering of molecules related to highly ordered gold cavities

We presented a controlled particles‐in‐cavity (PIC) pattern for surface‐enhanced Raman scattering (SERS) detection. The periodic gold cavity array was fabricated by electrodeposition using highly ordered polystyrene spheres as a template. The as‐prepared gold cavities can be used as a SERS active substrate with significant spectral enhancement and reproducibility, which was evaluated by SERS signals using 4‐mercaptobenzoic acid (4‐MBA) as probe molecules. The surface of these gold cavities was further functionalized with cetyltrimethylammonium bromide molecules, which may immobilize the 4‐MBA‐modified silver nanoparticles in the gold cavity to form a PIC structure via the electrostatic interaction. We have demonstrated that there exists a pH window for the immobilization of the nanoparticles inside cavities. Therefore, the silver nanoparticles can be selectively immobilized into the functionalized gold cavities under the optimized pH value of the media. Further enhancement of the Raman scattering of the labeled molecules can be achieved due to the interconnection between the silver nanoparticles and gold cavity. Copyright © 2013 John Wiley & Sons, Ltd.     

The Toxicity of Silver Nanoparticles Depends on Their Uptake by Cells and Thus on Their Surface Chemistry

A set of three types of silver nanoparticles (Ag NPs) are prepared, which have the same Ag cores, but different surface chemistry. Ag cores are stabilized with mercaptoundecanoic acid (MUA) or with a polymer shell [poly(isobutylene‐alt‐maleic anhydride) (PMA)]. In order to reduce cellular uptake, the polymer‐coated Ag NPs are additionally modified with polyethylene glycol (PEG). Corrosion (oxidation) of the NPs is quantified and their colloidal stability is investigated. MUA‐coated NPs have a much lower colloidal stability than PMA‐coated NPs and are largely agglomerated. All Ag NPs corrode faster in an acidic environment and thus more Ag(I) ions are released inside endosomal/lysosomal compartments. PMA coating does not reduce leaching of Ag(I) ions compared with MUA coating. PEGylation reduces NP cellular uptake and also the toxicity. PMA‐coated NPs have reduced toxicity compared with MUA‐coated NPs. All studied Ag NPs were less toxic than free Ag(I) ions. All in all, the cytotoxicity of Ag NPs is correlated on their uptake by cells and agglomeration behavior.     

A Correlative Analysis of Gold Nanoparticles Internalized by A549 Cells

Fluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light micro­scopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N‐labeled gold nanoparticles (3.3 × 10⁹ particles mL^?1, 0.02 μg Au mL^?1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.     

Three‐dimensional femtosecond laser micromachining of photosensitive glass for biomicrochips

Internal modification of transparent materials such as glass can be carried out using multiphoton absorption induced by a femtosecond (fs) laser. The fs‐laser modification followed by thermal treatment and successive chemical wet etching in a hydrofluoric (HF) acid solution forms three‐dimensional (3D) hollow microstructures embedded in photosensitive glass. This technique is a powerful method for directly fabricating 3D microfluidic structures inside a photosensitive glass microchip. We used fabricated microchips, referred to as a nanoaquarium, for dynamic observations of living microorganisms. In addition, the present technique can also be used to form microoptical components such as micromirrors and microlenses inside the photosensitive glass, since the fabricated structures have optically flat surfaces. The integration of microfluidics and microoptical components in a single glass chip yields biophotonic microchips, in other words, optofluidics, which provide high sensitivity in absorption and fluorescence measurements of small volumes of liquid samples.     

In Vitro Evaluation of Non‐Protein Adsorbing Breast Cancer Theranostics Based on 19F‐Polymer Containing Nanoparticles

Eight fluorinated nanoparticles (NPs) are synthesized, loaded with doxorubicin (DOX), and evaluated as theranostic delivery platforms to breast cancer cells. The multifunctional NPs are formed by self‐assembly of either linear or star‐shaped amphiphilic block copolymers, with fluorinated segments incorporated in the hydrophilic corona of the carrier. The sizes of the NPs confirm that small circular NPs are formed. The release kinetics data of the particles reveals clear hydrophobic core dependence, with longer sustained release from particles with larger hydrophobic cores, suggesting that the DOX release from these carriers can be tailored. Viability assays and flow cytometry evaluation of the ratios of apoptosis/necrosis indicate that the materials are non‐toxic to breast cancer cells before DOX loading; however, they are very efficient, similar to free DOX, at killing cancer cells after drug encapsulation. Both flow cytometry and confocal microscopy confirm the cellular uptake of NPs and DOX‐NPs into breast cancer cells, and in vitro ¹⁹F‐MRI measurement shows that the fluorinated NPs have strong imaging signals, qualifying them as a potential in vivo contrast agent for ¹⁹F‐MRI.     

Uptake and Intracellular Fate of Peptide Surface‐Functionalized Silica Hybrid Magnetic Nanoparticles In Vitro

Recently, the use of nanomaterials as intracellular targeting tools for theranostics has gained heightened interest. Despite the clear advantages posed by surface‐functionalized nanoparticles (NPs) in this regard, limited understanding currently exists due to difficulties in reliably synthesizing NPs with surface functionalizations adequate for use in such applications, as well as the manner of analytics used to assess the cellular uptake and intracellular localization of these NPs. In the present study, two key surface functionalities (a nuclear localization sequence (NLS) and integrin‐ligand (cRGD)) are attached to the surface of multifunctional, silica hybrid magnetic nanoparticles (SHMNPs) containing a polyethylene glycol (PEG) polymer coating using a well‐described, reliable, and reproducible microreactor set‐up. Subsequent analytical interpretation, via laser scanning confocal, transmission electron and dark‐field microscopy, as well as flow cytometry, of the interaction of SHMNPs‐PEG‐cRGD‐NLS with macrophage (J774A.1) and epithelial (HeLa) cells shows internalization of the SHMNPs‐PEG‐cRGD‐NLS in both cell types up to 24 h after 20 μg mL^?1 exposure, as well as increasing aggregation inside of vesicles over this time period. The findings of this study show that by incorporating a variety of state‐of‐the‐art analytical and imaging approaches, it is possible to determine the specific effectiveness of surface peptide and ligand sequences upon multifunctional SHMNPs.     

Self‐Assembly of Bidirectional‐Signal Nanoclusters for Two miRNAs Simultaneously Monitoring in Single Cancer Cells

A novel finding is herein reported that the bidirectional‐signal nanoclusters self‐assemble simultaneously on the nanoflowers as a result of four‐way folding (FWF) nanoprobes and DNA rolling circle replication reactions. The functionalized FWF nanoprobe containing the identification region for two targets monitoring and the trigger region for amplification signals is first used to activate the clustered amplification for two cancer‐related microRNAs (miRNAs) assays in single cells. Furthermore, the self‐assembled nanoclusters with two‐way amplification signals can provide more reliable information in situ in individual cell. Importantly, this new method can significantly distinguish cancer cells from normal cells and identify changes in the expression levels of cancer‐related miRNAs for single cells. These findings have exciting potential to provide new opportunities for detection and enhance the accuracy of early disease diagnosis.     

The estimation of a unique solution for steady-state diffuse optical tomography by applying mechanical pressure

The accuracy of diffuse optical tomography (DOT) highly depends on two important factors: first, the knowledge of the tissue optical heterogeneities for accurate modeling of light propagation, and second, the uniqueness of reconstructed values of optical properties. Previous studies illustrated that the inverse problem associated with steady-state DOT does not have unique solutions. In this study, we propose a simple method that can be applied to improve this challenging problem of steady-state DOT. In this method, we study the propagation of photons through compressed breast phantoms. The applied mechanical pressure can change the values of optical properties and this pressure dependence of optical properties as a set of constraint equations can be used to improve the inverse problem. The applied pressure can help us to restrict the distribution of possible values of depth and radius of defect inside breast phantom reconstructed by inverse problem.     

Optical Force Sensing with Cylindrical Microcontainers

Quantitative force sensing reveals essential information for the study of biological systems. Forces on molecules, cells, and tissues uncover functioning conditions and pathways. To analyze such forces, spherical particles are trapped and controlled inside an optical tweezers (OT) trap. Although these spherical particles are well‐established sensors in biophysics, elongated probes are envisioned for remote force sensing reducing heat damage caused by OT. There is thus a growing demand for force metrology with OT using complexly shaped objects, e.g., sac‐like organelles or rod‐like bacteria. Here, the employment of Zeolite‐L crystals as cylindrical force sensing probes inside a single optical trap is investigated. It is shown that cylindrical objects can be used as force probes since existing calibration assays can be performed with suitable corrections. Forces of active driving assays are compared with passive calibration methods. Finally, the investigations are extended to direct force measurements based on momentum calibration, in which the influence of rotation due to torque in a single optical trap is unveiled. Simulations reveal the relation between torque and the position of equilibrium in the trap. The results highlight the functionality of Zeolite‐L crystals as probes for force sensing, while opening perspectives for enhanced, accurate force metrology in biophotonics.