A novel shockwave-driven nano-motor composite microneedle transdermal delivery system for the localized treatment of osteoporosis: a basic science study.

BACKGROUND: Clinical protocols in osteoporosis treatment could not meet the requirement of increasing local bone mineral density. A local delivery system was brought in to fix this dilemma. The high-energy extracorporeal shock wave (ESW) can travel into the deep tissues with little heat loss. Hence, ESW-driven nanoparticles could be used for local treatment of osteoporosis. MATERIALS AND METHODS: An extracorporeal shock wave (ESW)--actuated nanomotor (NM) sealed into microneedles (MN) (ESW-NM-MN) was constructed for localized osteoporosis protection. The NM was made of calcium phosphate nanoparticles with a high Young's modulus, which allows it to absorb ESW energy efficiently and convert it into kinetic energy for solid tissue penetration. Zoledronic (ZOL), as an alternative phosphorus source, forms the backbone of the NM (ZOL-NM), leading to bone targeting and ESW-mediated drug release. RESULTS: After the ZOL-NM is sealed into hyaluronic acid (HA)--made microneedles, the soluble MN tips could break through the stratum corneum, injecting the ZOL-NM into the skin. As soon as the ESW was applied, the ZOL-NM would absorb the ESW energy to move from the outer layer of skin into the deep tissue and be fragmented to release ZOL and Ca2+ for anti-osteoclastogenesis and pro-osteogenesis. In vivo, the ZOL-NM increases localized bone parameters and reduces fracture risk, indicating its potential value in osteoporotic healing and other biomedical fields. CONCLUSION: The ESW-mediated transdermal delivery platform (ESW-NM-MN) could be used as a new strategy to improve local BMD and protect local prone-fracture areas.

Boron Neutron Capture Therapy-Derived Extracellular Vesicles via DNA Accumulation Boost Antitumor Dendritic Cell Vaccine Efficacy.

Radiated tumor cell-derived extracellular vesicles (RT-EVs) encapsulate abundant DNA fragments from irradiated tumor cells, in addition to acting as integrators of multiple tumor antigens. Accumulating evidence indicates these DNA fragments from damaged cells are involved in downstream immune responses, but most of them are degraded in cells before incorporation into derived RT-EVs, thus the low abundance of DNA fragments limits immune responses of RT-EVs. Here, this study found that different radiations affected fates of DNA fragments in RT-EVs. Boron neutron capture therapy (BNCT) induced DNA accumulation in RT-EVs (BEVs) by causing more DNA breaks and DNA oxidation resisting nuclease degradation. This is attributed to the high-linear energy transfer (LET) properties of alpha particles from the neutron capture reaction of 10B. When being internalized by dendritic cells (DCs), BEVs activated the DNA sensing pathway, resulting in functional enhancements including antigen presentation, migration capacity, and cytokine secretion. After vaccination of the BEVs-educated DCs (BEV@BMDCs), the effector T cells significantly expanded and infiltrated into tumors, suggesting robust anti-tumor immune activation. BEV@BMDCs not only effectively inhibited the primary tumor growth and metastasis formation but also elicited long-term immune memory. In conclusion, a successful DC vaccine is provided as a promising candidate for tumor vaccine.

Boron-Containing Mesoporous Silica Nanoparticles with Effective Delivery and Targeting of Liver Cancer Cells for Boron Neutron Capture Therapy.

Nanocarriers have been researched comprehensively for the development of novel boron-containing agents in boron neutron capture therapy (BNCT). We designed and synthesized a multifunctional mesoporous silica nanoparticle (MSN)-based boron-containing agent. The latter was coated with a lipid bilayer (LB) and decorated with SP94 peptide (SFSIIHTPILPL) on the surface as SP94-LB@BA-MSN. The latter incorporated boric acid (BA) into hydrophobic mesopores, coated with an LB, and modified with SP94 peptide on the LB. SP94-LB@BA-MSN enhanced nano interface tumor-targeting ability but also prevented the premature release of drugs, which is crucial for BNCT because adequate boron content in tumor sites is required. SP94-LB@BA-MSN showed excellent efficacy in the BNCT treatment of HepG-2 cells. In animal studies with tumor-bearing mice, SP94-LB@BA-MSN exhibited a satisfactory accumulation at the tumor site. The boron content reached 40.18 ± 5.41 ppm in the tumor site 4 h after injection, which was 8.12 and 15.51 times higher than those in mice treated with boronated phenylalanine and those treated with BA. For boron, the tumor-to-normal tissue ratio was 4.41 ± 1.13 and the tumor-to-blood ratio was 5.92 ± 0.45. These results indicated that nanoparticles delivered boron to the tumor site effectively while minimizing accumulation in normal tissues. In conclusion, this composite (SP94-LB@BA-MSN) shows great promise as a boron-containing delivery agent for the treatment of hepatocellular carcinoma using BNCT. These findings highlight the potential of MSNs in the field of BNCT.

Boron-Containing MOF Nanoparticles with Stable Metabolism in U87-MG Cells Combining Microdosimetry To Evaluate Relative Biological Effectiveness of Boron Neutron Capture Therapy.

Accurate prediction of the relative biological effectiveness (RBE) of boron neutron capture therapy (BNCT) is challenging. The therapy is different from other radiotherapy; the dynamic distribution of boron-containing compounds in tumor cells affects the therapeutic outcome considerably and hampers accurate measurement of the neutron-absorbed dose. Herein, we used boron-containing metal-organic framework nanoparticles (BMOFs) with high boron content to target U87-MG cells and maintain the concentration of the 10B isotope in cells. The content of boron in the cells could maintain 90% (60 ppm) within 20 min compared with that at the beginning; therefore, the accurate RBE of BNCT can be acquired. The effects of BNCT upon cells after neutron irradiation were observed, and the neutron-absorbed dose was obtained by Monte Carlo simulations. The RBE of BMOFs was 6.78, which was 4.1-fold higher than that of a small-molecule boron-containing agent (boric acid). The energy spectrum of various particles was analyzed by Monte Carlo simulations, and the RBE was verified theoretically. Our results suggested that the use of nanoparticle-based boron carriers in BNCT may have many advantages and that maintaining a stable boron distribution within cells may significantly improve the efficiency of BNCT.

3D culture boosting fullerenol nanoparticles to induce calreticulin exposure on MCF-7 cells for enhanced macrophage-mediated cell removal.

Calreticulin (CRT) on the cell surface that acts as an "eat me" signal is vital for macrophage-mediated programmed cell removal. The polyhydroxylated fullerenol nanoparticle (FNP) has appeared as an effective inducer to cause CRT exposure on cancer cell surface, but it failed in treating some cancer cells such as MCF-7 cells based on previous findings. Here, we carried out the 3D culture of MCF-7 cells, and interestingly found that the FNP induced CRT exposure on cells in 3D spheres via re-distributing CRT from endoplasmic reticulum (ER) to cell surface. Phagocytosis experiments in vitro and in vivo illustrated the combination of FNP and anti-CD47 monoclonal antibody (mAb) further enhanced macrophage-mediated phagocytosis to cancer cells. The maximal phagocytic index in vivo was about three times higher than that of the control group. Moreover, in vivo tumorigenesis experiments in mice proved that FNP could regulate the progress of MCF-7 cancer stem-like cells (CSCs). These findings expand the application of FNP in tumor therapy of anti-CD47 mAb and 3D culture can be used as a screening tool for nanomedicine.

Fabricated AIE-Based Probe to Detect the Resistance to Anoikis of Cancer Cells Detached from Tumor Tissue.

(1) Background: Resisting anoikis is a vital and necessary characteristic of malignant cancer cells, but there is no existing quantification method. Herein, a sensitive probe for assessing anoikis resistance of cancer cells detached from the extracellular matrix was developed based on the aggregation-induced emission (AIE) of AIEgens. It has been reported that detached cancer cell endocytose activated integrin clusters, and in the endosome these clusters recruit and activate phosphorylate focal adhesion kinase (pFAK) in the cytoplasm to induce signaling that supports the growth of detached cancer cells. (2) Methods: We established a lost nest cell model of cancer cells and determined their ability to resist anoikis. The colocalization of the activated integrin, pFAK, and endosomes in model cells was observed and calculated. (3) Results: The fluorescence signal intensity of the probe was significantly higher than that of the integrin antibody in the model cells and the fluorescence signal of probe signal was better overlapped with labeled pFAK by fluorescence in endosomes in model cells. (4) Conclusions: We developed a quantitative multi-parametric image analysis program to calculate fluorescent intensity of the probe and antibodies against pFAK and Rab5 in the areas of colocalization. A positive correlation of fluorescence signal intensity between the probe and pFAK on the endosome was observed. Therefore, the probe was used to quantitatively evaluate resisting anoikis of different cancer cell lines under the lost nest condition.

The Aggregation Induced Emission Probe of Detecting Enhanced Permeation and Retention Effects is Structured for Evaluating the Applicability of Nanotherapy to Different Tumor Individuals.

Enhanced permeation and retention (EPR) effect, the mechanism by which nanodrugs accumulate in tumors and acquire superior curative effect. The questions of these mechanisms occur because of limited clinical transformation of engineered nanomaterials after 30 years. The difference of EPR limits the therapeutic effect of nanodrugs in the individual patient. Evaluation of the EPR effect in the individual patient will aid in selecting patients who will accumulate higher amounts of nanotherapeutics and show better therapeutic efficacy. Based on varied TIMP1/MMP-9 in serum, an aggregation-induced emission luminogen probe was designed and constructed to detect and evaluate the EPR effect in model mouse. The result showed that the ratio of TIMP1/MMP-9 (in the range 0.2-1.2) and fluorescence intensity of the probe were negative linear correlation and the effects of BSA-rhodamine accumulation in tumor were individualized differences as well as correlated with the relative ratio of TIMP-1/MMP-9 in serum. Our data support the development of these biomarkers probes based on the personalized nanotherapy of tumor.

Intrinsic Biotaxi Solution Based on Blood Cell Membrane Cloaking Enables Fullerenol Thrombolysis In Vivo.

We report the construction of blood cell membrane cloaked mesoporous silica nanoparticles for delivery of nanoparticles [fullerenols (Fols)] with fibrinolysis activity which endows the active Fol with successful thrombolysis effect in vivo. In vitro, Fols present excellent fibrinolysis activity, and the Fol with the best fibrinolysis activity is screened based on the correlation between Fols' structure and their fibrinolysis activity. However, the thrombolytic effect in vivo is not satisfactory. To rectify the unsatisfactory situation and avoid the exogenous stimuli, a natural blood cell membrane cloaking strategy with loading the active Fol is chosen to explore as a novel thrombolysis drug. After cloaking, the therapeutic platform prolongs blood circulation time and enhances the targeting effect. Interestingly, compared with platelet membrane cloaking, red blood cell (RBC) membrane cloaking demonstrates stronger affinity with fibrin and more enrichment at the thrombus site. The Fol with RBC cloaking shows quick and efficient thrombolysis efficacy in vivo with less bleeding risk, more excellent blood compatibility, and better biosafety when compared with the clinical drug urokinase (UK). These findings not only validate the blood cell membrane cloaking strategy as an effective platform for Fol delivery on thrombolysis treatment, but also hold a great promising solution for other active nanoparticle deliveries in vivo.

Microfluidic Analysis for Separating and Measuring the Deformability of Cancer Cell Subpopulations.

Increased deformability and softness endow tumor cells with highly invasive and metastatic capabilities. We exploited these characteristics to fabricate a high-throughput microfluidic device to measure cell deformability and separate cancer cells. Driven by hydrodynamic forces, the cells with better deformability passed through the chip faster, whereas stiffer cells passed through the device over a longer time period. The MDA-MB-231 and MCF-7 cell lines were used to evaluate the device because their metastatic potentials were known. We found that MDA-MB-231 cells, which were softer and exhibited stronger deformability, passed through the device more quickly. HeLa cells were also successfully separated into softer and stiffer subpopulations, whose distinct mechanical properties were confirmed by atomic force microscopy. We also measured the expression of metastasis-associated proteins (epidermal growth factor receptor and integrin ß 1) and found that subpopulations with varied deformabilities had different expression levels. Our results suggested that this high-throughput microfluidic device could be used to screen and evaluate the curative effects of drug and cancer progression by simultaneously testing cell deformability and expression levels of metastasis-associated proteins in separated cell subpopulations.

Exploring the Interaction of Fullerenol with Key Digestive Proteases Using Raman-Based Frequency-Shift Sensing and Molecular Simulation Analysis.

Fullerenol has been demonstrated to be a potential anticancer nanodrug thanks to its excellent antioxidant properties. In this work, surface-enhanced Raman scattering (SERS)-based frequency shift method is employed to explore the interaction between fullerenol and the key digestive proteases (pepsin and trypsin) in the gastrointestinal tract. A dynamic adsorption process of fullerenol on pepsin/trypsin has been monitored by our detection platform. The binding sites of fullerenol to trypsin and pepsin are evidenced by introducing their inhibitors to the detection system, and the binding configurations/modes of fullerenol to pepsin and trypsin are precisely determined through computational simulations. The permeability of fullerenol through intestinal epithelial cells monolayer is also investigated and the results show that it can reach 6% in 6 h and 13% in 48 h, indicating its high oral bioavailability in human body. This work demonstrates the activity of fullerenol in the gastrointestinal tract and provides potential guidance for appropriate oral medicine design with the aid of a molecular docking method.

Au Nanoparticles Attenuate RANKL-Induced Osteoclastogenesis by Suppressing Pre-Osteoclast Fusion.

Osteoclasts are multinucleated terminal cells that originate from a hematopoietic monocyte/macrophage lineage. Excessive osteoclast formation in vivo can lead to bone metabolic diseases such as postmenopausal osteoporosis, multiple myeloma, rheumatoid arthritis, and lytic bone metastases of cancer cells. Au nanoparticles (AuNPs) are inorganic nanoparticles with outstanding biocompatibility. We assessed their effect on osteoclastogenesis and found that pre-osteoclast fusion induced by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colonystimulating factor (M-CSF) was suppressed by AuNPs. Cell migration and actin ring formation were also significantly inhibited. Finally, AuNPs reduced osteoclast bone absorption function. Interestingly, we observed altered fusogenic gene expression in treated pre-osteoclasts. Our results suggest that AuNPs have potential as a therapeutic agent for osteoclast-related bone metabolism diseases.

Nanoparticles with High-Surface Negative-Charge Density Disturb the Metabolism of Low-Density Lipoprotein in Cells.

Endocytosis is an important pathway to regulate the metabolism of low-density lipoprotein (LDL) in cells. At the same time, engineering nanoparticles (ENPs) enter the cell through endocytosis in biomedical applications. Therefore, a crucial question is whether the nanoparticles involved in endocytosis could impact the natural metabolism of LDL in cells. In this study, we fabricated a series of gold nanoparticles (AuNPs) (13.00 ± 0.69 nm) with varied surface charge densities. The internalized AuNPs with high-surface negative-charge densities (HSNCD) significantly reduced LDL uptake in HepG-2, HeLa, and SMMC-7721 cells compared with those cells in control group. Notably, the significant reduction of LDL uptake in cells correlates with the reduction of LDL receptors (LDL-R) on the cell surface, but there is no change in protein and mRNA of LDL-Rs. The cyclic utilization of LDL-R in cells is a crucial pathway to maintain the homoeostasis of LDL uptake. The release of LDL-Rs from LDL/LDL-R complexes in endosomes depended on reduction of the pH in the lumen. AuNPs with HSNCD hampered vacuolar-type H⁺-ATPase V1 (ATPaseV1) and ATPaseV0 binding on the endosome membrane, blocking protons to enter the endosome by the pump. Hence, fewer freed LDL-Rs were transported into recycling endosomes (REs) to be returned to cell surface for reuse, reducing the LDL uptake of cells by receptor-mediated endocytosis. The restrained LDL-Rs in the LDL/LDL-R complex were degraded in lysosomes.

Fullerenol nanoparticles suppress RANKL-induced osteoclastogenesis by inhibiting differentiation and maturation.

Bone health requires regulation of homeostatic equilibrium between osteoblasts and osteoclasts. The over-activation of osteoclasts can disrupt bone metabolism, resulting in osteoporosis and other bone-loss diseases. Fullerenol, a polyhydroxy derivative of fullerene, exhibits excellent biocompatibility. Here we show that fullerenol nanoparticles exert two functions: inhibition of osteoclastic differentiation and blockage of pre-osteoclast fusion to restructure osteoclast maturation and function. Experimentally, the nanoparticles reduced pre-osteoclast migration and inhibited ruffled border formation to block their maturation. In addition, fullerenol dose-dependently restricted the differentiation of bone marrow macrophage cells (BMMs) to form osteoclasts following treatment with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB (RANKL) to activate NF-κB and mitogen activating protein kinase (MAPK) signaling pathways. It is possible that the very small size of fullerenol allows it to directly cross the cellular membrane to access the cytoplasm and regulate osteoclastogenesis from BMMs. Our results suggest that fullerenol could be used to treat bone-loss diseases such as osteoporosis.

Study on orally delivered paclitaxel nanocrystals: modification, characterization and activity in the gastrointestinal tract.

Drug nanocrystals (NCs) can improve the solubility and bioavailability of insoluble drugs for oral administration. However, the biocompatibility and mechanisms of transmittance of drug NCs through the intestinal epithelial tissue are still not well understood. In this work, the physico-chemical properties and interactions with biomolecules in oral delivery pathways, as well as the transmittance through mimical intestinal epithelial cells, of NCs of paclitaxel (PTX) are investigated. PTX was previously demonstrated to be an effective anti-cancer drug. It is found that maximum 1% (w/v) poly(styrenesulfonate) is sufficient to keep PTX NCs monodisperse in varied biological environments and presents no significant interaction with extracellular biomolecules for at least 24 h. The concentration of PTX NCs is kept carefully controlled to avoid serious toxicity to cells (10 µg ml-1 in our experiments but this also depends on NC size). The transmittance of PTX NCs through mimical intestinal epithelial reached 25% in 6 h, demonstrating its comparatively high oral bioavailability in the human body. This work demonstrates the great potential of PTX NC treated in oral delivery.

Endocytosed nanoparticles hold endosomes and stimulate binucleated cells formation.

BACKGROUND: Nanotechnology developed rapidly in cellular diagnosis and treatment, the endocytic system was an important pathway for targeting cell. In the research of developing macrophages as drug carriers or important therapeutic targets, an interesting phenomenon, internalized nanoparticles induced to form binucleated macrophages, was found although the particles dose did not cause obvious cytotoxicity. RESULTS: Under 25 µg/ml, internalized 30 nm polystyrene beads(30 nm Ps nanoparticles) induced the formation of binucleated macrophages when they entered into endosomes via the endocytic pathway. These internalized 30 nm Ps nanoparticles (25 µg/ml) and 30 nm Au-NPs (1.575 ng/ml) also induced markedly rise of binucleated cell rates in A549, HePG-2 and HCT116. This endosome, aggregated anionic polystyrene particles were dispersed and bound on inner membrane, was induced to form a large vesicle-like structure (LVLS). This phenomenon blocked transport of the particles from the endosome to lysosome and therefore restricted endosomal membrane trafficking through the transport vesicles. Early endosome antigen-1 and Ras-related protein-11 expressions were upregulated; however, the localized distributions of these pivotal proteins were altered. We hypothesized that these LVLS were held by the internalized and dispersed particles decreasing the amount of cell membrane available to support the completion of cytokinesis. In addition, altered distributions of pivotal proteins prevented transfer vesicles from fusion and hampered the separation of daughter cells. CONCLUSIONS: 30 nm Ps nanoparticles induced formation of LVLS, blocked the vesicle transport in endocytic system and the distributions of regular proteins required in cytokinesis which led to binucleated cells of macrophages. Markedly raised binucleated rate was also observed in human lung adenocarcinoma epithelial cell line(A549), human hepatoma cell line(HePG-2) and human colorectal cancer cell line(HCT116) treated by 30 nm Ps nanoparticles and Au-NPs.

Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation.

The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met-p16). The probe, paired with Met-p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

Hypoxia-regulated lncRNAs in cancer.

Hypoxic regions are common in solid tumors and have an impact on tumor progression and on the therapeutic response. However, the underlying mechanism for hypoxic tumor microenvironment has not been entirely elucidated. Recently, long noncoding RNAs (lncRNAs) are being increasingly recognized to contribute to carcinogenesis through diverse mechanisms. To date, several lncRNAs have been described in hypoxia-associated cancer process, implying a potential role in maintaining cellular homeostasis and enabling an adaptive survival under hypoxic stress conditions. While it has been widely accepted that a complex cellular network of gene products, such as protein and miRNA, take part in hypoxic cancer progression, it remains largely elusive how lncRNAs participate in it. In this review, we introduce an update view of lncRNAs, focusing on hypoxia-related lncRNAs. We hereby summarize the cause and consequence of hypoxia-modulated lncRNAs in cancer as well as their functional mechanisms, highlighting the specific roles of lncRNAs in hypoxia response in cancer.

YAP and TAZ Take Center Stage in Cancer.

The Hippo pathway was originally identified and named through screening for mutations in Drosophila, and the core components of the Hippo pathway are highly conserved in mammals. In the Hippo pathway, MST1/2 and LATS1/2 regulate downstream transcription coactivators YAP and TAZ, which mainly interact with TEAD family transcription factors to promote tissue proliferation, self-renewal of normal and cancer stem cells, migration, and carcinogenesis. The Hippo pathway was initially thought to be quite straightforward; however, recent studies have revealed that YAP/TAZ is an integral part and a nexus of a network composed of multiple signaling pathways. Therefore, in this review, we will summarize the latest findings on events upstream and downstream of YAP/TAZ and the ways of regulation of YAP/TAZ. In addition, we also focus on the crosstalk between the Hippo pathway and other tumor-related pathways and discuss their potential as therapeutic targets.

On-demand generation of singlet oxygen from a smart graphene complex for the photodynamic treatment of cancer cells.

Graphene oxide (GO) has been proven to be a highly efficient long-range quencher for various fluorescence processes, which intrinsically work through a photophysical mechanism similar to that of singlet oxygen generation (SOG). Under our hypothesis that GO may be capable of quenching the SOG process, here we design and synthesize a novel nanocomplex consisting of GO, a photosensitizer and an aptamer. We demonstrate that GO is an ideal SOG controller, which can reversibly quench and recover SOG depending on the interaction intensity between GO and a photosensitizer. Additionally, it can simultaneously act as a carrier for the efficient loading and delivery of the photosensitizers to cancer cells. Thus, during the delivery process, SOG of the nanocomplex can be completely inhibited by the quenching capacity of the GO even though there is light present; however, when the nanocomplex enters into cancer cells where target molecules are present, SOG is triggered by a target binding event and singlet oxygen is reversibly released from the nanocomplex, ultimately inducing significant cell death in the presence of light. This proof-of-concept study provides a new chemical strategy for creating a highly selective photodynamic therapy with low toxicity, using hydrophilic GO-based systems.