Dual-targeting multifunctional hyaluronic acid ligand-capped gold nanorods with enhanced endo-lysosomal escape ability for synergetic photodynamic–photothermal therapy of cancer

Au nanorods (AuNRs) have attracted considerable interest as drug delivery systems because of their enhanced cell internalization and stronger drug-loading ability. In addition, the incorporation of photodynamic therapy (PDT) and photothermal therapy (PTT) into one nanosystem presents great promise to defect multiple drawbacks in cancer therapy. Herein, we fabricated a multifunctional and dual-targeting nanoplatform based on hyaluronic acid-grafted-(mPEG/triethylenetetramine-conjugated-lipoic acid/tetra(4-carboxyphenyl)porphyrin/folic acid) polymer ligand capped AuNRs (AuNRs@HA-g-(mPEG/Teta-co-(LA/TCPP/FA)) for combined photodynamic–photothermal therapy of cancer. The prepared nanoparticles displayed high TCPP loading capacity and excellent stability in different biological media. Furthermore, AuNRs@HA-g-(mPEG/Teta-co-(LA/TCPP/FA)) not only could produce a localized hyperthermia to conduct PTT, but also generate cytotoxic singlet oxygen (¹O₂) to perform PDT under laser irradiation. Confocal imaging results disclosed that this nanoparticle endowing the specific function of polymeric ligand could enhance cellular uptake, accelerate endo/lysosomal escape, as well as produce higher reactive oxygen species. Importantly, this combination therapy strategy could also induce higher anticancer potential than PDT or PTT only against MCF-7 tumor cells in vitro. Therefore, this work presented an AuNRs-based therapeutic nanoplatform with great potential in dual-targeting and photo-induced combination therapy of cancer.     

The Effect of Aspect Ratio of Gold Nanorods on Cell Imaging with Two-Photon Excitation

To make the gold nanorod (AuNR) a better photoluminescence (PL) probe for cell imaging under two-photon excitation (TPE), the effect of the aspect ratio of AuNRs was studied. The AuNRs with the aspect ratios of 2.7, 3.2, 4.1, and 4.5 and correlated longitudinal surface plasmon resonance (LSPR) bands of 710, 760, 820, and 870 nm were compared. The approach of two-photon excited PL was used to measure the two-photon absorption cross section (TPACS) of these AuNRs in aqueous solutions. Under TPE of an 800-nm femtosecond laser, the TPACS of AuNRs with an aspect ratio of 3.2 was found to be the highest (about 3?×?10⁹ GM), and that of AuNRs (aspect ratio of 2.7) was only 1.5?×?10⁹ GM. The probe function of these two AuNRs was further compared in cell imaging studies using the human liver cancer cell (QGY) as the cell model. Both TPE PL image and confocal reflectance image of AuNR-loaded cells were acquired comparatively in measurements. The brightness and contrast of confocal reflectance images for these two AuNRs in cells are similar. In contrast, the PL images of cellular AuNRs (2.7) under TPE of 800 nm are weak but that of cellular AuNRs (3.2) is much better. These results show that when the LSPR band of AuNRs is coincided with the excitation wavelength, the TPACS of these AuNRs will be enhanced ensuring a good quality of cell imaging under TPE. The LSPR band is correlated to the aspect ratio of AuNRs. Therefore, in cell imaging studies with TPE, the aspect ratio effect of AuNRs should be taken into consideration.     

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA–Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA–Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA–Se NPs in MCF-7 cells. The results indicated that the 70-nm FA–Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA–Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca²+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA–Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA–Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA–Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.     

Investigation of Biophysical Mechanisms in Gold Nanoparticle Mediated Laser Manipulation of Cells Using a Multimodal Holographic and Fluorescence Imaging Setup

Laser based cell manipulation has proven to be a versatile tool in biomedical applications. In this context, combining weakly focused laser pulses and nanostructures, e.g. gold nanoparticles, promises to be useful for high throughput cell manipulation, such as transfection and photothermal therapy. Interactions between laser pulses and gold nanoparticles are well understood. However, it is still necessary to study cell behavior in gold nanoparticle mediated laser manipulation. While parameters like cell viability or perforation efficiency are commonly addressed, the influence of the manipulation process on other essential cell parameters is not sufficiently investigated yet. Thus, we set out to study four relevant cell properties: cell volume and area, ion exchange and cytoskeleton structure after gold nanoparticle based laser manipulation. For this, we designed a multimodal imaging and manipulation setup. 200 nm gold nanoparticles were attached unspecifically to canine cells and irradiated by weakly focused 850 ps laser pulses. Volume and area change in the first minute post laser manipulation was monitored using digital holography. Calcium imaging and cells expressing a marker for filamentous actin (F-actin) served to analyze the ion exchange and the cytoskeleton, respectively. High radiant exposures led to cells exhibiting a tendency to shrink in volume and area, possibly due to outflow of cytoplasm. An intracellular raise in calcium was observed and accompanied by an intercellular calcium wave. This multimodal approach enabled for the first time a comprehensive analysis of the cell behavior in gold nanoparticle mediated cell manipulation. Additionally, this work can pave the way for a better understanding and the evaluation of new applications in the context of cell transfection or photothermal therapy.     

Multiple effects of electroporation on the adhesive behaviour of breast cancer cells and fibroblasts

Background

Recently electroporation using biphasic pulses was successfully applied in clinical developments for treating tumours in humans and animals. We evaluated the effects of electrical treatment on cell adhesion behaviour of breast cancer cells and fibroblasts. By applying bipolar electrical pulses we studied short- and long-lived effects on cell adhesion and survival, actin cytoskeleton and cell adhesion contacts in adherent cancer cells and fibroblasts.

Methods

Two cancer cell lines (MDA-MB-231 and MCF-7) and one fibroblast cell line 3T3 were used. Cells were exposed to high field intensity (200 - 1000 V/cm). Cell adhesion and survival after electrical exposure were studied by crystal violet assay and MTS assay. Cytoskeleton rearrangement and cell adhesion contacts were visualized by actin staining and fluorescent microscope.

Results

The degree of electropermeabilization of the adherent cells elevated steadily with the increasing of the field intensity. Adhesion behaviour of fibroblasts and MCF-7 was not significantly affected by electrotreatment. Interestingly, treating the loosely adhesive cancer cell line MDA-MB-231 with 200 V/cm and 500 V/cm resulted in increased cell adhesion. Cell replication of both studied cancer cell lines was disturbed after electropermeabilization. Electroporation influenced the actin cytoskeleton in cancer cells and fibroblasts in different ways. Since it disturbed temporarily the actin cytoskeleton in 3T3 cells, in cancer cells treated with lower and middle field intensity actin cytoskeleton was well presented in stress fibers, filopodia and lamellipodia. The electrotreatment for cancer cells provoked preferentially cell-cell adhesion contacts for MCF-7 and cell-ECM contacts for MDA-MB- 231.

Conclusions

Cell adhesion and survival as well as the type of cell adhesion (cell-ECM or cell-cell adhesion) induced by the electroporation process is cell specific. The application of suitable electric pulses can provoke changes in the cytoskeleton organization and cell adhesiveness, which could contribute to the restriction of tumour invasion and thus leads to the amplification of anti-tumour effect of electroporation-based tumour therapy.     

Pteroyl-gamma-glutamate-cysteine synthesis and its application in folate receptor-mediated cancer cell targeting using folate-tethered liposomes

Cell membrane-associated folate receptors are selectively overexpressed in certain human tumors. The high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. Folate-tethered liposomes bearing pteroyl-gamma-glutamate-cysteine-polyethylene glycol (PEG)-distearoylphosphatidylethanolamine (DSPE) as the targeting component are under investigation as mediators of drug and gene delivery to cancer cells that overexpress folate receptors. Pteroyl-gamma-glutamate-cysteine synthesis is one of the crucial starting steps in the preparation of pteroyl-gamma-glutamate-cysteine-PEG-DSPE. However, published methods for the synthesis of pteroyl-gamma-glutamate-cysteine provide low yields and are not easily reproducible. Therefore, we developed a modified synthetic method for the removal of the N(10)-trifluoroacetyl group after cleavage/deprotection that is reliable, is easily reproducible, and has high yield (38%) compared with an unreliable yield of 3-20% with the earlier methods. Folate-tethered liposomes containing calcein or doxorubicin were prepared using pteroyl-gamma-glutamate-cysteine-PEG-DSPE as the targeting component along with nontargeted liposomes with PEG-DSPE. The results of the uptake of calcein and cytotoxicity of doxorubicin in human cervical cancer HeLa-IU(1) cells and human colon cancer Caco-2 cells demonstrated that folate-tethered liposomes were efficient in selective delivery to cancer cells overexpressing folate receptors. The improvement in yield of the targeting component can significantly facilitate "scale up" of folate receptor-mediated liposomal cancer therapy to the preclinical and clinical levels of investigations.     

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm2 and power density of 45 mW/cm2. Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton.     

Aptamer‐conjugated mesoporous polydopamine for docetaxel targeted delivery and synergistic photothermal therapy of prostate cancer

ObjectivesIt is imperative to develop efficient strategies on the treatment of prostate cancer. Here, we constructed multifunctional nanoparticles, namely AS1411@MPDA‐DTX (AMD) for targeted and synergistic chemotherapy/photothermal therapy of prostate cancer.Materials and MethodsMesoporous polydopamine (MPDA) nanoparticles were prepared by a one‐pot synthesis method, DTX was loaded through incubation, and AS1411 aptamer was modified onto MPDA by the covalent reaction. The prepared nanoparticles were characterized by ultra‐micro spectrophotometer, Fourier transform infrared spectra, transmission electron microscope, and so on. The targeting ability was detected by selective uptake and cell killing. The mechanism of AMD‐mediated synergistic therapy was detected by Western blot and immunofluorescence.ResultsThe prepared nanoparticles can be easily synthesized and possessed excellent water solubility, stability, and controlled drug release ability, preferentially in acidic context. Based on in vitro and in vivo results, the nanoparticles can efficiently target prostate cancer cells, promote DTX internalization, and enhance the antitumor effects of chemo‐photothermal therapy strategies under the NIR laser irradiation.ConclusionsAs a multifunctional nanoplatform, AS1411@MPDA‐DTX could efficiently target prostate cancer cells, promote DTX internalization, and synergistically enhance the antiprostate cancer efficiency by combining with NIR irradiation.     

Label-free colorimetric sensor for ultrasensitive detection of heparin based on color quenching of gold nanorods by graphene oxide

A novel label-free colorimetric strategy was developed for ultrasensitive detection of heparin by using the super color quenching capacity of graphene oxide (GO). Hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorods (AuNRs) could easily self-assembly onto the surface of GO through electrostatic interaction, resulting in decrease of the surface plasmon resonance (SPR) absorption and consequent color quenching change of the AuNRs from deep to light. Polycationic protamine was used as a medium for disturbing the electrostatic interaction between AuNRs and GO. The AuNRs were prevented from being adsorbed onto the surface of GO because of the stronger interaction between protamine and GO, showing a native color of the AuNRs. On the contrary, in the presence of heparin, which was more easily to combine with protamine, the AuNRs could self-assembly onto the surface of GO, resulting in the native color disappearing of AuNRs. As the concentration of heparin increased, the color of AuNRs would gradually fade until almost colorless. The amounts of self-assembly AuNRs were proportional to the concentration of heparin, and thereby the changes in the SPR absorption and color had been used to monitor heparin levels. Under optimized conditions, good linearity was obtained in a range of 0.02-0.28 μg/mL (R=0.9957), and a limit of detection was 5 ng/mL. The simultaneous possession of high sensitivity and selectivity, simplicity, rapidity, and visualization enabled this sensor to be potentially applicable for ultrasensitive and rapid on-site detection toward trace heparin.     

Probing the behaviors of gold nanorods in metastatic breast cancer cells based on UV-vis-NIR absorption spectroscopy

In this work, behaviors of positively-charged AuNRs in a highly metastatic tumor cell line MDA-MB-231 are examined based on UV-vis-NIR absorption spectroscopy in combination with inductively coupled plasma mass spectrometry (ICP-MS), transmission electron microscopy (TEM) and dark-field microscopic observation. It is found that characteristic surface plasmon resonance (SPR) peaks of AuNRs can be detected using spectroscopic method within living cells that have taken up AuNRs. The peak area of transverse SPR band is shown to be proportionally related to the amount of AuNRs in the cells determined with ICP-MS, which suggests a facile and real time quantification method for AuNRs in living cells. The shape of longitudinal SPR band in UV-vis-NIR spectrum reflects the aggregation state of AuNRs in the cells during the incubation period, which is proved by TEM and microscopic observations. Experimental results reveal that AuNRs are internalized by the cells rapidly; the accumulation, distribution and aggregation of AuNRs in the cells compartments are time and dose dependent. The established spectroscopic analysis method can not only monitor the behaviors of AuNRs in living cells but may also be helpful in choosing the optimum laser stimulation wavelength for anti-tumor thermotherapy.     

Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL⁻¹ (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.     

Invasion inhibition by a MEK inhibitor correlates with the actin-based cytoskeleton in lung cancer A549 cells

Metastasis remains the primary cause of lung cancer. The molecules involved in metastasis may be candidates for new targets in the therapy of lung cancer. The MEK/ERK signaling pathway has been highlighted in a number of studies on invasiveness and metastasis. In this paper, we show that the MEK inhibitor U0126 induces flattened morphology, remodels the actin-based cytoskeleton, and potently inhibits chemotaxis and Matrigel invasion in the human lung cancer A549 cell line. Furthermore, downregulation of ERK by small interfering RNA significantly inhibits the invasion of A549 cells and induces stress fiber formation. Taken together, our findings provide the first evidence that the inhibition of invasion of lung cancer A549 cells by inhibiting MEK/ERK signaling activity is associated with remodeling of the actin cytoskeleton, suggesting a novel link between MEK/ERK signaling-mediated cell invasion and the actin-based cytoskeleton.     

综述——基于纳米材料的生物检测与医学诊断技术：吲哚菁绿纳米颗粒在癌症诊断和治疗中的应用

吲哚菁绿(ICG)是一种传统的临床近红外(NIR)荧光染料,同时能够高效吸收激光用于光热和光动力治疗．但是ICG在水溶液中的不稳定性及在体内的快速清除限制了它的应用．纳米技术的快速发展为ICG的进一步开发应用提供了新材料和新思路．本文主要介绍ICG纳米颗粒在肿瘤近红外诊断及光热和光动力治疗领域研究的最新进展．     

Gold nanorods-conjugated TiO<Subscript>2</Subscript> nanoclusters for the synergistic combination of phototherapeutic treatments of cancer cells

Background

Recently, a combination of photodynamic therapy (PDT) and photothermal therapy (PTT) to generate reactive oxygen species (ROS) and heat to kill cancer cells, respectively has attracted considerable attention because it gives synergistic effects on the cancer treatment by utilizing the radiation of nontoxic low-energy photons such as long wavelength visible light and near IR (NIR) penetrating into subcutaneous region. For the effective combination of the phototherapies, various organic photosensitizer-conjugated gold nanocomplexes have been developed, but they have still some disadvantages due to photobleaching and unnecessary energy transfer of the organic photosensitizers.

Results

In this study, we fabricated novel inorganic phototherapeutic nanocomplexes (Au NR–TiO₂ NCs) by conjugating gold nanorods (Au NRs) with defective TiO₂ nanoparticle clusters (d-TiO₂ NP clusters) and characterized their optical and photothermal properties. They were observed to absorb a broad range of visible light and near IR (NIR) from 500 to 1000 nm, exhibiting the generation of ROS as well as the photothermal effect for the simultaneous application of PDT and PTT. The resultant combination of PDT and PTT treatments of HeLa cells incubated with the nanocomplexes caused a synergistic increase in the cell death compared to the single treatment.

Conclusion

The higher efficacy of cell death by the combination of PDT and PTT treatments with the nanocomplexes is likely attributed to the increases of ROS generation from the TiO₂ NCs with the aid of local surface plasma resonance (LSPR)-induced hot electrons and heat generation from Au NRs, suggesting that Au NR–TiO₂ NCs are promising nanomaterials for the in vivo combinatorial phototherapy of cancer.

     

Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines

Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell lines derived from the normal human colon, HCEC, NCM356 and NCM460, were grown for 32–34 days in media containing 25, 50, 75 or 150 nM folic acid, and the expression of genes involved in cell-cycle checkpoints, intracellular signaling, folate uptake and cell adhesion and migration was determined. Expression of Folate Receptor 1 was increased with decreasing media folate in all cell lines, as was p53, p21, p16 and β-catenin. With decreasing folate, the expression of both E-cadherin and SMAD-4 was decreased in NCM356. APC was elevated in NCM356 but unchanged in the other lines. No changes in global methylation were detected. A significant increase in p53 exon 7–8 strand breaks was observed with decreasing folate in NCM460 cells. The changes observed are consistent with DNA damage-induced activation of cell-cycle checkpoints and cellular adaptation to folate depletion. Folate-depletion-induced changes in the Wnt/APC pathway as well as in genes involved in cell adhesion, migration and invasion may underlie observed relationships between folate status and cancer risk.     

靶向纳米探针介导的近红外激光热疗

肿瘤靶向治疗的研究是当今生物医学界的研究热点.本研究采用整合素αvβ3单克隆抗体作为肿瘤靶向分子,以单壁碳纳米管(SWNT)作为运输载体,同时利用单壁碳纳米管在近红外区的光吸收特性,开展靶向肿瘤光热治疗.实验结果表明,这种整合素αvβ3单抗标记的碳纳米管探针对高表达αvβ3的U87MG细胞具有高靶向选择性和靶向光杀伤性...     

Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeleton

Progesterone plays a role in breast cancer development and progression but the effects on breast cancer cell movement or invasion have not been fully explored. In this study, we investigate the actions of natural progesterone and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin cytoskeleton remodeling and on breast cancer cell movement and invasion. In particular, we characterize the nongenomic signaling cascades implicated in these actions. T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices in the presence of both progestins. Exposure to the hormones triggers a rapid remodeling of the actin cytoskeleton and the formation of membrane ruffles required for cell movement, which are dependent on the rapid phosphorylation of the actin-regulatory protein moesin. The extra-cellular small GTPase RhoA/Rho-associated kinase (ROCK-2) cascade plays central role in progesterone- and MPA-induced moesin activation, cell migration and invasion. In the presence of progesterone, progesterone receptor A (PRA) interacts with the G protein G alpha(13), while MPA drives PR to interact with tyrosine kinase c-Src and to activate phosphatidylinositol-3 kinase, leading to the activation of RhoA/ROCK-2. In conclusion, our findings manifest that progesterone and MPA promote breast cancer cell movement via rapid actin cytoskeleton remodeling, which are mediated by moesin activation. These events are triggered by RhoA/ROCK-2 cascade through partially differing pathways by the two compounds. These results provide original mechanistic explanations for the effects of progestins on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.