Qin Tu

Folate-Decorated Hybrid Polymeric Nanoparticles for Chemically and Physically Combined Paclitaxel Loading and Targeted Delivery

In this study, folate-functionalized hybrid polymeric nanoparticles (NPs) were prepared as carriers of low water solubility paclitaxel for tumor targeting, which were composed of monomethoxy-poly(ethylene glycol)-b-poly(lactide)-paclitaxel (MPEG-PLA-paclitaxel) and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-folate (TPGS-FOL). NPs with various weight ratios of MPEG-PLA-paclitaxel and TPGS-FOL were prepared using a solvent extraction/evaporation method, which can also physically encapsulate paclitaxel. The size, size distribution, surface charge, and morphology of the drug-loaded NPs were characterized using a Zetasizer Nano ZS, scanning electron microscope (SEM), and atomic force microscopy (AFM). The encapsulation and drug loading efficiencies of these polymeric NPs are analyzed using high-performance liquid chromatography (HPLC) at 227 nm. The combination of covalent coupling and physical encapsulation is found to improve the loading of paclitaxel in NPs greatly. The in vitro antitumor activity of the drug-loaded NPs is assessed using a standard method of transcriptional and translational (MTT) assays against HeLa and glioma C6 cells. When the cells were exposed to NPs with the same paclitaxel weights, cell viability decreases in relation to the increase in TPGS-FOL in drug-loaded NPs. To investigate drug-loaded NP cellular uptake, the fluorescent dye coumarin-6 is utilized as a model drug and enveloped in NPs with 0 or 50% TPGS-FOL. Confocal laser scanning microscopy (CLSM) analysis shows that cellular uptake is lower for coumarin-6-loaded NPs with 0% TPGS-FOL than those with 50% TPGS-FOL. However, no difference for NIH 3T3 cells with normally expressed folate receptors is found. Results from in vitro antitumor activity and cellular uptake assay demonstrate that folic acid promotes drug-loaded NP cellular uptake through folate receptor-mediated endocytosis (RME). All of these results demonstrate that folate-decorated hybrid polymeric NPs are potential carriers for tumor-targeted drug delivery.

Atomic force microscope study of tumor cell membranes following treatment with anti-cancer drugs

Integrity of the cell membrane is a basic requirement for maintaining the biological characteristics of a cell. In this study, changes in the morphology and ultrastructure of HeLa (human cervical carcinoma), HepG2 (human hepatocellular liver carcinoma), and C6 (rat glioma) cells were studied by atomic force microscopy (AFM) both before and after treatment with the anti-cancer drugs, colchicine or cytarabine. In response to both drugs, the microstructure of the cell membrane of all three cell types displayed similar changes; that is, with increases in drug concentration and reaction time, the degree of morphological changes on the surface of cell membrane increased. These changes included increases in the fluctuation of the surface components of the cell membrane, increase in shrinkage, or even the appearance of pores. Cell viability was maintained, as determined by optical microscope observation of gross cell morphology and by MTT assay results. Analysis of the cell membrane root-mean-square (RMS) roughness showed that under the action of colchicine and cytarabine, RMS values for the cell membranes of all three tumor cell types were positively correlated to the drug concentration and reaction time. This research has great significance for the visual diagnosis of early stage apoptosis in tumor cells in response to anti-cancer drugs, as well as in the studies on the interaction between drugs and cells. The use of AFM can be a rapid and sensitive visual method for studying the interaction between cells and drug.

Preparation and in vitro properties of redox-responsive polymeric nanoparticles for paclitaxel delivery

Rice-like polymeric nanoparticles (NPs) composed of a new redox-responsive polymer, poly(ethylene glycol)-b-poly(lactic acid) (MPEG-SS-PLA), were prepared to carry paclitaxel (PTX) for glutathione (GSH)-regulated drug delivery. The PTX-loaded MPEG-SS-PLA NPs were fabricated using an optimized oil-in-water emulsion/solvent evaporation method. The size and morphology of the prepared NPs were characterized by scanning electron microscopy (SEM). The SEM results demonstrate that the NPs were dispersed as individual particles and were rice-shaped. The PTX loading efficiency, in vitro release, and stability of the NPs were analyzed by high-performance liquid chromatography (HPLC). The HPLC results revealed that the NPs released almost 90% PTX within 96 h when GSH presented at intracellular concentrations, whereas only a very small PTX amount was released at plasma GSH levels. The in vitro cytotoxicities of the NPs against A549, MCF-7, and HeLa carcinoma cells were assessed using a standard methyl thiazolyl tetrazoliun (MTT) assay. The MTT assay results show that the NPs caused concentration- and time-dependent changes in cell viability. To investigate the cellular uptake of the PTX-loaded NPs, visual endocytosis assay was performed using the fluorescent dye coumarin-6 as a model drug. The endocytosis assay results reveal rapid penetration and intracellular accumulation of coumarin-6-loaded NPs, as well as rapid coumarin-6 dispersion from the NPs. Overall, these findings establish that the NPs containing the synthesized redox-responsive polymer MPEG-SS-PLA can be used as potential carrier systems for antitumor drug delivery.

Surface modification of PDMS by surface-initiated atom transfer radical polymerization of water-soluble dendronized PEG methacrylate

The current paper reports the synthesis of a highly hydrophilic, antifouling dendronized poly(3,4,5-tris(2-(2-(2-hydroxylethoxy)ethoxy)ethoxy)benzyl methacrylate) (PolyPEG) brush using surface initiated atom transfer radical polymerization (SI-ATRP) on PDMS substrates. The PDMS substrates were first oxidized in H(2)SO(4)/H(2)O(2) solution to transform the Si-CH(3) groups on their surfaces into Si-OH groups. Subsequently, a surface initiator for ATRP was immobilized onto the PDMS surface, and PolyPEG was finally grafted onto the PDMS surface via copper-mediated ATRP. Various characterization techniques, including contact angle measurements, attenuated total reflection infrared spectroscopy, and X-ray photoelectron spectroscopy, were used to ascertain the successful grafting of the PolyPEG brush onto the PDMS surface. Furthermore, the wettability and stability of the PDMS-PolyPEG surface were examined by contact angle measurements. Anti-adhesion properties were investigated via protein adsorption, as well as bacterial and cell adhesion studies. The results suggest that the PDMS-PolyPEG surface exhibited durable wettability and stability, as well as significantly anti-adhesion properties, compared with native PDMS surfaces. Additionally, our results present possible uses for the PDMS-PolyPEG surface as adhesion barriers and anti-fouling or functional surfaces in biomedical applications.

High-throughput microfluidic system for long-term bacterial colony monitoring and antibiotic testing in zero-flow environments

In this study, a high-throughput microfluidic system is presented. The system is comprised of seven parallel channels. Each channel contains 32 square-shaped microchambers. After simulation studies on samples loaded into the microchambers, and the solute exchange between the microchambers and channels, the long-term culture of Escherichia coli (E. coli) HB101 in the microchambers is realized. Using the principle that L-arabinose (L-Ara) can induce recombinant E. coli HB101 pGLO to synthesize green fluorescent protein (GFP), the real-time analysis of GFP expression in different initial bacterial densities is performed. The results demonstrate that higher initial loading densities of the bacterial colony cause bacterial cell to enter log-phase proliferation sooner. High or low initial loading densities of the bacterial cell suspension induce the same maximum growth rates during the log-phase. Quantitative on-chip analysis of tetracycline and erythromycin inhibition on bacterial cell growth is also conducted. Bacterial morphology changes during antibiotic treatment are observed. The results show that tetracycline and erythromycin exhibit different inhibition activities in E. coli cells. Concentrations of 3 μg/mL tetracycline can facilitate the formation of long filamentous bacteria with the average length of more than 50 μm. This study provides an on-chip framework for bacteriological research in a high-throughput manner and the development of recombinant bacteria-based biosensors for the detection of specific substances.

Photocatalyzed surface modification of poly(dimethylsiloxane) with polysaccharides and assay of their protein adsorption and cytocompatibility.

An improved approach for the surface modification of poly(dimethylsiloxane) (PDMS) using carboxymethyl cellulose (CMC), carboxymethyl beta-1,3-dextran (CMD), and alginic acid (AA) was investigated. The PDMS substrates were first oxidized in a H(2)SO(4)/H(2)O(2) solution to transform the Si-CH(3) groups on their surfaces into Si-OH groups. Then methacrylate groups were grafted onto the substrates through a silanization reaction using 3-(trimethoxysilyl)propyl methacrylate. Sequentially, cysteamine was conjugated onto the silanized surfaces by the reaction between the thiol and methacrylate groups under 254 nm UV exposure. Afterward, the amino-terminated PDMS substrates were sequentially reacted with CMC, CMD, and AA in the presence of N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, resulting in the grafting of polysaccharides onto PDMS surfaces. The composition and chemical state of the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS). In addition, the stability and dynamic characteristics of the polysaccharide-grafted PDMS substrates were investigated by XPS and temporal contact angle experiments. A protein adsorption assay using bovine serum albumin (BSA), chicken egg albumin, lysozyme, and RNase-A showed that the introduction of CMD and AA can reduce the adsorption of negatively charged BSA and chicken egg albumin, but increase the adsorption of the positively charged lysozyme and RNase-A. However, CMC-modified PDMS surfaces showed protein-repelling properties, regardless of whether the protein was positively or negatively charged. A cell culture and migration study of glioma C6, MKN-45, MCF-7, and HepG-2 cells revealed that the polysaccharide-modified PDMS greatly improved the cytocompatibility of native PDMS.

Cancer stem cell labeling using poly(L-lysine)-modified iron oxide nanoparticles

Cell labeling using magnetic nanoparticles is an increasingly used approach in noninvasive behavior tracking, in vitro separation of cancer stem cells (CSCs), and CSC-based research in cancer therapy. However, the impact of magnetic labeling on the biological properties of targeted CSCs, such as self-renewal, proliferation, multi-differentiation, cell cycle, and apoptosis, remains elusive. The present study sought to explore the potential effects on biological behavior when CSCs are labeled with superparamagnetic iron oxide (SPIO) nanoparticles in vitro. The glioblastoma CSCs derived from U251 glioblastoma multiforme were labeled with poly(L-lysine) (PLL)-modified γ-Fe(2)O(3) nanoparticles. The iron uptake of glioblastoma CSCs was confirmed through prussian blue staining, and was further quantified using atomic absorption spectrometry. The cellular viability of the SPIO-labeled glioblastoma CSCs was assessed using a fluorescein diacetate and propidium iodide double-staining protocol. The expressed specific markers and multi-differentiation of SPIO-labeled glioblastoma CSCs were comparatively assessed by immunocytochemistry and semi-quantitative RT-PCR. The effects of magnetic labeling on cell cycle and apoptosis rate of glioblastoma CSCs and their differentiated progenies were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of glioblastoma CSCs and their differentiated progenies were not affected by SPIO labeling compared with their unlabeled counterparts. Moreover, the magnetically labeled CSCs displayed an intact multi-differentiation potential, and could be sub-cultured to form new tumor spheres, which indicates the CSCs capacity for self-renewal. In addition, cell cycle distribution, apoptosis rate of the magnetically labeled glioblastoma CSCs, and their differentiated progenies were not impaired. Therefore, the SPIO-labeled CSCs could be a feasible approach in conducting further functional analysis of targeted CSCs.

Antifouling properties of poly(dimethylsiloxane) surfaces modified with quaternized poly(dimethylaminoethyl methacrylate).

A quaternized poly(dimethylaminoethyl methacrylate)-grafted poly(dimethylsiloxane) (PDMS) surface (PDMS-QPDMAEMA) was successfully prepared in this study via solution-phase oxidation reaction and surface-initiated atom transfer radical polymerization (SI-ATRP) using dimethylaminoethyl methacrylate (DMAEMA) as initial monomer. PDMS substrates were first oxidized in H(2)SO(4)/H(2)O(2) solution to transform the SiCH(3) groups on their surfaces into SiOH groups. Subsequently, a surface initiator for ATRP was immobilized onto the PDMS surface, and DMAEMA was then grafted onto the PDMS surface via copper-mediated ATRP. Finally, the tertiary amino groups of PolyDMAEMA (PDMAEMA) were quaternized by ethyl bromide to provide a cationic polymer brush-modified PDMS surface. Various characterization techniques, including contact angle measurements, attenuated total reflection infrared spectroscopy, and X-ray photoelectron spectroscopy, were used to ascertain the successful grafting of the quaternized PDMAEMA brush onto the PDMS surface. Furthermore, the wettability and stability of the PDMS-QPDMAEMA surface were examined by contact angle measurements. Antifouling properties were investigated via protein adsorption, as well as bacterial and cell adhesion studies. The results suggest that the PDMS-QPDMAEMA surface exhibited durable wettability and stability, as well as significant antifouling properties, compared with the native PDMS and PDMS-PDMAEMA surfaces. In addition, our results present possible uses for the PDMS-QPDMAEMA surface as adhesion barriers and antifouling or functional surfaces in PDMS microfluidics-based biomedical applications.

Small molecule-initiated light-activated semiconducting polymer dots: an integrated nanoplatform for targeted photodynamic therapy and imaging of cancer cells.

Photodynamic therapy (PDT) is a noninvasive and light-activated method for cancer treatment. Two of the vital parameters that govern the efficiency of PDT are the light irradiation to the photosensitizer and visual detection of the selective accumulation of the photosensitizer in malignant cells. Herein, we prepared an integrated nanoplatform for targeted PDT and imaging of cancer cells using folic acid and horseradish peroxidase (HRP)-bifunctionalized semiconducting polymer dots (FH-Pdots). In the FH-Pdots, meta-tetra(hydroxyphenyl)-chlorin (m-THPC) was used as photosensitizer to produce cytotoxic reactive oxygen species (ROS); fluorescent semiconducting polymer poly[2-methoxy-5-((2-ethylhexyl)oxy)-p-phenylenevinylene] was used as light antenna and hydrophobic matrix for incorporating m-THPC, and amphiphilic Janus dendrimer was used as a surface functionalization agent to conjugate HRP and aminated folic acid onto the surface of FH-Pdots. Results indicated that the doped m-THPC can be simultaneously excited by the on-site luminol-H2O2-HRP chemiluminescence system through two paths. One is directly through chemiluminescence resonance energy transfer (CRET), and the other is through CRET and subsequent fluorescence resonance energy transfer. In vitro PDT and specificity studies of FH-Pdots using a standard transcriptional and translational assay against MCF-7 breast cancer cells, C6 glioma cells, and NIH 3T3 fibroblast cells demonstrated that cell viability decreased with increasing concentration of FH-Pdots. At the same concentration of FH-Pdots, the decrease in cell viability was positively relevant with increasing folate receptor expression. Results from in vitro fluorescence imaging exhibited that more FH-Pdots were internalized by cancerous MCF-7 and C6 cells than by noncancerous NIH 3T3 cells. All the results demonstrate that the designed semiconducting FH-Pdots can be used as an integrated nanoplatform for targeted PDT and on-site imaging of cancer cells.

Investigation of hypoxia-induced myocardial injury dynamics in a tissue interface mimicking microfluidic device.

Myocardial infarction is a major cause of morbidity and mortality worldwide. However, the methodological development of a spatiotemporally controllable investigation of the damage events in myocardial infarction remains challengeable. In the present study, we describe a micropillar array-aided tissue interface mimicking microfluidic device for the dynamic study of hypoxia-induced myocardial injury in a microenvironment-controllable manner. The mass distribution in the device was visually characterized, calculated, and systematically evaluated using the micropillar-assisted biomimetic interface, physiologically relevant flows, and multitype transportation. The fluidic microenvironment in the specifically functional chamber for cell positioning and analysis was successfully constructed with high fluidic relevance to the myocardial tissue. We also performed a microenvironment-controlled microfluidic cultivation of myocardial cells with high viability and regular structure integration. Using the well-established culture device with a tissue-mimicking microenvironment, a further on-chip investigation of hypoxia-induced myocardial injury was carried out and the varying apoptotic responses of myocardial cells were temporally monitored and measured. The results show that the hypoxia directionally resulted in observable cell shrinkage, disintegration of the cytoskeleton, loss of mitochondrial membrane potential, and obvious activation of caspase-3, which indicates its significant apoptosis effect on myocardial cells. We believe this microfluidic device can be suitable for temporal investigations of cell activities and responses in myocardial infarction. It is also potentially valuable to the microcontrol development of tissue-simulated studies of multiple clinical organ/tissue disease dynamics.