The role of surface modification for TiO2 nanoparticles in cancer cells.

Titanium dioxide nanoparticles (TiO2 NPs) have a potential in the field of biological application. However, its poor dispersibility in water hampered its applications. In this study, 3-phosphonopropionic acid and 3-aminopropyl-triethoxysilane were respectively used for surface modification on TiO2 NPs with negative and positive surface charges (denoted as TiO2-COOH and TiO2-NH2). Zeta potentials of the prepared samples with high absolute value demonstrate the great improvement in their dispersibility. In terms of viability experiment, both TiO2-COOH and TiO2-NH2 showed low cytotoxicity. The cellular uptake efficiency and the uptake pathways of TiO2-COOH and TiO2-NH2 for cancer cells were studied. The exocytosis of TiO2-NH2 was also observed in the experiment.

Targeting and Photodynamic Killing of Cancer Cell by Nitrogen-Doped Titanium Dioxide Coupled with Folic Acid.

Titanium dioxide (TiO2) has attracted wide attention as a potential photosensitizer (PS) in photodynamic therapy (PDT). However, bare TiO2 can only be excited by ultraviolet illumination, and it lacks specific targeting ligands, which largely impede its application. In our study, we produced nitrogen-doped TiO2 and linked it with an effective cancer cell targeting agent, folic acid (FA), to obtain N-TiO2-FA nanoconjugates. Characterization of N-TiO2-FA included Zeta potential, absorption spectra and thermogravimetric analysis. The results showed that N-TiO2-FA was successfully produced and it possessed better dispersibility in aqueous solution than unmodified TiO2. The N-TiO2-FA was incubated with human nasopharyngeal carcinoma (KB) and human pulmonary adenocarcinoma (A549) cells. The KB cells that overexpress folate receptors (FR) on cell membranes were used as FR-positive cancer cells, while A549 cells were used as FR-negative cells. Laser scanning confocal microscopy results showed that KB cells had a higher uptake efficiency of N-TiO2-FA, which was about twice that of A549 cells. Finally, N-TiO2-FA is of no cytotoxicity, and has a better photokilling effect on KB cells under visible light irradiation. In conclusion, N-TiO2-FA can be as high-value as a PS in cancer targeting PDT.

Enhancement of the photokilling effect of aluminum phthalocyanine in photodynamic therapy by conjugating with nitrogen-doped TiO2 nanoparticles.

As a second-generation photodynamic therapy (PDT) photosensitizer, aluminum phthalocyanine chloride tetrasulfonate (Pc) has gained great attention due to its high absorption at the red light region. Yet, its application in PDT is strongly limited by its low cellular uptake efficiency. In this report, nitrogen-doped TiO2 nanoparticles (N-TiO2) conjugated with Pc are synthesized by a two-step surface modification method. The N-TiO2-Pc products are characterized by Zeta potential, transmission electron microscopy and UV-vis absorption spectroscopy. The cellular uptake, intracellular distribution, cytotoxicity and the photokilling effect of the nanoparticles are studied on different cancer cell lines. Compared with Pc, the absorption spectrum of N-TiO2-Pc expands from red to UV region, resulting in a higher production of reactive oxygen species under visible light irradiation. In addition, the cellular uptake of Pc is largely improved by its carrier N-TiO2. The photokilling efficiency of N-TiO2-Pc is over ten times higher than that of Pc. The results suggest that N-TiO2-Pc is an excellent candidate as a photosensitizer in PDT.

Study of polycation-capped Mn:ZnSe quantum dots as a novel fluorescent probe for living cells.

Transition metal manganese ion (Mn(2+)) doped zinc selenide quantum dots (Mn:ZnSe D-Dots) have been considered as a new material for fluorescent probes in biological labeling. However, this application is limited by the low membrane permeability of D-Dots. In this work, Mn:ZnSe D-Dots were capped with the polycation Sofast to label living cells. For the first time, the efficiency of cellular uptake in living cells is significantly enhanced. Various molar ratios of Sofast to D-Dots were explored and compared to obtain the optimal reaction conditions between Sofast and D-Dots for preparing Sofast/D-Dots nano-compound. A comparison on the fluorescence labeling ability of living cells were made between Sofast/D-Dots and pure D-Dots. Results from laser scanning confocal microscope show that Sofast/D-Dots complexes enter the cells more efficiently than pure D-Dots, even with a lower concentration and shorter incubation time. The cytotoxicities of D-Dots and Sofast/D-Dots were also studied. It was found that Sofast/D-Dots have a much lower cytotoxicity than cadmium-containing quantum dots (i.e. CdTe and CdTe/ZnS). Our results suggest that the non-heavy-metal-containing Sofast/D-Dots complexes have a great potential in the application of biological labeling, especially of long-time bioimaging in living cells.

Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation.

The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT.

Study on the visible-light-induced photokilling effect of nitrogen-doped TiO2 nanoparticles on cancer cells.

Nitrogen-doped TiO2 (N-TiO2) nanoparticles were prepared by calcining the anatase TiO2 nanoparticles under ammonia atmosphere. The N-TiO2 showed higher absorbance in the visible region than the pure TiO2. The cytotoxicity and visible-light-induced phototoxicity of the pure- and N-TiO2 were examined for three types of cancer cell lines. No significant cytotoxicity was detected. However, the visible-light-induced photokilling effects on cells were observed. The survival fraction of the cells decreased with the increased incubation concentration of the nanoparticles. The cancer cells incubated with N-TiO2 were killed more effectively than that with the pure TiO2. The reactive oxygen species was found to play an important role on the photokilling effect for cells. Furthermore, the intracellular distributions of N-TiO2 nanoparticles were examined by laser scanning confocal microscopy. The co-localization of N-TiO2 nanoparticles with nuclei or Golgi complexes was observed. The aberrant nuclear morphologies such as micronuclei were detected after the N-TiO2-treated cells were irradiated by the visible light.

Liposome encapsulation of thiol-capped CdTe quantum dots for enhancing the intracellular delivery.

Although water soluble thiol-capped quantum dots (QDs) have been widely used as photoluminescence (PL) probes in various applications, the negative charges on thiol terminals limit the cell uptake hindering their applications in cell imaging. The commercial liposome complex (Sofast®) was used to encapsulate these QDs forming the liposome vesicles with the loading efficiency as high as about 95%. The cell uptakes of unencapsulated QDs and QD loaded liposome vesicles were comparatively studied by a laser scanning confocal microscope. We found that QD loaded liposome vesicles can effectively enhance the intracellular delivery of QDs in three cell lines (human osteosarcoma cell line (U2OS); human cervical carcinoma cell line (Hela); human embryonic kidney cell line (293 T)). The photobleaching of encapsulated QDs in cells was also reduced comparing with that of unencapsulated QDs, measured by the PL decay of cellular QDs with a continuous laser irradiation in the microscope. The flow cytometric measurements further showed that the enhancing ratios of encapsulated QDs on cell uptake are about 4-8 times in 293 T and Hela cells. These results suggest that the cationic liposome encapsulation is an effective modality to enhance the intracellular delivery of thiol-capped QDs.

Enhancement of intracellular delivery of anti-cancer drugs by the Tat peptide.

The arginine-rich cationic Tat peptides have been reported to enhance the intracellular delivery of macromolecules, including DNA, RNA, and proteins. In this work an arginine cationic peptide derived from the HIV-1 Tat protein was conjugated with noncovalent bonds to sulfonated aluminum phthalocyanine (AlPcS, a photosensitizer for the light-activated photodynamic cancer therapy), doxorubicin (DOX, a chemotherapeutic agent), or quantum dots (QDs, often used as carriers for the delivery of anticancer drugs). The fluorescence of intracellular conjugates of AlPcS-Tat, DOX-Tat, and QDs-Tat was studied by means of confocal laser scanning microscopy in the human nasopharyngeal carcinoma KB cells and cervical carcinoma Hela cells in vitro. The Tat peptide with noncovalent links can enhance at least a twofold of intracellular delivery of AlPcS, DOX, and QDs via an endocytotic pathway in the two tumor cell lines. This finding may suggest that the Tat peptide-mediated intracellular delivery of anticancer drugs may have the potential for improving efficacy of cancer therapy.

Conjugates of folic acids with BSA-coated quantum dots for cancer cell targeting and imaging by single-photon and two-photon excitation.

Bovine serum albumin (BSA)-coated CdTe/ZnS quantum dots (BSA-QDs) were selected to conjugate with folic acid (FA), forming FA-BSA-QDs. This study aims to develop these small FA-BSA-QDs (less than 10 nm) for the diagnosis of cancers in which the FA receptor (FR) is overexpressed. The enhancement of cellular uptake in FR-positive human nasopharyngeal carcinoma cells (KB cells) for FA-BSA-QDs was found by means of confocal fluorescence microscopy under single-photon and two-photon excitation. The uptake enhancement for FA-BSA-QDs was further evaluated by flow-cytometric analysis in 10(4) KB cells, and was about 3 times higher than for BSA-QDs on average. The uptake enhancement was suppressed when KB cells had been pretreated with excess FA, reflecting that the enhancement was mediated by the association of FR at cell membranes with FA-BSA-QDs. When human embryonic kidney cells (293T) (FR-negative cells) and KB cells, respectively, were incubated with FA-BSA-QDs (1 µM) for 40 min, the FA-BSA-QD uptake by 293T cells was much weaker than that by KB cells, demonstrating that FA-BSA-QDs could undergo preferential binding on FR-positive cancer cells. These characteristics suggest that FA-BSA-QDs are potential candidates for cancer diagnosis.

Plasmonic gold nanorods can carry sulfonated aluminum phthalocyanine to improve photodynamic detection and therapy of cancers.

Hexadecyltrimethylammonium bromide-coated gold nanorods (AuNRs) with positive charges were effectively bound to negatively charged sulfonated aluminum phthalocyanine (AlPcS), a photosensitizer for photodynamic detection and therapy, due to the electrostatic force, with a loading content of 10(4) AlPcS molecules per rod. A 5-fold increase in the AlPcS fluorescence of the AlPcS-AuNRs complex was seen. The excitation fluorescence spectrum of the AlPcS-AuNRs with a typical 520 nm band fits well with the resonance band of AuNR surface plasmons, suggesting that such increased AlPcS fluorescence is produced from the strong surface plasmons of AuNRs. The intracellular distribution of AlPcS-AuNRs was studied in the QGY liver cancer cells by respectively imaging the AlPcS fluorescence and AuNRs reflectance with a confocal microscope. Furthermore, the AlPcS-AuNRs-loaded cells were photodynamically damaged after being exposed to red light in a light-dose-dependent manner. In contrast, no phototoxicity of the cells was seen after incubation with the same amount of free AlPcS, indicating that the AlPcS-AuNRs can enhance the AlPcS-mediated photodynamic effect. In addition, the loaded AlPcS can be photothermally released from AuNRs in the cells by the irradiation with an 800 nm femtosecond laser, demonstrating the potential for controlled drug release.

Study on the intracellular fate of Tat peptide-conjugated quantum dots by spectroscopic investigation.

The photoluminescence (PL) spectrum of water-soluble thiol-capped CdTe quantum dots (QDs) conjugated with Tat peptide in solution showed a remarkable redshift as compared to that of unconjugated QDs. After cellular uptake of the Tat-QDs conjugates, the micro-PL spectrum of Tat-QDs in lysosomes showed a spectral blueshift, which was most probably due to the fact that Tat peptide was digested by the enzymes, leaving the Tat-detached QDs in lysosomes. The reasons for the spectral changes have been discussed in detail.

Poly (N-isopropylacrylamide)-coated multifunctional nanoparticles for cell tracking.

OBJECTIVE: The objective of this work was to explore a new modality of cell tracking that uses multifunctional nanoparticles. The tracking of transfused cells in vivo is an important step to study the therapeutic course and mechanism of cell therapeutics. BACKGROUND DATA: Several methods of cell tracking have been developed. Our novel method uses multifunctional nanoparticles to track cells via both fluorescence and magnetic resonance. MATERIALS AND METHODS: Poly (N-isopropylacrylamide) (PNIPAM)-coated multifunctional nanoparticles containing both CdTe quantum dots (QDs) and Fe(3)O(4) magnetic particles were used to label Chinese hamster ovary (CHO) cells. The labeled cells were measured by confocal fluorescence microscope in vitro at the photoluminescent band (600 nm) of QDs. When these labeled cells were injected into the mouse, the in vivo images were detected by magnetic resonance imaging (MRI). RESULTS: The nanoparticles easily bound to the plasma membrane of CHO cells after incubation at 37 degrees C. The surface PNIPAM of nanoparticles is a well-known thermoresponsive polymer with a volume phase transition temperature. It is hydrophilic at temperatures below critical solution temperatures (32-34 degrees C) and becomes hydrophobic at higher temperatures. The cellular binding of nanoparticles was stable and nontoxic, and the photoluminescence of nanoparticles could still be seen after 48 h in labeled cells. In addition, the labeled cells can be manipulated by an external magnet. The magnetic resonance images showed that these labeled cells also can be measured in vivo in mice. CONCLUSIONS: PNIPAM-coated multifunctional nanoparticles showed potential for labeling cells and for tracking cells both in vitro and in vivo with the use of fluorescence and magnetic resonance. This new modality of cell tracking has the merits of simplicity and reliability.

Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells.

UNLABELLED: Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11671-009-9307-9) contains supplementary material, which is available to authorized users.

The environmental influence on the photoluminescence behavior of thiol-capped CdTe quantum dots in living cells.

Water-soluble colloidal semiconductor quantum dots (QDs) as a new class of fluorescent probes with excellent optical properties have attracted considerable attention in bioapplications. Because of the large surface-to-volume ratio of QDs, especially in the case of QDs passivated with organic ligands, the photoluminescence (PL) of QDs is sensitive to the environmental conditions. Generally, the PL behavior of intracellular QDs is more complicated than that in solutions due to the complex cellular environment. In this review, the environmental influence on the PL behavior of intracellular thiol-capped CdTe QDs, such as the time-dependent spectral blueshift, photobleaching and lifetime shortening, is introduced and its mechanisms are discussed.

Thiol-capped CdTe quantum dots with two-photon excitation for imaging high autofluorescence background living cells.

To effectively image living cells with quantum dots (QDs), particularly for those cells containing high content of native fluorophores, the two-photon excitation (TPE) with a femto-second 800 nm laser was employed and compared with the single-photon excitations (SPE) of 405 nm and 488 nm in BY-2 Tobacco (BY-2-T) and human hepatocellular carcinoma (QGY) cells, respectively. The 405 nm SPE produced the bright photoluminescence (PL) signals of cellular QDs but also induced a strong autofluorescence(AF) from the native fluorophores like flavins in cells. The AF occupied about 30% and 13% of the total signals detected in QD imaging channel in the BY-2-T and QGY cells, respectively. With the excitation of 488 nm SPE, the PL signals were lower than those excited with the 405 nm SPE, although the AF signals were also reduced. The 800 nm TPE generated the best PL images of intracellular QDs with the highest signal ratio of PL to AF, because the two-photon absorption cross section of QDs is much higher than that of the native fluorophores. By means of the TPE, the reliable cellular imaging with QDs, even for the cells having the high AF background, can be achieved.

Light distribution in the erythrocyte under laser irradiation: a finite-difference time-domain calculation.

In medical applications of low power laser irradiations, safety is one of the most concerning problems since the light focused by the biological object itself may cause damage of living organisms. The light distributions in an erythrocyte with the shape of native biconcave, oblate spheroid, or disk sphere under the irradiation of a plane light of 632.8 nm were studied with a numerical calculation method of finite-difference time domain. The focusing effect by either the biconcave erythrocyte, oblate spheroid, or disk sphere erythrocyte was found to be so remarkable that the light intensities at the focused areas close to the erythrocyte membrane were about 10 times higher than that of the incident light when the light irradiated along the erythrocyte plane. This focusing effect became weak and even disappeared when the irradiation direction deviated from the erythrocyte plane for more than an angle of 15 degrees. Because the highest light intensity in the erythrocyte can be about one order of magnitude higher than that of the incident light, this factor should be taken into account for laser safety in medical applications.

Fluorescence detection of protoporphyrin IX in living cells: a comparative study on single- and two-photon excitation.

Photodynamic therapy (PDT) involves a combination of a lesion-localizing photosensitizer with light and has been established as a new modality for some medical indications. Much evidence has shown the correlation between subcellular localization of a photosensitizer with its photodynamic efficiency. However, the fluorescence of most photosensitizers in cells is weak and easily photobleached. We compare the effect of single-photon excitation (SPE) with that of two-photon excitation (TPE) on fluorescence detection of protoporphyrin IX (PpIX), a potent photosensitizer, in the PLC hepatoma cells in vitro. By using laser scanning confocal fluorescence microscopy, both fluorescence images and spectra of intracellular PpIX are studied with SPE of 405- and 488-nm lasers, and TPE of 800-nm femtosecond laser. The 405-nm laser is more efficient at exciting PpIX fluorescence than the 488-nm laser, but causes a considerable photobleaching of the PpIX fluorescence and induces weak autofluorescence signals of native flavins in the cells as well. The 800-nm TPE is found to significantly improve the quality of PpIX fluorescence images with negligible PpIX photobleaching and minimized endogenous autofluorescence, indicating the potential of 800-nm TPE for studying cellular localization of porphyrin photosensitizers for PDT.

Photochemical instability of thiol-capped CdTe quantum dots in aqueous solution and living cells: process and mechanism.

The process and mechanism of photochemical instability of thiol-capped CdTe quantum dots (QDs) in aqueous solution were experimentally studied. After laser irradiation, the corresponding Raman bands of the Cd-S bond decreased obviously, indicating bond breaking and thiol detachment from the QD surfaces. Meanwhile, a photoinduced aggregation of QDs occurred with the hydrodynamic diameter increased to hundreds of nanometers from an initial 20 nm, as detected with dynamic light scattering measurements. The bleaching of the photoluminescence of QDs under laser irradiation could be attributed to the enhanced nonradiative transfer in excited QDs caused by increased surface defects due to the losing of thiol ligands. Singlet oxygen (1O2) was involved in the photooxidation of QDs, as revealed by the inhibiting effects of 1O2 quenchers of histidine or sodium azide (NaN3) on the photobleaching of QDs. The linear relationship in Stern-Volmer measurements between the terminal product and the concentration of NaN3 demonstrated that 1O2 was the main pathway of the photobleaching in QD solutions. By comparing the photostability of QDs in C2C12 cells with and without NaN3 treatment, the photooxidation effect of 1O2 on photobleaching of cellular QDs was confirmed.

Tracking of mercury ions in living cells with a fluorescent chemodosimeter under single- or two-photon excitation.

Tracking of Hg2+ in solutions as well as in living cells was conducted with a fluorescent chemodosimeter by measuring the spectral shift of its fluorescence under single- or two-photon excitation. The spectral hypsochromic shifts of this chemodosimeter when reacting with Hg2+ were found to be about 50 nm in acetonitrile/water solutions and 32 nm in Euglena gracilis 277 living cells. This chemodosimeter shows high sensitivity and selectivity, and is not influenced by the pH values. It can signal Hg2+ in solutions down to the ppb range under either single-photon excitation (SPE) at 405 nm or two-photon excitation (TPE) at 800 nm. However, with low cellular chemodosimeter concentrations, the SPE spectra were disturbed by the auto-fluorescence from the native fluorophore in the cell, while the TPE spectra were still of high quality since the two-photon absorption cross section of this chemodosimeter is much larger than that of the native fluorophores in the cell.