The Herbal Medicine Suanzaoren (Ziziphi Spinosae Semen) for Sleep Quality Improvements: A Systematic Review and Meta-analysis.

BACKGROUND: Sleep disturbances are common and bothersome among cancer and noncancer populations. Suanzaoren (Ziziphi Spinosae Semen) is commonly used to improve sleep, yet its efficacy and safety are unclear. METHODS: We systematically searched PubMed, Cochrane Library, and EMBASE from inception through October 5, 2021, to identify randomized trials of Suanzaoren. We included randomized trials comparing Suanzaoren to placebo, medications, cognitive behavioral therapy (CBT), or usual care for improving sleep outcomes in cancer and noncancer patients with insomnia or sleep disturbance. We performed a risk of bias analysis following Cochrane guidelines. Depending on heterogeneity, we pooled studies with similar comparators using fixed- and random-effects models. RESULTS: We included participants with insomnia disorder (N = 785) or sleep disturbance (N = 120) from 9 trials. Compared with placebo, Suanzaoren led to significant subjective sleep quality improvements in participants with insomnia and patients with sleep disturbance combined (standard mean difference -0.58, 95% CI -1.04, -0.11; P < .01); Compared with benzodiazepines or CBT, Suanzaoren was associated with a significant decrease in insomnia severity (mean difference -2.68 points, 95% CI -5.50, -0.22; P = .03) at 4 weeks in the general population and cancer patients. The long-term effects of Suanzaoren were mixed among trials. Suanzaoren did not increase the incidence of major adverse events. The placebo-controlled studies had a low risk of bias. CONCLUSION: Suanzaoren is associated with short-term patient-reported sleep quality improvements among individuals with insomnia or sleep disturbance. Due to the small sample size and variable study quality, the clinical benefits and harms of Suanzaoren, particularly in the long term, should be further assessed in a sufficiently powered randomized trial. REGISTRATION: PROSPERO CRD42021281943.

SARS-CoV-2 nsp5 Exhibits Stronger Catalytic Activity and Interferon Antagonism than Its SARS-CoV Ortholog.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an enormous threat to economic activity and public health worldwide. Previous studies have shown that the nonstructural protein 5 (nsp5, also called 3C-like protease) of alpha- and deltacoronaviruses cleaves Q231 of the NF-κB essential modulator (NEMO), a key kinase in the RIG-I-like receptor pathway, to inhibit type I interferon (IFN) production. In this study, we found that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleaved NEMO at multiple sites (E152, Q205, and Q231). Notably, SARS-CoV-2 nsp5 exhibited a stronger ability to cleave NEMO than SARS-CoV nsp5. Sequence and structural alignments suggested that an S/A polymorphism at position 46 of nsp5 in SARS-CoV versus SARS-CoV-2 may be responsible for this difference. Mutagenesis experiments showed that SARS-CoV-2 nsp5 (S46A) exhibited poorer cleavage of NEMO than SARS-CoV-2 nsp5 wild type (WT), while SARS-CoV nsp5 (A46S) showed enhanced NEMO cleavage compared with the WT protein. Purified recombinant SARS-CoV-2 nsp5 WT and SARS-CoV nsp5 (A46S) proteins exhibited higher hydrolysis efficiencies than SARS-CoV-2 nsp5 (S46A) and SARS-CoV nsp5 WT proteins in vitro. Furthermore, SARS-CoV-2 nsp5 exhibited stronger inhibition of Sendai virus (SEV)-induced interferon beta (IFN-ß) production than SARS-CoV-2 nsp5 (S46A), while introduction of the A46S substitution in SARS-CoV nsp5 enhanced suppression of SEV-induced IFN-ß production. Taken together, these data show that S46 is associated with the catalytic activity and IFN antagonism by SARS-CoV-2 nsp5. IMPORTANCE The nsp5-encoded 3C-like protease is the main coronavirus protease, playing a vital role in viral replication and immune evasion by cleaving viral polyproteins and host immune-related molecules. We showed that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleave the NEMO at multiple sites (E152, Q205, and Q231). This specificity differs from NEMO cleavage by alpha- and deltacoronaviruses, demonstrating the distinct substrate recognition of SARS-CoV-2 and SARS-CoV nsp5. Compared with SARS-CoV nsp5, SARS-CoV-2 nsp5 encodes S instead of A at position 46. This substitution is associated with stronger catalytic activity, enhanced cleavage of NEMO, and increased interferon antagonism of SARS-CoV-2 nsp5. These data provide new insights into the pathogenesis and transmission of SARS-CoV-2.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease.

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Porcine Deltacoronavirus nsp5 Cleaves DCP1A To Decrease Its Antiviral Activity.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The nonstructural protein nsp5, also called 3C-like protease, is responsible for processing viral polyprotein precursors in coronavirus (CoV) replication. Previous studies have shown that PDCoV nsp5 cleaves the NF-κB essential modulator and the signal transducer and activator of transcription 2 to disrupt interferon (IFN) production and signaling, respectively. Whether PDCoV nsp5 also cleaves IFN-stimulated genes (ISGs), IFN-induced antiviral effector molecules, remains unclear. In this study, we screened 14 classical ISGs and found that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a (pDCP1A) through its protease activity. Similar cleavage of endogenous pDCP1A was also observed in PDCoV-infected cells. PDCoV nsp5 cleaved pDCP1A at glutamine 343 (Q343), and the cleaved pDCP1A fragments, pDCP1A1-343 and pDCP1A344-580, were unable to inhibit PDCoV infection. Mutant pDCP1A-Q343A, which resists nsp5-mediated cleavage, exhibited a stronger ability to inhibit PDCoV infection than wild-type pDCP1A. Interestingly, the Q343 cleavage site is highly conserved in DCP1A homologs from other mammalian species. Further analyses demonstrated that nsp5 encoded by seven tested CoVs that can infect human or pig also cleaved pDCP1A and human DCP1A, suggesting that DCP1A may be the common target for cleavage by nsp5 of mammalian CoVs.IMPORTANCE Interferon (IFN)-stimulated gene (ISG) induction through IFN signaling is important to create an antiviral state and usually directly inhibits virus infection. The present study first demonstrated that PDCoV nsp5 can cleave mRNA-decapping enzyme 1a (DCP1A) to attenuate its antiviral activity. Furthermore, cleaving DCP1A is a common characteristic of nsp5 proteins from different coronaviruses (CoVs), which represents a common immune evasion mechanism of CoVs. Previous evidence showed that CoV nsp5 cleaves the NF-κB essential modulator and signal transducer and activator of transcription 2. Taken together, CoV nsp5 is a potent IFN antagonist because it can simultaneously target different aspects of the host IFN system, including IFN production and signaling and effector molecules.

Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.

Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CLpros) from CoVs (also called main proteases) are essential for viral replication and are also involved in polyprotein cleavage and immune regulation, making them attractive and effective targets for the development of antiviral drugs. Herein, the 3CLpro from the porcine epidemic diarrhea virus, an enteropathogenic CoV, was used as a model to identify novel crucial residues for enzyme activity. First, we established a rapid, sensitive, and efficient luciferase-based biosensor to monitor the activity of PDEV 3CLproin vivo. Using this luciferase biosensor, along with confirming the well-known catalytic residues (His41 and Cys144), we identified 4 novel proteolytically inactivated mutants of PDEV 3CLpro, which was also confirmed in mammalian cells by biochemical experiments. Our molecular dynamics (MD) simulations showed that the hydrogen bonding interactions occurring within and outside of the protease's active site and the dynamic fluctuations of the substrate, especially the van der Waals contacts, were drastically altered, a situation related to the loss of 3CLpro activity. These data suggest that changing the intermolecular dynamics in protein-substrate complexes eliminates the mechanism underlying the protease activity. The discovery of novel crucial residues for enzyme activity in the binding pocket could potentially provide more druggable sites for the design of protease inhibitors. In addition, our in-depth study of the dynamic substrate's envelope model using MD simulations is an approach that could augment the discovery of new inhibitors against 3CLpro in CoVs and other viral 3C proteases.-Zhou, J., Fang, L., Yang, Z., Xu, S., Lv, M., Sun, Z., Chen, J., Wang, D., Gao, J., Xiao, S. Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.

Assessing activity of Hepatitis A virus 3C protease using a cyclized luciferase-based biosensor.

Hepatitis A is an acute infection caused by Hepatitis A virus (HAV), which is widely distributed throughout the world. The HAV 3C cysteine protease (3Cpro), an important nonstructural protein, is responsible for most cleavage within the viral polyprotein and is critical for the processes of viral replication. Our group has previously demonstrated that HAV 3Cpro cleaves human NF-κB essential modulator (NEMO), a kinase required in interferon signaling. Based on this finding, we generated four luciferase-based biosensors containing the NEMO sequence (PVLKAQ↓ADIYKA) that is cleaved by HAV 3Cpro and/or the Nostoc punctiforme DnaE intein, to monitor the activity of HAV 3Cpro in human embryonic kidney cells (HEK-293T). Western blotting showed that HAV 3Cpro recognized and cleaved the NEMO cleavage sequence incorporated in the four biosensors, whereas only one cyclized luciferase-based biosensor (233-DnaE-HAV, 233DH) showed a measurable and reliable increase in firefly luciferase activity, with very low background, in the presence of HAV 3Cpro. With this biosensor (233DH), we monitored HAV 3Cpro activity in HEK-293T cells, and tested it against a catalytically deficient mutant HAV 3Cpro and other virus-encoded proteases. The results showed that the activity of this luciferase biosensor is specifically dependent on HAV 3Cpro. Collectively, our data demonstrate that the luciferase biosensor developed here might provide a rapid, sensitive, and efficient evaluation of HAV 3Cpro activity, and should extend our better understanding of the biological relevance of HAV 3Cpro.

Porcine deltacoronavirus nsp5 inhibits interferon-ß production through the cleavage of NEMO.

Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-ß), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-ß production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses.

Laser beam controlled drug release from Ce6-gold nanorod composites in living cells: a FLIM study.

A new method to image drug release from drug-nanoparticle composites in living cells was established. The composites of silica coated gold nanorods (AuNR@SiO2) and chlorine e6 (Ce6) photosensitizers (AuNR@SiO2-Ce6) were formed by electrostatic force with a Ce6 loading efficiency of 80%. The strong resonance absorptions of AuNR@SiO2-Ce6 in the near-infrared (NIR) region enabled the effective release of Ce6 from AuNR@SiO2-Ce6 by 780 nm CW laser irradiation. The 780 nm laser beam was applied to not only control the releasing amount of Ce6 from cellular AuNR@SiO2-Ce6 by adjusting the irradiation dose (time), but also to spatially confine the Ce6 release in cells by focusing the laser beam on the target sites. Furthermore, the fluorescence lifetime of Ce6 was found to change drastically from 0.9 ns in the AuNR@SiO2-Ce6 complex to 6 ns after release, and therefore fluorescence lifetime imaging microscopy (FLIM) was introduced to image the photo-induced Ce6 release in living cells. Finally, the controllable killing effect of photodynamic cancer therapy (PDT) using AuNR@SiO2-Ce6 was demonstrated by changing the released amount of Ce6, which indicates that AuNR@SiO2-Ce6 is promising for targeted tumour PDT.

The aggregation enhanced photoluminescence of gold nanorods in aqueous solutions.

The photoluminescence (PL) properties of single gold nanorod (AuNR) under one-photon excitation (OPE) have been reported recently. In this work, the PL of AuNRs in aqueous solutions were studied with OPE of 514 or 633 nm to characterize the emissions of transverse and longitudinal surface Plasmon resonance (TSPR and LSPR) bands, because the AuNRs aqueous solution was frequently used in bio-medical applications. We found that under 514 nm OPE the TSPR emissions of four groups of AuNRs with different aspect ratios in aqueous solutions were all strong dominating the PL emission with the quantum yield (QY) of 10(-4), which is at least three orders of magnitude higher than that of single AuNR. We further found that the aggregate was the basic form of AuNRs in aqueous solution and living cells, measured by the elastic light scattering and transmission electron microscopy measurements. The Plasmon coupling particularly the TSPR coupling between the neighbored AuNRs in aggregates enhanced the PL and increased the QY, because the conjugation of the rod side to side was a main aggregate mode. Under 633 nm OPE, only LSPR emissions of AuNRs aqueous solutions occurred with the QY level of 10(-5) which is very similar to that of singe AuNR, because of the negligible LSPR coupling.

Carbon nanodots featuring efficient FRET for two-photon photodynamic cancer therapy with a low fs laser power density.

The 5,10,15,20-tetrakis(1-methyl 4-pyridinio) porphyrins (TMPyP), a photosensitizer used for photodynamic therapy of cancers (PDT), were linked to carbon dots (CDots) to form the conjugates of CDot-TMPyP by the electrostatic force. The 415 nm emission band of CDots was well overlapped with the absorption band of TMPyP, so that the Cdots in conjugates can work as donor to transfer the energy to TMPyP moiety by fluorescence resonance energy transfer (FRET) with an FRET efficiency of 45%, determined by the fluorescence lifetime change between the free CDots and conjugated CDots. The two-photon absorption cross section (TPACS) of TMPyP is as low as 110 GM and the TMPyP thus be not suitable for two-photon PDT. Whereas the CDots have high TPACS, and their TPACS are excitation wavelength dependent with the maximum value of 15000 GM at 700 nm. Therefore, the conjugates of CDot-TMPyP were explored for two-photon excitation (TPE) PDT. The two-photon image of CDot-TMPyP in Hela cells was clearly seen under the excitation of a 700 nm femto-second (fs) laser. The singlet oxygen production of CDot-TMPyP was also much higher than that of TMPyP alone under TPE of a 700 nm fs laser. The in vitro PDT killing was further achieved with CDot-TMPyP by TPE of the 700 nm fs laser. Particularly herein the low power density of fs laser from unfocused laser beam was successfully used to carry out the TPE PDT, because of the high TPACS of CDots. These results demonstrate that the CDot-TMPyP conjugates are promising for TPE PDT and needed to investigate further.

Two orders of magnitude fluorescence enhancement of aluminum phthalocyanines by gold nanocubes: a remarkable improvement for cancer cell imaging and detection.

The metal-enhanced fluorescence (MEF) by metal nanoparticles is a useful technique for fluorescence detections in biological systems. The MEF effects with gold nanorods (AuNRs) and nanocubes (AuNCs) for fluorescence enhancements of sulfonated aluminum phthalocyanine (AlPcS), a commonly used and clinical approved photosensitizer for photodynamic therapy of cancers, were studied in this work. For the AuNRs which have the low aspect ratios with the corresponding longitudinal surface plasma resonance (LSPR) band in the region of 600-750 nm, the fluorescence quenching of conjugated AlPcS was found. Whereas for the AuNRs that have the LSPR bands of 800-900 nm, the MEF of AlPcS was obtained with the enhancing factor of 2-6 times, respectively. Using AuNCs, a great enhancement of AlPcS fluorescence was achieved with an enhancing factor of 150 times. Using two cancer cell lines as in vitro models, an outstanding fluorescence enhancement of AlPcS-AuNCs conjugates in cells, relative to AlPcS alone, was obtained under one-photon excitation (OPE) of 405 nm. Moreover, the bright fluorescence image of AlPcS-AuNCs in cells was also achieved under the two-photon excitation (TPE) of an 800 nm femtosecond laser. The high-quality cell imaging with either OPE or TPE demonstrated the potential of AlPcS-AuNCs in cancer cell detections.

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.

Conjugates of folic acids with zinc aminophthalocyanine for cancer cell targeting and photodynamic therapy by one-photon and two-photon excitations.

To improve the photodynamic detection and therapy of cancers (PDT), folic acid (FA) was conjugated with zinc tetraaminophthalocyanine (ZnaPc) to form ZnaPc-FA. The uptake efficiency of ZnaPc-FA to a FR-positive (folate receptor overexpressed) KB cell line (human nasopharyngeal epidermal carcinoma) was much higher than that of ZnaPc demonstrating an enhanced binding ability of ZnaPc-FA to KB cells. When KB cells were pretreated with free FA followed by incubation of ZnaPc-FA, the high uptake rate of ZnaPc-FA disappeared which demonstrated the special binding function of the FA terminal of ZnaPc-FA on KB cells. The confocal fluorescence images further showed that the affinity of ZnaPc-FA to FR-negative A549 cells (human lung epithelial carcinoma cancer cells) was very low, confirming that ZnaPc-FA can only target FR-positive cancers. The two-photon absorption cross-section of ZnaPc-FA was also higher than that of sulfonated aluminum phthalocyanine (AlPcS), an approved PS for clinical applications. With a 780 nm femto-second (fs) laser, the fluorescence image of ZnaPc-FA in KB cells under two-photon excitation (TPE) can be clearly seen, and the two-photon induced singlet oxygen in ZnaPc-FA solution was found to be proportional to the irradiation dose of the fs laser. The PDT damaging effect of ZnaPc-FA on KB cells was much effective relative to AlPcS under common one-photon excitation, and the killing efficacy of ZnaPc-FA under TPE was 10-fold higher than that of AlPcS. These results suggest that ZnaPc-FA is a promising candidate for PDT improvements and particularly for TPE PDT.

Single-step noninjection synthesis of highly luminescent water soluble Cu+ doped CdS quantum dots: application as bio-imaging agents.

Novel highly luminescent Cu(+) doped CdS quantum dots (QDs) were directly synthesized in aqueous phase through a facile single-step noninjection method. Due to their bright red fluorescence, ultrasmall size, and good biocompatibility, as-prepared CdS:Cu(+) QDs have potential as probes in bio-imaging.

The use of femto-second lasers to trigger powerful explosions of gold nanorods to destroy cancer cells.

Gold nanorods (AuNRs) with an aspect ratio of 3-4 exhibit large cross sections for single and multi photon light absorption processes in the near infrared region due to surface plasmon resonances. 800 nm laser pulses with the 150 fsec pulse duration (fs laser) can trigger explosions of AuNRs. The fs laser pulses at 20 W/mm(2) equivalent continuous wave (cw) power density blasted AuNRs in QGY human carcinoma cells as confirmed using transmission electron microscopy, while a cw laser at the same power density and dose did not. Cell survival studies further demonstrated that the cw laser at a dose of 15 J/mm(2) resulted in the death of 15% of AuNRs-loaded cells, probably due to a photothermal effect, while the fs laser at only 1.5 J/mm(2) killed more than 90% of AuNRs-loaded cells, indicating that the fs laser-triggered explosions of intracellular AuNRs are powerful enough to instantaneously kill tumour cells.

The conjugates of gold nanorods and chlorin e6 for enhancing the fluorescence detection and photodynamic therapy of cancers.

Gold nanorods (AuNRs) were conjugated with chlorin e6 (Ce6), a commonly used photosensitizer, to form AuNRs-Ce6 by electrostatic binding. Due to the strong surface plasmon resonance coupling, the fluorescence of conjugated Ce6 was enhanced 3-fold and the production of singlet oxygen was increased 1.4-fold. AuNRs-Ce6 were taken up by the HeLa and KB cell lines more easily than free Ce6, enhancing the intracellular delivery of Ce6. The increased cellular amount of Ce6 leads to a 3-fold more efficient photodynamic killing of these two cell lines. This demonstrates the potential of this approach to improve photodynamic detection and therapy of cancers.

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.

Femto-second laser beam with a low power density achieved a two-photon photodynamic cancer therapy with quantum dots.

Focusing the femto-second (fs) laser beam on the target was the usual way to carry out a two-photon excitation (TPE) in previous photodynamic therapy (PDT) studies. However, focusing the laser deep inside the tissues of the tumor is unrealistic due to tissue scattering, so that this focusing manner seems unfit for practical TPE PDT applications. In this work, we prepared a conjugate of quantum dots (QDs) and sulfonated aluminum phthalocyanine (AlPcS) for TPE PDT, because QDs have a very high two-photon absorption cross section (TPACS) and thus QDs can be excited by an unfocused 800 nm fs laser beam with a low power density and then transfer the energy to a conjugated AlPcS via fluorescence resonance energy transfer (FRET). The FRET efficiency of the QD-AlPcS conjugate in water was as high as 90%, and the FRET process of the cellular QD-AlPcS was also observed in both KB and HeLa cells under TPE of a 800 nm fs laser. The singlet oxygen (1O2) products were produced by the QD-AlPcS under the TPE of the unfocused 800 nm fs laser via FRET mediated PDT. Moreover, the QD-AlPcS can effectively destroy these cancer cells under the irradiation of the 800 nm unfocused fs laser beam with a power density of 92 mW mm-2, and particularly the killing efficiency of the TPE is comparable to that of the commonly used one-photon excitation (OPE) at visible wavelengths. These results highlight the potential of QD-AlPcS for TPE PDT with a near infrared wavelength.

Quantum dot-aluminum phthalocyanine conjugates perform photodynamic reactions to kill cancer cells via fluorescence resonance energy transfer.

Sulfonated aluminum phthalocyanines (AlPcSs), commonly used photosensitizers for photodynamic therapy of cancers (PDT), were conjugated with amine-dihydrolipoic acid-coated quantum dots (QDs) by electrostatic binding, achieving 70 AlPcSs per QD. The AlPcS-QD conjugates can utilize the intense light absorptions of conjugated QDs to indirectly excite AlPcSs producing singlet oxygen via fluorescence resonance energy transfer (FRET), demonstrating a new excitation model for PDT. The AlPcS-QD conjugates easily penetrated into human nasopharyngeal carcinoma cells and carried out the FRET in cells, with efficiency around 80%. Under the irradiation of a 532-nm laser, which is at the absorption region of QDs but not fit for the absorption of AlPcSs, the cellular AlPcS-QD conjugates can destroy most cancer cells via FRET-mediated PDT, showing the potential of this new strategy for PDT.

Conjugation of sulfonated aluminum phthalocyanine to doxorubicin can improve the efficacy of photodynamic cancer therapy.

Sulfonated aluminum phthalocyanine (AlPcS), a widely used photosensitizer for photodynamic therapy of cancer, was conjugated to doxorubicin (Dox), a chemotherapy drug, through electrostatic binding. The fluorescence resonance energy transfer from Dox to AlPcS showed the formation of AlPcS-Dox conjugates, as the fluorescence intensity of conjugated Dox was decreased and that of the AlPcS moiety was enhanced. This AlPcS-Dox conjugation was further confirmed by electrophoresis. The AlPcS-Dox conjugates enhanced the cellular uptake of AlPcS three times more than unconjugated AlPcS in both human hepatocellular carcinoma cell line 7701 and rat basophilic leukemia cell line. Moreover, the photodynamic killing effect of the conjugates was markedly increased as compared with that of AlPcS alone or the cytotoxicity of Dox alone, showing an enhanced effect of the AlPcS-Dox conjugates. These results indicate that the conjugation of a photosensitizer with a chemotherapy drug may improve photodynamic cancer therapy.