s2fieldsofstudy: Chemistry Biology

text:
Artemisinin Inhibits Chloroplast Electron Transport Activity: Mode of Action

Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo), behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the QB; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth.

Introduction
Nature produces a large array of bioactive materials and compounds with unexploited properties. These products have been used more often as structural leads for the discovery and development of natural product based herbicides or pesticides. Often these products have novel sites of action, and even if unsuitable for commercial use, the identification of a new target site is valuable for designing synthetic herbicides [1].
An example of such a natural product is artemisinin, a sesquiterpenoid lactone with a highly reactive endoperoxide bridge from Artemisia annua L., commonly used as a potential antimalarial drug [2]. The compound and several of its structural analogous like, artemisinic acid, arteannuin B, deoxyartemisinin, artesunic acid and sodium artelinate have been reported to possess phytotoxic potential [3][4][5][6][7][8]. It is also shown to inhibit seedling growth in a number of mono and dicotyledonous plants [3]. However, its mode of action and the mechanistic details still remain undeciphered.
Some allelochemicals have been recognized to disrupt the electron transport activity both in chloroplast and mitochondria; including alteration in their membrane architecture [9]. The mitochondrial electron transport chain in yeast has been identified as the major target of this compound as antimalarial agent [10]. Artemisinin, as argued by Li et al. [10] can disrupt the normal electron flow in yeast mitochondria by depolarizing its membrane potential and interacts with NADH-dehydrogenase complex (complex-I).
The chloroplast and mitochondria perform largely a similar kind of function; producing high energy compounds for use in other cellular metabolic activities. Many of the electron transport components between these two organelles are structurally and functionally comparable, like cytochrome system, quinone moieties, ATP-synthesizing complex etc. Further, the chloroplast lamellar system also harbors a mitochondrial homologous NADH-dehydrogenase [11], the chloroplast NADH-dehydrogenase that is engaged in respiratory function of the chloroplast (chloro-respiration).
The discovery of artemisinin interaction with the electron transport chain in yeast and malarial mitochondria systems [10,12], and considering that the process of photosynthesis, particularly the thylakoid photofunction, being highly susceptible for a large range of herbicides [13], we examined the phytotoxic effect of artemisinin on the chloroplast photoelectron transport activity both under in vitro and in vivo experimental conditions.
The results from our in vitro and in vivo studies suggest the presence of two different modes of action of the compound on photosynthetic electron transport. Artemisinin is primarily an energy transfer inhibitor of isolated thylakoid membranes. However, the in vivo studies indicate that the compound impairs the thylakoid electron flow as an inhibitor of secondary quinone (Q B ) of PSII (Photosystem II). The inhibition was determined to be caused by a yet uncharacterized artemisinin-metabolite rather than its direct interaction with thylakoid membrane. Thus, artemisinin may act as a natural prophytotoxin.

Results
Inhibitory effect of artemisinin on electron transport activity: an in vitro study To find out the potency of artemisinin as an inhibitor of thylakoid photofunction, the effect of artemisinin on electron transport rate supported by the transfer of electrons from water to FeCN (potassium ferricyanide) was examined. The extent of inhibition was assessed relative to DMSO (dimethyl sulfoxide) supplemented reaction rates as a control ( Fig. 1; trace '0'), which was identical to the rate obtained without addition of DMSO. The degree of inhibition was concentration dependent and showed the onset of a saturating tendency around 744 mM; the highest concentration tested in this report. About 65-70% inhibition of control activity was observed at this concentration of artemisinin (inset fig. 1).
The inhibitory effect of the compound was further evaluated under phosphorylating and uncoupled conditions of electron flow. Two different uncouplers known to dissipate the formation of high-energy state through different means were used in this study. The NH 4 Cl (ammonium chloride), an amine type of uncoupler is known to arrest the acidification (chemical potential, DpH) of thylakoid lumen [14] and GS (gramicidin-S) is a pore forming ionophore [15] known to collapse the electrical potential (DY) of high energy state of the membrane. In presence of NH 4 Cl or GS, the extent of artemisinin-mediated (744 mM) inhibition of basal electron transport was reduced by nearly 50% (Fig. 2, A). Addition of ADP (adenosine diphosphate) and iP (inorganic phosphate), as expected, stimulated the basal electron transport rate. However, a decline in the artemisinin-mediated inhibition to 45-50% as against 65-70% in basal conditions was observed under phosphorylating condition (Fig. 2, B). Although the phosphorylating electron transport rate was further stimulated in presence of uncouplers, the toxicity of artemisinin, however drastically reduced it to the tune of 15-20% in presence of NH 4 Cl and about 20-25% with GS (Fig. 2, B). To gain insight about the effect of artemisinin in vivo, rice plants grown under greenhouse conditions were sprayed with artemisinin as described in materials and methods. Dark adapted leaves when exposed to a strong saturating light (3000 mmol photon m 22 s 21 , red light of 650 nm) for 1 ms, the Chl. a fluorescence emission rapidly increases from an initial O level to a maximum P through transient steps, the J and I. The O-J-I-P uphill rise in fluorescence (F O , F J , F I and F P ) sequence represents the successive reduction of the electron acceptor pools of PSII [16][17][18]. We followed the notations as defined by Strasser et al. [17] to explain the redox significance of these transients. Artemisinin administration induced a small but discernible higher F J and a higher F I . The major difference noted due to artemisinin treatment, was the disappearance of the F I -F P transient (Fig. 3, A). It is known that F J is very sensitive to changes in the redox state of the PQ-pool. The F I -F P phase is correlated to the transfer of electrons through PSI as shown by DBMIB (dibromothymoquinone) treatment [19], primarily an antagonist to cytochrome b 563 but having limited effect on Q B site as well [20,21]. These alternations in fluorescence characteristics may have arisen due to reduced level of electron flow through PSI, accompanied with changes in the redox state of PQ.
A small but discernible rise in F O -F J fluorescence intensity suggests the existence of a small effect on Q A reduction in dark adapted leaves. However, a faster rise in Chl. fluorescence from F I to F P level in artemisinin treated leaves suggest a slow reduction of Q B resulting in formation of lower pool of PQH 2 (plastoquinol), which in turn alters the overall redox chemistry of Q A to Q B to PQH 2 in PSII complex. DCMU (3-(3,4-dichlorophenyl)-1,1dimethylurea) treatment that inhibits the electron flow from Q A to Q B by binding to Q B , results in the transformation of three step fluorescence rise O-J-I-P into an O-J rise only due to the accumulation of Q A 2 . Comparison of the fluorescence induction curves of control and artemisinin treated leaves showed similar sensitivity to DCMU (Fig. 3, B). These results indicate that the electron flow from water to Q A is insensitive to artemisinin toxicity and the compound renders its toxic affect in the span of electron flow beyond Q A . The room temperature (25uC) and 77 K fluorescence emission spectral analysis (See Fig. S1, Supporting Information) also suggest that the compound has no effect on the energy transfer from core antenna to PSII reaction centre Chl. [22]. Therefore, to decipher the effect of the compound on the span of electron flow beyond Q A , we studied the slow Chl. a fluorescence transient, which gives the signature for various photosynthetic events [23]. Chl. a slow fluorescence kinetics The control rice leaves showed multiphasic Chl. a fluorescence induction. Artemisinin treatment to leaves, however, induced an alteration in the relative amplitude of these transients (Fig. 4). A major difference was marked in the quenching of fluorescence from F p to F t level. The rise in fluorescence at F p , in artemisinin treated leaves is followed by a decline to the't' level but having a much higher amplitude than the control leaf sample. This characteristic phenomenon of alteration in Chl. a transient by artemisinin was also observed in spinach and pea (data not shown).
The F p to F t decay is manifestly far more complex because of the participation of multiple reactions to quench the fluorescence. The principal causes of this decay relate to gradual re-oxidation of Q A by PSI (photochemical quenching, the qP), the energization of the thylakoid membrane due to proton translocation (non-photochemical or energy dependent quenching, the qE) and possibly the state transition (qT) [24]. The normal deviation of fluorescence decay from F p to F t in artemisinin treated leaves as compared to control leave suggest an interruption of electron flow in a site(s) beyond the Q A site of electron acceptance.
The estimated Chl. concentration in both control and artemisinin treated leaves were comparable suggesting that artemisinin treatment had no affect on the Chl. pigment synthesis (See Fig. S2, Supporting Information). We, therefore, further analyzed the electron flow in thylakoid membranes, isolated from artemisinin treated leaves by dissecting the electron transport chain into smaller segments with the use of site specific artificial electron acceptors, donors and inhibitors [25,26].

Identification of the target site of artemisinin in the electron transport chain
Thylakoids were isolated from leaves of control and artemisinin sprayed rice plants as described in materials and methods. The FeCN supported whole chain electron transport rate in chloroplasts of artemisinin treated leaves showed a substantially low activity as compared to the rate obtained in control leaf chloroplasts. From various batches of thylakoid preparations, it was inferred that the electron transport rate in chloroplast of treated leaves was reduced by 80-85% as compared to control (Fig. 5, A). Similar extent of inhibition was noted, when FeCN was replaced with MV (methylviologen), another end terminal electron acceptor, which also intercepts electron from the reducing side of PSI (data not shown). Of the total reduction in full chain electron transport, a major reduction in rate of about 65-70% was accounted for pBQ (para-benzoquinone) supported PSII catalyzed electron transport reaction (Fig. 5, B). The PSI catalyzed electron flow from DCIPH 2 (reduced-dichlorophenolindophenol) to MV was reduced by 10-15% in artemisinin treated leaves as compared to control rate (Fig. 5, C).
Unlike the in vitro situation, the inhibitory affect was persistent to a comparable extent both in loosely coupled (basal) and chemically uncoupled thylakoids (Fig. 6), which specifically suggests that, the compound acts primarily as an electron transport inhibitor rather than as an energy transfer inhibitor.
The lipophilic electron acceptor pBQ is known to intercept electrons after Q A in a DCMU sensitive manner, mostly from the plastoquinol pool (PQH 2 ) and also from Q B (bound form) site of PSII complex [27,28]. These sites can be resolved by analyzing the kinetic parameters of electron transport reaction (K m and V max ), the reciprocal plot of rate (O 2 -evolving activity) against increasing concentrations of pBQ ( Fig. 7 A, B and C). Similar to an earlier report [27], the plastoquinol pool (PQH 2 ) with pBQ maintained a high affinity (low K m ) with low V max against the Q B that possess a low affinity (high K m ) with high V max value in thylakoids prepared from control leaf (Fig. 7, B). However, artemisinin treatment alters the relative pBQ binding affinity (K m ) and the V max of both these sites in an opposite manner (Fig. 7, C) with a significant reduction in their respective V max amplitude (see the inset table in Fig. 7). The electron transport reaction was further examined as a function of increasing concentration of DQ (duroquinone) that supports PSII catalyzed reaction by intercepting electron only from PQH 2 pool [27,29]. Thylakoids prepared from artemisinin treated leaves is found to support a substantially low rate of electron flow from water oxidation complex to DQ as compared to thylakoids of control leaf (Fig. 8). The affinity (K m ) of DQ for reduced PQ did not change in the thylakoids from the artemisinin treated plants (104-106 mM) despite that, the electron transport rate was 5 times higher in the absence of the sesquiterpenoid lactone (Fig. 8 inset). This indicates that the available pool size of the substrate (PQH 2 ) in artemisinin treated leaves is significantly low as compared to control; a condition that might have arisen as or due to inability of conversion of bound quinone to free pool of reduced PQ.
Based on the in vitro and in vivo experimental conditions, two contrasting situations about the interaction of artemisinin with thylakoid photofunction were observed. These two conditions also differed significantly on the required concentration of the compound to exhibit its maximum inhibitory affect. The effective concentration under in vivo condition (see materials and methods) may still be at a lower side considering the penetration efficiency of the compound, spray drift and also the leaf density per plant. However, all our explanations are based on the external sprayed quantity of the compound. Such large discrepancies in artemisinin concentration dependency and differential mode of action under in vitro (energy transfer inhibitor) and in vivo (electron transport inhibitor) experimental conditions suggest that the inhibition of electron flow under in vivo experimental condition may not be a direct consequence of interaction of artemisinin as was the case for in vitro situation. Therefore, it is presumed at this stage, that the in vivo damage to thylakoid function is possibly routed through some artemisinin-metabolite, rather than direct interaction of the compound per se. Evidences for the formation of a putative artemisininmetabolite In search of an artemisinin-metabolite that could be of importance in instituting the in vivo inhibitory affect, we examined the electron transport activity in freshly prepared thylakoids following its incubation with the supernatant prepared from artemisinin treated leaves (see materials and methods).
An inhibition in FeCN supported electron flow was clearly evident in thylakoids incubated with the supernatant obtained from artemisinin treated leaves compared to its counterpart following incubation (See Fig. S3, Supporting Information).  The biological activity of the supernatant was checked by fold diluting the supernatant. It was marked that the extent of inhibition was reduced upon dilution of the extract (See Fig. S4, Supporting Information). Further, the supernatant from the 50006g centrifugation was ultracentrifuged as described in materials and methods section. Electron transport rate was analysed by incubating control chloroplasts with both fractions. Samples incubated with the supernatant showed no change in the rates (Fig. 9, A), while about 40-45% decline in electron transport rate was evident in samples incubated with the re-suspended pellet obtained from artemisinin treated leaves (Fig. 9, B).

Discussion
Much interest is drawn towards the compound, artemisinin, due to its herbicidal activity [3, 4, 30 and 31] in addition to its medicinal use. Although the compound has earlier been tested for growth regulating activity, there is no report on the structural moieties essential for its bioactivity and mechanism of action. We report here the biological activity of artemisinin tested both under in vitro and in vivo conditions using thylakoid/chloroplast photofunction as assay system. Artemisinin inhibited electron transport rate when added to isolated thylakoids. The assessment of in vitro experimental results suggest that artemisinin is effective in developing its maximal inhibitory affect in loosely coupled (basal) followed by fully coupled (phosphorylating) thylakoid electron transport reactions and has a significant low reactivity in chemically uncoupled thylakoids. Therefore, under in vitro system the compound behaves more as an energy transfer inhibitor [25] than as a potent inhibitor of electron transport. However, the apparent requirement of a high concentration of artemisinin (744 mM) does not explain the difference in inhibition (of about 65-70%) as shown in this report with that of previous reports on growth regulatory effects in plants as well as in yeast cells. At 33 mM artemisinin, inhibited the germination of lettuce and annual wormwood, and growth of roots and shoots in lettuce, redroot pigweed, pitted morning glory, annual wormwood and common purslane [6]. Also at a concentration of 80 mM, artemisinin arrests the growth of yeast cells [10]. An attempt was, therefore, made here to reassess the effect of artemisinin on the photoelectron transport activity in thylakoids isolated from leaves of plants externally treated with the compound.
In order to elucidate the relationship between the concentration and biological activity of the compound, it was necessary to use a test system that required minimal amount of the compound and still showed biological activity. Therefore, we sprayed rice plants with artemisinin at concentrations 1.86 mmol of artemisinin/plant (see materials and methods) and reassessed its toxicity. Chl. a fluorescence transient was measured to evaluate the effect of artemisinin on the photochemical efficiency of PSII. The O-J-I-P transient depicts the rate of reduction kinetics of various components of PSII. Addition of artemisinin resulted in the disappearance of F I -F P transient which has been interpreted as the result of slower reduction of Q B due to presence of a smaller plastoquinol pool in comparison to the control. A small but relative increase in variable fluorescence as a function of artemisinin was indicative of a loss in Q A 2 re-oxidation capacity. Further, the presence of comparable DCMU mediated induction of fluorescence emission suggest that artemisinin has no affect on the span of electron flow from water to Q A . The above conclusion was further supported from the slow fluorescence transient obtained following in vivo exposure of rice leaves to artemisinin. The ground level fluorescence (F O ) and the maximal fluorescence (F m ) yield did not change appreciably, but there were pronounced changes in the relative decay from F P to F t . This deviation represents the slow rate of electron flow from Q B to intersystem electron transport components.
To investigate the contribution of artemisinin towards the inhibition of electron flow, thylakoids were isolated from leaves of control and artemisinin sprayed plants and evaluated for their respective electron transport rate in the presence of site specific electron donors and acceptors, and also uncouplers. This sesquiterpene lactone was found to behave as a strong inhibitor of electron flow, potentially a specific inhibitor to Q B functioning. The Q B quinione moiety in thylakoid membrane remains housed in a 32 kDa peptide (D1 polypeptide) of PSII core complex, which otherwise is also known as the herbicidal binding protein. Many synthetic products like DCMU, atrazine and also natural compounds such as fisherellin, grandinol, sorgoleone etc. [32,1] have been shown to interact with this protein impairing the electron flow beyond Q A . The detailed mechanism of interaction at molecular level, either by forming an inhibitory complex with the protein (chemical modification) or with the quinone moiety (steric hindrance) for some of these compounds has also been deciphered [32].
Both the in vitro and in vivo scenarios presented different outcomes in our study, thus emphasizing that the mode of action of the compound in plant system (in vivo) is not a direct interaction of the compound with the thylakoid electron transport event, but illustrates the presence of a putative artemisinin-inhibitorymetabolite. The metabolite on homogenization comes out of the cellular component and is present in the supernatant. The supernatant was tested for inhibition in normal chloroplasts. Results from incubation studies show that the supernatant obtained from homogenization of artemisinin treated leaves is capable of inhibiting electron flow to about 65-70% (See Fig. S3, Supporting Information). Further, the metabolite is confirmed to be particulate in nature as ultracentrifugation data reveal its presence in the pellet. The pelleted fraction is mostly comprised of cell organelles like mitochondria, golgi and other membrane structures, but less of nuclei and plastids. Here, further experiments are needed to exactly decipher the organelle specific location and association of the artemisinin-metabolite. However, the compound does get degraded in the cell free extract, as storage of the supernatant leads to decline in its inhibitory effect (chloroplasts incubated with supernatant stored for 24 h at 4uC does not show any inhibition). We also observed that long term administration of artemisinin leads to the death of plant (See Fig.  S5, Supporting Information).
For artemisinin, as described in this report, we have so far been successful in only identifying the mode (site) of its action on the photosynthetic electron transport. The significance of addressing the action of a natural phytotoxin often lies in the novelty of their site and mechanism of action. Although, as mentioned earlier their use may not be commercially viable, the identification of a new target site is always valuable in the design of new synthetic herbicides. Therefore, artemisinin could be selected as a suitable lead for development as an effective herbicide and/or for the design of new biodegradable agent with agrochemical applications [1]. However in absence of the structural features of the proposed putative artemisinin-inhibitory-metabolite, the intrinsic mechanism of its interaction either with the protein or with the Q B moiety of PSII reaction centre remain to be worked out to fully understand the mechanism of action of artemisinin.

Materials
Purified artemisinin was obtained as gift from Ipca Laboratories Ltd., (India). DMSO was used to make stock solution of artemisinin. The electron acceptors like FeCN, DCIP, MV, DQ, pBQ were obtained from Sigma. The electron transport inhibitor DCMU from Sigma was recrystallized from 95% ethanolic solution. The buffers used were also procured from Sigma. Other chemicals used in the experiment were of analytical grade.

Plant materials
Fresh spinach (Beta palonga) was obtained from local market. Rice (Oryza sativa var. Safari) plant was grown in soil under greenhouse conditions of light, temperature and humidity. The leaves were thoroughly washed in distilled water before using them for experimental purpose.

Plant growth and artemisinin treatment
Twenty one day old rice plants were sprayed with artemisinin twice at a 24 h interval. One mililiter of 74.4 mM artemisinin in 1% (V/V) DMSO, 0.1% (V/V) TWEEN-20 was diluted 100 fold with water and used to treat 40 plants (,1.86 mmol of artemisinin/plant). Control plants were sprayed with the same solution without artemisinin. The 4 th (quaternary) leaf was harvested 20 h after the 2 nd spray and used for all experiments. The individual effect of the surfactant and DMSO-plus-surfactant was first evaluated against water control. Since no effect was obtained with these variations, we have routinely used the DMSO plus the surfactant sprayed leaves as control against artemisinin treated leaves and in figures the same has been depicted as DMSO (control).

Chlorophyll a fluorescence measurement in intact leaves
The room temperature Chl. a fluorescence technique was used to study the photosynthetic activity in intact leaf samples. The fast Chl. a transients (O-J-I-P) in millisecond (ms) time span was studied with plant efficiency analyzer (Handy PEA fluorimeter) following Strasser et al. [17], while the relative thylakoid electron transport, membrane energization and carbon fixation activities were evaluated from slow fluorescence transients, obtained using FMS2 instrument (Hansatech) as per the detail outlined in Baker [23]. The readouts from Handy PEA were analyzed using biolyzerhp3 software.  Isolation of thylakoid membrane Thylakoids were isolated from spinach and rice leaves following [33] with some modifications. Leaves were homogenized in cold grinding buffer [50 mM tricine-NaOH (pH 7.5), 400 mM sucrose, 5 mM MgCl 2 and 5 mM KCl]. The homogenate was filtered through Miracloth and the filtrate was centrifuged at 50006g for 5 min. The chloroplast pellet was washed in 5 mM tricine-NaOH (pH 7.5) buffer containing, 400 mM sucrose and 2 mM MgCl 2 and centrifuged at 3006g for 1 min. The supernatant was recentrifuged at 50006g for 5 min. The pellet (thylakoids) was finally taken up in a minimal volume of suspending buffer [20 mM tricine-NaOH (pH 7.5), 100 mM sucrose and 5 mM MgCl 2 ]. Chlorophyll concentration in methanolic extract was measured spectrophotometrically [34].

Electron transport measurement
Photosystem II, PSI and whole chain (PSII plus PSI) catalyzed electron transport activities were measured as oxygen (O 2 ) evolution or uptake in a Clarke type O 2 electrode assembly (Hansatech) at 2261uC using site-specific artificial electron acceptors, donors, and inhibitors [25,26]. The light was ratesaturating (500 mE m 22 s 21 ). Phosphorylating electron transport was measured with addition of 1 mM ADP and 5 mM K 2 HPO 4 (iP) to basal electron transport components (25 mM hepes-NaOH (pH-7.5), 100 mM sucrose, 5 mM MgCl 2 and 20 mg Chl. ml 21 ). NH 4 Cl and GS were used at a final concentration of 5 mM and 10 mM respectively as and when required. The whole chain electron flow was assayed in presence of 2 mM FeCN as terminal electron acceptor of PSI. pBQ and DQ (400 mM) were used to measure PSII catalyzed electron flow. To measure PSI catalyzed electron flow, DCIPH 2 (1 mM DCIP plus 2 mM ascorbate) was used as electron donor to cytochrome b 6 /f region and 1 mM MV as transient electron acceptor. The PSI reaction mixture additionally contained 2 mM sodium azide (catalase inhibitor) and 10 mM DCMU (PSII inhibitor).
All experiments were repeated with 3 to 4 individual batches of thylakoid preparations. Experiments in intact plant samples were also made from different batches of plants. Although the absolute values of electron transport activity among various batches varied between 10-12%, the phenomenological observations were consistent.

Putative artemisinin-metabolite analysis
Leaf tissue (700 mg) each from control and artemisinin treated rice leaves were homogenized in 6 ml of the grinding buffer, filtered and the filtrate was centrifuged at 50006g for 5 min. Supernatant was kept in ice for further experimentation. Freshly isolated thylakoids (100 mg Chl. equivalent) from spinach were incubated with 1 ml of the supernatant obtained from artemisinin treated leaves for different time intervals (supernatant obtained from homogenized tissue sprayed with DMSO were taken as control for all experiments). The thylakoids were then spun down (50006g for 5 min), washed once with washing buffer, resuspended in suspension buffer and assayed for their respective electron transport activities. In order to further characterize the nature of the inhibitory-metabolite, the supernatant from 50006g centrifugation was ultracentrifuged at 1,81,7006g (50,000 rpm in the Sorvall TFT 80.4 rotor) for 1 h at 4uC. The supernatant was collected carefully and the pellets were resuspended in 1 ml of the suspending buffer. Isolated chloroplasts (100 mg Chl. equivalent) from spinach were incubated in the fractions for 30 and 60 min.
The pelleted thylakoids, following the treatment, were assayed for whole chain electron transport activity with FeCN as electron acceptor. Figure S1 Room temperature (25uC) and 77 K fluorescence spectra of isolated thylakoid from control (DMSO) and artemisinin treated (Artemisinin) leaves. The Chl. concentration was 3 mg ml 21 . 40% glycerol was used in the chloroplast suspension medium for low temperature (77 K) spectral measurements.


extfieldsofstudy:
Medicine
Materials Science

text:
Effects of light irradiation upon photodynamic therapy based on 5-aminolevulinic acid–gold nanoparticle conjugates in K562 cells via singlet oxygen generation

Purpose As a precursor of the potent photosensitizer protoporphyrin IX (PpIX), 5-aminolevulinic acid (5-ALA), was conjugated onto cationic gold nanoparticles (GNPs) to improve the efficacy of photodynamic therapy (PDT). Methods Cationic GNPs reduced by branched polyethyleneimine and 5-ALA were conjugated onto the cationic GNPs by creating an electrostatic interaction at physiological pH. The efficacy of ALA-GNP conjugates in PDT was investigated under irradiation with a mercury lamp (central wavelength of 395 nm) and three types of light-emitting diode arrays (central wavelengths of 399, 502, and 621 nm, respectively). The impacts of GNPs on PDT were then analyzed by measuring the intracellular PpIX levels in K562 cells and the singlet oxygen yield of PpIX under irradiation. Results The 2 mM ALA-GNP conjugates showed greater cytotoxicity against K562 cells than ALA alone. Light-emitting diode (505 nm) irradiation of the conjugates caused a level of K562 cell destruction similar to that with irradiation by a mercury lamp, although it had no adverse effects on drug-free control cells. These results may be attributed to the singlet oxygen yield of PpIX, which can be enhanced by GNPs. Conclusion Under irradiation with a suitable light source, ALA-GNP conjugates can effectively destroy K562 cells. The technique offers a new strategy of PDT.

Introduction
Photodynamic therapy (PDT) is a minimally invasive therapeutic modality approved for cancer treatment and some other nononcological disorders. 1 In PDT, a photosensitizer (PS) with photosensitizing properties is selectively accumulated in malignant tissue. When the malignant tissue is exposed to light with a specific wavelength, the PS is activated from its ground singlet state to an excited singlet state, after which it undergoes intersystem crossing to an excited triplet state. Some PS molecules, when they are in the excited state, release energy through physical processes. This energy produces fluorescence and phosphorescence that can be used for diagnosing disease. Other PS molecules are transferred to biological substrates and molecular oxygen to generate reactive oxygen species (ROS) and singlet oxygen, which are responsible for tumor regression and the cytotoxicity of neoplastic cells.
Improving the efficacy of PDT requires three elements: a PS, a light source, and tissue oxygenation. 2 Although the concentration of oxygen in tissue depends solely on tissue status, 3 studies should explore novel photosensitizers and more suitable light resources to improve the effect of photodynamic therapy.
The first generation PSs used in PDT were hematoporphyrin and derivative hematoporphyrin. Although some of these compounds have been approved in application, they are far from an ideal PS due to their significant limiting factors for chemotherapeutics and radiotherapy. 1 For these reasons, research on PDT has been focused on the development of alternative new-generation PSs with improved physical, chemical, and therapeutic properties.
The pro-photosensitizing metabolite 5-aminolevulinic acid (ALA) has attracted significant attention. 4 ALA is a natural precursor of protoporphyrin IX (PpIX) in the heme biosynthetic pathway. Under normal physiological conditions, heme synthesis from glycine through PpIX is regulated by negative feedback control of the enzyme ALA synthase by free heme; thus, the concentration of ALA or PpIX in normal tissue is sufficiently low to preclude cell damage that can occur by photochemical reactions. In contrast, in tumor tissues, the regulatory feedback system is bypassed as the administration of exogenous ALA causes excess cellular accumulation of downstream metabolites. 5 Due to the importance of heme synthesis in ALA-PDT, we chose the K562 human erythroleukemic cell, which is a well-known model for heme synthesis because erythroid cells produce 95% of total heme, 6 in order to significantly enhance the efficacy of ALA-PDT. The detailed mechanism of ALA-PDT cytotoxic effects on K562 cell line has been described previously. 7 The hydrophilic nature of ALA may limit its ability to penetrate skin or cell membranes and thereby restrict the effectiveness of ALA-PDT. 8 Gold nanoparticles (GNPs) have high available surface activity 9 and good biocompatibility, 10,11 which allows exploitation of their unique chemical and physical properties for transporting and unloading pharmaceuticals. 12 The use of GNPs as carriers for PS delivery represents a promising approach to enhance the efficacy of PDT. [13][14][15][16][17] As nanosized particles, GNPs can be delivered to and accumulate in tissues with permeable vasculature due to the enhanced permeability and retention (EPR) effect. 18 Given that tumor tissues are known to exhibit highly enhanced vascular permeability, 19 GNPs can passively accumulate in tumor tissues through the rich permeable vasculature. 20 In addition to the EPR effect, some studies have indicated that cationic GNPs show facilitated binding to negative tumor cell membranes. 14 Moreover, GNPs can adsorb onto leukemia K562 cells through the weak interaction between the nanoparticles and mercapto or primary amine groups on the cell membrane. 21 Accordingly, we chose cationic GNPs as carriers to deliver drugs to bind to leukemia K562 cells, which have a negative surface charge. 22 In addition, in recent demonstrations combining nanomaterials with PSs, GNPs not only facilitated delivery of PS to tumor cells but were also better for enhancing the singlet oxygen yield of PpIX. 13,14 The ability of PpIX to generate singlet oxygen is one of the most important factors for evaluating the PDT effect. 23 Thus, a compatible light source can be used to excite the optical properties of GNPs to improve the singlet oxygen yield of a PS.
In this study, we synthesized cationic GNPs by in situ chemical reduction of HAuCl 4 with the use of branched polyethyleneimine (BPEI) as a reductant and stabilizer. 24 We then conjugated 5-ALA onto the cationic GNPs through an electrostatic interaction under physiological pH (pH 7.2-7.6). We also investigated the efficacy of ALA-GNP conjugates under irradiation by LEDs and a mercury lamp, and measured the singlet oxygen yield of PpIX to analyze the influence of GNPs on the PS.

Synthesis and characterization of ALA-gNP conjugates
Different molar ratios of 0.1% HAuCl 4 and BPEI (molecular weight = 25,000) were mixed and stirred in ultrapure water at 80°C to prepare a series of GNPs, and solutions were collected until the color changed from yellow to dark red upon completion of the reduction reaction. The GNPs were submit your manuscript | www.dovepress.com Dovepress Dovepress then washed with ultrapure water and purified twice by centrifugation at 25,000 × g for 30 min at 4°C. 5-ALA was dissolved in 20 mM HEPES to adjust the pH value to 7.2-7.4, after which it was filtered with a 0.2-µm filter. The colloidal GNPs were also filtered with a 0.2-µm filter. Subsequently, the ALA solution and GNP colloidal solution were mixed at a number of ratios to prepare the conjugates.
Ultraviolet-visible (UV-Vis) absorption spectra were measured using a DU-640 UV-Vis spectrophotometer (JASCO, Tokyo, Japan). Transmission electron microscopy (TEM) images were acquired on a JEM-200CX transmission electron microscope (Hitachi, Tokyo, Japan). The average diameter of the GNPs and conjugates were examined using a ZetaSizer Nano ZS90 (Malvern Instruments, Malvern, UK). This device was also used to measure the zeta-potential after GNP and conjugate solutions were further adjusted to pH 7.4 with 1 N NaOH.

Light source
Light-emitting diodes (LEDs) have been used for PDT due to their great advantages of convenience, stability, and broadspectrum properties. 25 We thereby used LEDs in this study. A mercury lamp irradiation system, which caused significant photodamage to tumor cells in research on ALA-PDT, 26 was also adopted to compare the PDT results with those after LED irradiation. Preliminary studies have indicated that a low light dose irradiation can induce cell death via a slower mechanism with its gentler thermal effect. Chen et al reported that a light dose of 1.44 J/cm 2 on cells incubated with gold nanoshells was optimal for reducing cell viability. 27 Here, we used an LED source (Hangke Co, Hangzhou, China) and a 400-W mercury arc lamp (Yaming Co, Shanghai, China) with the same light dose of 3.6 J/cm 2 , but different values of full width at half-maximum to comprehensively explore the efficacy of ALA-GNP conjugates in PDT.
Mercury lamp irradiation was generated with a Spiegel condenser illuminated by a mercury lamp (Yaming Co, Shanghai, China), filtered with interference filters (Haiguang Co, Shanghai, China) of 410 nm (bandwidth = 10 nm), and attenuated by a lens. An energy fluence rate of 1 mW/cm 2 was obtained by adjusting the distance between the lens and sample cells. Previous work on ALA in our lab has demonstrated that irradiation with 410 nm caused maximum photodamage to the membrane and mitochondrial function of HL60 cells. 26 We adopted three types of LEDs. Purple LEDs (central wavelength = 398 nm) were chosen to contrast with the mercury lamp. Green LEDs (central wavelength = 505 nm), which fit the absorption wavelength of GNPs, can excite photic properties such as surface plasmon resonance. Red LEDs (central wavelength = 621 nm), which have better penetration depth, display significant efficacy in PDT. 28 The LED light sources were created with a diode array system containing 96 LED units with maximum emission. The energy fluence rates of these light sources were adjusted to 1 mW/cm 2 using a variable resistor in series.
The energy fluence rates of these light sources were measured using a 3-sigma laser power meter (Coherent Inc, Portland, OR). The emission spectra of the light sources ( Figure 1) were detected using a HR4000 high-resolution spectrometer (Ocean Optics Inc, Dunedin, FL).

Cell culture
We used the chronic myelogenous leukemia cell line K562 (provided by Northwest University, Xi'an, China). RPMI1640 supplemented with 10% FBS and 1% penicillinstreptomycin was used as the cell-culture medium. The cells were cultured at a humidified atmosphere of 37°C with 5% CO 2 in a HERAcell 150 Copper CO 2 incubator (Thermo Fisher Scientific) and subcultured every 3 days.

Photodynamic therapy in vitro
To five 96-well plates, we added the same cells and drugs as follows: 100 µL serum-free cell suspensions per well (cell density of 10 6 /mL) were seeded in several 96-well plates. ALA, ALA-GNP conjugates, and GNPs were freshly prepared at three concentrations (3, 2, and 1 mM each) and respectively added to the cell suspensions in different wells for the drug groups. A drug-free cell suspension was used for the control group. Every group had three duplicate samples. All 96-well plates containing cells were incubated with the medium containing different reagents for 4 hours under dark conditions. Four plates were irradiated by the mercury lamp and three types of LEDs for 1 hour each. At the same time, another 96-well plate containing cells was used as the control under dark treatment.

Measurement of PpIX formation and singlet oxygen generation
For PpIX detection, K562 cell suspensions (cell density of 10 6 /mL) were seeded in 6-well plates. The medium of the cell-seeded well plates was then replaced with a serumfree medium containing 2 mM ALA, GNPs, or ALA-GNP conjugates. Untreated cells were used as the drug-free control. Cells were treated with the drug-containing medium under dark conditions. Homogeneous aliquots of cells were withdrawn from 6-well plates at 0, 3, 4, 6, and 8 hours, respectively. Cell suspensions were washed twice with PBS to remove free drug lysed in SDS lysis buffer, and then shaken for 10 minutes. Cell lysates were centrifuged at 12,000 × g for 5 minutes, and the fluorescence intensity was measured using an F-4500 fluorescence spectrophotometer (Hitachi). The excitation waveband was 410 nm, and the emission spectra were scanned from 420 to 700 nm. The peak fluorescence value at 631 nm was used for kinetic measurements.
SOSGR was used as the singlet oxygen-tracking agent. Given that SOSGR is cell-impermeable, 100 µL of GNP solution was directly mixed with 50 µL of 200 µM PpIX in cell-free solution to study the effect of GNPs on singlet oxygen generation of PpIX. Next, 20 µL of 100 µM SOSGR and ultrapure water were added to obtain the final 1000-µL sample solution. The PpIX alone, GNP alone, and drug-free groups were also prepared in the same manner. Irradiation treatment was performed by exposing the sample to red LEDs and green LEDs for different durations. The fluorescence measurement of SOSGR was performed using an F-4500 fluorescence spectrophotometer (Hitachi) at an excitation wavelength of 488 nm.

Statistical analysis
The experiments were performed at least three times. Data are shown as the mean ± standard error of independent experiments. The means were compared using Student's t-test, and differences were considered statistically significant at P , 0.001.

Characterization of cationic gNPs
BPEI is a type of polyelectrolyte that serves as a reductant, stabilizer, and linker for preparing GNPs. 24 The secondary amine of BPEI has a strong reducing ability, readily forming ion pairs with AuCl 4 anions to produce zero-valent gold atoms. 29 The gold atoms can then collide with each other to form GNPs. In contrast, the amine groups of BPEI, which associate with the particle surface, prevent further growth of GNPs. Therefore, when BPEI and HAuCl 4 are mixed at a suitable ratio, GNPs with low-dispersing and uniform distribution can be prepared. In addition, due to the positive charge of the amine group on BPEI, this type of GNP is positively charged.
The absorption spectra of GNPs showed little difference when mole ratio of HAuCl 4 and BPEI were changed ( Figure 2). With increased BPEI, absorbance peaks showed a small red shift around 530 nm. This absorbance peak was caused by transverse surface plasma absorption of GNPs. Moreover, longitudinal surface plasma absorption of ∼700 nm did not exist, 30 demonstrating that the majority of the GNPs were uniform sphere-like particles. 31 However, when the molar concentration of BPEI was increased to eight times that of HAuCl 4 , the absorbance peak showed a significant red shift, far away from the characteristic absorption peak of GNPs. Thus, excess BPEI served as a linker bridging different particles into dimer or trimer aggregates. In addition, the average diameter of GNPs increased as the BPEI concentration increased, as shown in Figure 3. When the mixture ratios of HAuCl 4 and BPEI were between 0.5:1 and 1:4, the size distributions of the GNPs were uniform. When the ratio reached 1:8, the average size of GNPs achieved micron levels with a dispersing and nonuniform distribution.
To conjugate more ALA onto cationic GNPs, we expected that GNPs would have more positive potential to attract ALA with negative charge. As illustrated in Figure 4, the zeta-potential increased with BPEI concentration within a certain range. However, when excessive BPEI (1:8) induced aggregates, the zeta-potential became negative. We chose to prepare ALA-GNP conjugates at a 1:4 ratio because these kinds of GNPs are similar in size to 1:3 conjugates, but are more positively charged and thus conjugate ALA more readily.
Characterization of ALA-gNP conjugates ALA, a zwitterion, can have either an end group of COO − or NH3 + , depending on the solution pH. 32 At physiological pH (7.2-7.6), its net negative charge enables conjugation to cationic GNPs through static electronic interactions. 14 ALA was added to GNPs at various concentrations and the corresponding zeta-potentials were obtained at a solution pH of 7.4, as shown in Figure 5. As ALA concentration was increased, the zeta-potential of the GNPs decreased. This also confirmed that ALA had conjugated onto GNPs.
The stability of ALA-GNPs based on the sharp decrease in the zeta-potential indicates that the particles may be unstable.
However, the amine groups of BPEI, which associate with the particle surface, caused steric hindrance to prevent the aggregation of conjugates. 33 This was confirmed by measurements of the size distribution, as shown in Figure 6. Even when the concentration of the added ALA was increased to 8 mM, the average size of the conjugations did not change significantly. Thus, the addition of ALA did not induce the aggregation of conjugates.

Dark toxicity
The dark toxicity was investigated to determine whether the GNPs and ALA-GNP conjugates would affect the viability of K562 cells. The cell viability of dark treatment is shown in Figure 7, where cell viability is the optical density value of various drug treated groups in serum-free medium for 24 hours divided by that of the groups for 0 hours.   There were no significant differences between the drugtreated groups and the untreated group.
PDT using ALA-gNP conjugates As described earlier, PSs and light sources are both key elements that influence the efficacy of PDT. Therefore, we incubated K562 cell lines with ALA-GNP conjugates, ALA, or GNPs, and irradiated them with light sources to comprehensively analyze the effects of ALA-GNP conjugates and light conditions. In order to understand the interference of additional drugs, cell viability was characterized based on a normalized value, the OD value of the light-treatment group divided by that of the corresponding dark control group. Cell viabilities after irradiation treatment are shown in Figure 8. ALA-GNP conjugates showed greater cytotoxicity against tumor cells than ALA alone under irradiation with various light sources. Moreover, GNPs alone had no significant effect on cell viability under irradiation. However, different light sources influenced the efficacy of PDT differently. For the control group without drug treatment, cell viability after purple LED irradiation decreased to 91.28%, which was lower than that after irradiation with a red or green LED. UV exposure itself may induce cell death and potentially harm normal cells. This conjecture is supported by the observation that the cell viability after exposure to filtered 410-nm light from the mercury lamp decreased to 85.89%. Therefore, the purple LED and mercury lamp was not the optimal light source due to its UV effects on normal cells.
In addition to comparing the different effects of irradiation with various light sources for the control group, the effects of drug treatment on cell viability were also significantly different. As shown in Figure 8, although red and green LEDs did not significantly influence cell viability for the control group, cell viability with drug treatment was different after irradiation with these LED light sources. Comparing the control group with 2 mM 5-ALA-GNP treatment, cell viability decreased to 29.87% for 2 mM 5-ALA-GNPs under irradiation with green LEDs, but cell viability only decreased to 70.31% for 2 mM 5-ALA-GNPs under irradiation with red LEDs.
As shown in Figure 8C, although cell viability decreased to 51.62% for 1 mM 5-ALA-GNP treatment under irradiation with green LEDs in PDT, cell viability decreased with increasing concentrations of 5-ALA-GNPs and ALA. The group treated with 2 mM and 3 mM 5-ALA-GNP showed better inactivation than the group treated with the same concentration of ALA. Hence, green LEDs are the optimal light sources for enhancing the effects of 5-ALA-GNPs in PDT, with the group given 2 mM 5-ALA-GNPs showing the greatest inactivation under irradiation with green LEDs.

PpIX formation and detection
To determine the factors by which GNPs enhance the inactivating effects of ALA, we first investigated whether GNPs could affect PpIX formation. Medium containing 2 mM ALA, GNPs, or ALA-GNP conjugates was added to K562 cells with identical cell densities and incubated for different times. After thorough washing with PBS to remove free drug, the cells were lysed and centrifuged. Figure 9, which shows the fluorescence measurement of the intracellular PpIX formed from 5-ALA, indicated that the intracellular accumulation of PpIX in K562 was time-dependent. Maximum accumulation was observed at approximately 4-6 h after the addition of 5-ALA or 5-ALA-GNPs. Although the fluorescence intensity of the conjugate group was higher than that of the ALA group after 6 hours of incubation, this cannot explain why irradiation under red light has a lower inactive effect than other light sources. Therefore, enhancing the uptake of ALA with GNPs was not the only factor that resulted in a

Singlet oxygen measurement
We considered whether the photosensitization of PpIX was improved by GNPs due to PpIX producing more singlet oxygen. ALA is a precursor of the photosensitizer PpIX, which is the reaction agent in PDT. Therefore, we directly used PpIX to detect the influence of GNPs on singlet oxygen generation. Singlet oxygen is a short-lived species and can easily lose its energy by emitting light or internal conversion into heat. 34 Given the difficulty of directly measuring singlet oxygen generation, we used SOSGR, which emits green fluorescence at 525 nm in the presence of singlet oxygen, as the singlet oxygen sensor. The intensity of the green fluorescent signal could then be correlated with singlet oxygen concentration without signals from other reactive oxygen species. Figure 10 shows the time-dependent fluorescence intensity of SOSGR with various treatments. GNPs clearly enhance the singlet oxygen generation of PpIX under exposure to green LEDs. The fluorescence response of SOSGR to GNPs is absent in controls with only GNPs or no drugs. However, singlet oxygen generation is limited under red LED exposure. These results provide a possible explanation for the better efficacy of ALA-GNP conjugates under irradiation with green LEDs in PDT.

Discussion
In this study, cationic GNPs were synthesized simply by heating an aqueous solution of HAuCl 4 and the polyelectrolyte BPEI without the addition of additional reductants. By controlling the molar ratio of HAuCl 4 and BPEI at 1:4, optimal GNPs with a zeta-potential of +43.0 mV were obtained to conjugate with ALA ( Figure 4). Due to steric hindrance of BPEI, ALA was conjugated with GNPs Tumor cells have a lower negative zeta-potential in the cell membrane than do healthy cells, which should enhance the binding of positively charged ALA-GNPs to negatively charged K562 cells. 35 Additionally, ALA displays high selectivity for tumor cells due to enhanced selectivity of PpIX accumulation in malignant lesions. 36 Given this dual selectivity for tumor cells, ALA-GNPs should have better targeting effects than other photosensitive drugs.
In addition to identifying the optimum selectivity for tumor cells, the results of PDT indicated that GNPs can enhance the inactivation effects of ALA. One reason may be that GNPs act as water-soluble and biocompatible carriers that allow delivery of ALA through the cell membrane to synthesize PpIX, consistent with the observation that PpIX formation is detected. Hence, when the same concentration of ALA and ALA-GNPs were added to the cells, more ALA was synthesized into PpIX in the 5-ALA-GNP treatment group, and the accumulated PpIX induced significantly more photosensitization. 37 However, this cannot explain why irradiation with different light sources produces different inactivation effects. For efficient photochemical reactions, matching the PS absorption with light emission spectra is crucial for obtaining higher photon absorption yields. 38 The absorption of the PS is an important parameter of the "total effective fluence." 39 Its absorption spectrum indicates that PpIX has the highest  absorption peak at 404 nm, the second highest peak at 548 nm, and the lowest peak at 642 nm in phosphate buffer (pH 7). 40 Some studies have demonstrated that blue or green light is more potent than red light in activating PpIX. 39,41 The same PDT result in our study (Figure 8) also demonstrated that the mercury lamp (central wavelength = 395 nm) and two types of LEDs (central wavelength = 399 nm, 502 nm) were more effective than the red LED (central wavelength = 621 nm). The reason that most researchers have chosen a red light for PDT relates to the depth tissue, as an increased amount of red light is absorbed at greater depths. In addition, Mosely has calculated that green light gives the highest effective fluence before the tumor extends to 2.0 mm. 39 Although the absorption of PpIX can explain why red light does not have high effective fluence, it cannot fully explain the ALA-GNP conjugates in our study. According to the absorption spectrum of PpIX, the absorption peak at 548 nm is lower than that at 404 nm. However, the mercury lamp (central wavelength = 395 nm) and LED (central wavelength = 502 nm) show similar levels of K562 cell destruction by ALA-GNP conjugates. According to the PDT mechanism, 42,43 an important factor responsible for the destruction of target cells is singlet oxygen. 2 Comparing the singlet oxygen measurements in our study, we postulate that the emission spectrum of green light fits with the absorption peak of GNPs ( Figure 2) to induce an increase in the net system absorption of PpIX and enhance the photocurrent between GNPs and PpIX after light irradiation. Through nonradiative energy transfer and coupling to the surface plasmons of GNPs, more PSs are excited to their triplet and singlet states, which results in more singlet oxygen generation. 44 However, the emission spectra of other light sources do not fit with the absorption peak of GNPs. Hence, they cannot cause a strong surface plasmon effect to occur in GNPs.

Conclusion
This study aimed to combine PDT with nanotechnology by using a novel ALA-GNP conjugation technique to improve PDT efficacy. Through a comprehensive study on the preparation of ALA-GNP conjugates, light source adoption, and cell viability measurements, we conclude that ALA can be conjugated to GNPs and that the conjugation leads to a significant improvement in PDT efficacy. Using these conjugates as delivery vehicles, GNPs induced more ALA transport into the cell, thereby enhancing the effect of ALA-PDT because the hydrophilic nature of ALA limits its ability to penetrate cell membranes. In addition, GNPs noticeably enhanced singlet oxygen generation, which also led to a high efficacy of tumor cell destruction. This enhancement may result from the surface plasmon resonance that leads to increased net system absorption and triplet yield of PSs.
Based on parallel experiments with four light sources, we concluded that UV light has an inhibitory action on cell viability, possibly by causing normal cell death. Green light is optimal for conjugation-PDT; it has the best efficacy because green light itself does not harm cells.
ALA-GNP conjugation offers a new modality for efficient destruction of tumor cells. With the cooperation of a suitable light source with matching wavelength and sufficient power, ALA-GNP conjugation has great potential for enhancing cancer treatment with PDT. Nevertheless, significant advancement in related areas requires a full understanding of ALA-GNP accumulation in cell compartments. In addition, the role of plasmonic properties of GNPs in conjugation-PDT requires further investigation.


extfieldsofstudy: Chemistry Medicine

text:
1-Fluoro-3,3-dimethyl-1,3-dihydro-1λ3-benzo[d][1,2]iodoxole

The asymmetric unit of the title compound, C9H10FIO, contains two independent molecules which are weakly bound by intermolecular O⋯I interactions [3.046 (4) and 2.947 (4) Å]. The two covalent I—F bonds are slightly longer than the two I—O bonds.

Experimental Crystal data

Data collection
Enraf-Nonius CAD-4 diffractometer Absorption correction: scan (NRCVAX;Gabe et al., 1989] T min = 0.337, T max = 0.422 3408 measured reflections 3408 independent reflections 2833 reflections with I > 2(I) 1 standard reflections every 100 reflections intensity decay: none Refinement R[F 2 > 2(F 2 )] = 0.030 wR(F 2 ) = 0.073 S = 1.05 3408 reflections 217 parameters H-atom parameters constrained Á max = 0.64 e Å À3 Á min = À1.22 e Å À3 Table 1 Selected bond lengths (Å ). Hypervalent iodine compounds have received a growing attention in recent years. This is not surprising considering that these reagents are polyvalent electrophiles and mild oxidants (Zhdankin & Stang, 2002;Wirth, 2005). In this family, haloiodanes are interesting yet under exploited electrophilic halogen sources. A research project currently underway in our group aims to exploit haloiodanes as electrophilic halogen sources. We developed a synthesis to obtain the title compound in order to evaluate and compare its reactivity with its chloro and bromo analogs. This is the first reported synthesis of the title compound.
In the crystal structure, two independent molecules, as shown in Fig. 1, are weakly bound by O···I interactions  (Braddock et al., 2006) analogs. This is consistent with the trans effect behavior described in a variety of hypervalent λ 3 -iodane compounds (Ochiai et al., 2006). In contrast to the bromo analog, the title compound was found to be completely unreactive for the fluorination of anisole. While the title compound is a stable solid, caution must be taken when drying the crude solution. The use of anhydrous MgSO 4 to dry the solution results in the displacement of the fluorine by a sulfate dianion. Drying by co-evaporation with benzene prevents this side reaction.
A more in-depth study of the reactivity of this novel fluoroiodane is currently underway.
Experimental 2-(2-Iodophenyl)-propan-2-ol (164 mg, 0.63 mmol) was dissolved in MeCN (3 ml) and SelectFluor (289 mg, 0.81 mmol) was added in one portion. The reaction was then stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure. The crude product was dissolved in CH 2 Cl 2 (10 ml), washed once with water (10 ml), and concentrated under reduced pressure. The crude product was dried by coevaporation with benzene. Crystals were grown by slow diffusion of a pentane solution on a CH 2 Cl 2 solution of the title compound at room temperature.

Refinement
The hydrogen atoms were placed at idealized calculated geometric positions and refined isotropically using a riding model.  The asymmetric unit of the title compound, showing 30% probability displacement ellipsoids. The I···O interactions are shown by dotted lines. H atoms are depicted as circles of arbitrary size.  (Flack, 1992) program was used for centering, indexing, and data collection. One standard reflection was measured every 100 reflections, no decay was observed during data collection. The data were corrected for absorption by empirical methods based on psi scans and reduced with the NRCVAX (Gabe, 1989) programs. They were solved using SHELXS97 (Sheldrick, 2008) and refined by full-matrix least squares on F2 with SHELXL97 (Sheldrick, 2008). The non-hydrogen atoms were refined anisotropically. Refinement of F 2 against ALL reflections. The weighted Rfactor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and Rfactors based on ALL data will be even larger.


Physicochemical Parameters for Biological and Synthetic Surfactants Used as Detergents

: Biological and synthetic surfactants were compared in terms of their ability to reduce interfacial tension, change the thermodynamic characteristics of a pre-conditioned surface, and to modify the rheological properties of their respective formulations at two different temperatures. Both classes of surfactants were able to reduce the interfacial tension of their formulations to a similar level. However, the biosurfactants were more effective than the synthetics surfactants. Biosurfactants also altered the surface properties of stainless steel, rendering it hydrophilic. Microbial adhesion to stainless steel conditioned with biosurfactants was found to be thermodynamically unfavorable for all microbial strains tested. A linear relationship between shear stress and shear rate was obtained across a range of experimental conditions for all surfactant mixtures, indicating that all formulations behaved as Newtonian fluids.

Introduction
The contamination of surfaces by spoilage and pathogenic microorganisms is a concern in the food industry. The development of biofilms in food processing environments results in product spoilage and possible risks to public health, in addition to create a number of serious problems for industrial fluid processing operations [1][2][3]. The presence of organisms such as Salmonella spp., Listeria monocytogenes and Escherichia coli in food processing environments can be a persistent source of contamination [4].
To reduce or eliminate microorganisms found on food contact surfaces, cleaning and disinfection procedures using physical and chemical methods have been extensively used over the years [5]. An interesting strategy is the pre-treatment of surfaces using surface-active compounds, also known as surfactants.
Surfactants can be classified into two main groups: synthetic surfactants and biosurfactants. Synthetic Nitschke et al. [13] investigated the effect of the biosurfactant surfactin on the adhesion of the food pathogens Listeria monocytogenes, Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces. The pre-conditioning of stainless steel surfaces with surfactin caused a reduction in the number of adhered cells of Enterobacter sakazakii and Listeria monocytogenes. The most significant result was obtained with L. monocytogenes, where the number of adhered cells was reduced by 10 2 CFU/cm 2 . On polypropylene surfaces, surfactin significantly decreased the adhesion of all strains.
In the literature, there are no references that describe the thermodynamic properties of surfaces that have been pre-conditioned with surfactants. This work aimed to compare biological and synthetic surfactants in terms of their ability to reduce interfacial tension, change the hydrophobicity of pre-conditioned surface and alter the rheological properties of their respective formulations at two different temperatures.

Materials
A rhamnolipid biosurfactant, consisting of a mixture of mono-and di-rhamnolipids from the glycolipid group produced by Pseudomonas aeruginosa was obtained from Jeneil Biosurfactant Co. (Saukville, Winconsin, USA) [14]. The molecular formulas for mono-and di-rhamnolipids are C 26 H 48 O 9 and C 32 H 58 O 13, respectively ( Fig. 1). The critical micelle concentration (CMC) was found to be 0.035 g/L by conductivity measurements performed at various dilutions [15].
The synthetic surfactant used was dodecylbenzene sodium sulfonate, and its CMC is 0.4 g/L.

Detergent Formulations
The detergent formulations used in the following experiments are shown in Table 1. The sodium carbonate concentration was selected to match the concentration typically used in the food industry for cleaning procedures. The concentrations chosen for other surfactants were above their respective CMCs to ensure the formation of micelles.

Interfacial Tension Measurements of the Detergent Formulations
The interfacial tension of each detergent formulation was measured by the pendant drop method using a goniometer (DSA 100, Kruss ® , Germany). The surface tension measurement was carried out at 25 ± 1 °C. To calibrate the instrument, the surface tension of pure distilled Milli-Q water (Millipore, Illinois, USA) was measured before each set of experiments. Coupons (10 mm  10 mm  0.05 mm) of stainless steel #4 were first cleaned by washing with a neutral liquid detergent and water, followed by rinsing with distilled water. Subsequently, they were put in a muffle furnace for 2 h at 200 °C to remove organic materials. After that, the coupons were immersed in detergent formulations (Table 1) for 24 h at 20-25 °C. Control coupons without treatment were maintained in distilled water under the same conditions.
Surface hydrophobicity was determined based on contact angle measurements of the stainless steel with water, formamide and α-bromonaphthalene using a goniometer. The total Gibbs free energy of interaction (∆G sws TOT ) and the total free energy of adhesion between stainless steel and different bacteria (∆G adhesion ) were determined by appropriate formulas [17].

Hydrophobicity of the Bacterial Strains
One milliliter of each bacterial culture was stored at -80 °C in a nutrient broth (Merck, São Paulo, Brazil) containing glycerol (80:20). A working culture of each strain, containing approximately 10 7 CFU/mL, was prepared by inoculating 10 mL of Brain Hearth Infusion (BHI, Merck, São Paulo, Brazil) with 100 µL of frozen culture, followed by incubation at 32 °C for 24 h. The culture was sub-cultured three times before use. The number of microorganisms in each suspension was obtained by total count using Plate Count Agar (PCA, Merck, São Paulo, Brazil) at 32 °C for 24 h.
Contact angles for microorganism surfaces were determined on a layer of vegetative cells using the drop method [18]. First, the strains were activated twice in BHI broth (Merck, São Paulo, Brazil), creating suspensions with approximately 10 7 CFU/mL. The suspensions were centrifuged at 12,000 g for 10 min and washed three times in 0.1 M phosphate buffered saline (PBS). The cell pellet was suspended in the same buffer, and the suspension was filtered using a cellulose acetate membrane (0.45 µm pore size, 27 mm in diameter, Millipore, Brazil) with negative pressure. During the filtration, 30 mL of pure water (Milli-Q) was added. The membranes were transferred to Petri plates containing 1% (v/v) of agar and 10% (v/v) of glycerol. The membranes were cut into three parts for contact angle measurements.
Each surface was classified as hydrophobic or hydrophilic according to the determination of the total free energy of interaction (∆G sws TOT ). The total free energy of interaction among molecules of a surface (s) immersed in water (w) was determined by the sum of the apolar and polar free energies of interaction, ∆G sws LW and ∆G sws AB , respectively: Here, γ LW is the interfacial tension arising from Lifshitz-van der Waals forces; γ + is the interfacial tension arising from the electron acceptor component of the acid-base interaction; γis the interfacial tension of the electron donor component of the acid-base interaction; and s and w indicate surface and water, respectively [17].
When ∆G sws TOT > 0, the surface is considered hydrophilic; conversely, if ∆G sws TOT < 0, then the surface is considered hydrophobic. Bacteria were used to determine the total Gibbs free energy of adhesion (∆G adhesion ) between conditioned stainless steel and each strain.

Determination of the Total Gibbs Free Energy of Adhesion (∆G adhesion )
From the values of the individual interfacial tension components, it is possible to determine the ∆G adhesion between two surfaces: microbial cells (b) and food processing surfaces (s): Because the free energy is related to the interfacial tension, ∆G adhesion can be represented by the following: Here,  bs is the interfacial tension between the bacterial surfaces and the adhesion surface;  bl is the interfacial tension between the bacterial surfaces and the liquid; and  sl is the interfacial tension between the adhesion surfaces and the liquid.
The ∆G adhesion values allow for an evaluation of the thermodynamics of the adhesion process: if ∆G adhesion < 0, then the process is favorable; if ∆G adhesion > 0, then the process is unfavorable.
All measurements were performed in duplicate with three repetitions.

Rheological Measurements of the Detergent Formulations
Rheological measurements were performed using a Modular Advanced Rheometer System (HAAKE MARS, Thermo Electron Corp., Germany). The experiments were carried out at 25 °C and 70 °C for the different surfactant formulations, which correspond to manual cleaning temperatures and cleaning-in-place temperatures, respectively. Steady rheological measurements were performed with double gap cylinder system (DG41) geometry, using a gap size set to 0.8 mm. Flow curves were evaluated by measuring the shear rates from 0 to 1,200 s -1 . N ew samples were used for measurements at each experimental temperature. Rheological parameter models were calculated from data using ThermoHaake RheoWin Pro 297 Software by the least squares method.

Interfacial Tension of Detergent Formulations
We observed that all surfactant formulations had interfacial tensions that were reduced relative to the control. However, formulations B, C and D produced greater reductions in interfacial tension than formulation A (Table 2). Although there is not a significant difference (P > 0.05) among the interfacial tensions of formulations B, C and D, biosurfactants are clearly advantageous as they are effective at low concentrations. It is important to emphasize that formulations C and D employed biosurfactant concentrations 50-fold and 10-fold lower, respectively, than the synthetic surfactant concentration in formulation B. Normally, sodium carbonate is used in the food industry to clean surfaces. In this work, we verified that the addition of a biosurfactant to a sodium carbonate formulation reduced its interfacial tension, thereby making the hygiene procedure easier and more efficient.
The results of this experiment were similar to the findings of Pornsunthorntawee et al. [6]. These researchers found that, in the case of the crude biosurfactant produced by Pseudomonas aeruginosa, the surface tension rapidly decreased as the concentration of biosurfactants increased. Furthermore, they found that a minimum surface tension of 29.0 mN/m was obtained at an initial surfactant concentration greater than 0.02%. In our experiment formulation D with a rhamnolipid concentration of 0.02% (Table 2) decreased the interfacial tension to 28.09 mN/m (46%) when compared with formulation A.

Hydrophobicity of Conditioned Stainless Steel Surfaces
The conditioning of stainless steel changed its hydrophobicity. The coupons of stainless steel that were immersed in water (control formulation) were classified as hydrophobic because the ∆G sws TOT value was negative, whereas coupons of stainless steel conditioned with the detergent formulations were considered hydrophilic (∆G sws TOT > 0). In particular, treatment with a 0.004% formulation of rhamnolipid (Formulation C) resulted in a more hydrophilic surface (42.48 MJ/m 2 ) ( Table 3). According to thermodynamic principles, when TOT sws G  is negative, there is a net cohesive attraction between the molecules of a surface when immersed in water. Thus, for a hydrophobic material, TOT sws G  has a negative sign. In other words, molecules of a hydrophobic surface, when immersed in water, prefer to interact with each other rather than form an interface with water, whereas a positive value of TOT sws G  shows that the molecules of a surface prefer to interact with adjacent water molecules rather than with themselves. Such a surface is hydrophilic [19].
The contact angle measurements are interpreted directly in terms of the surface energy of the adsorbed layer and provide access to equilibrium or steady-state adsorption because angle measurements do not perturb adsorption dynamics. The treatment of stainless steel with biosurfactants causes its surface properties to change [13]. The addition of a synthetic or biological surfactant alters the wettability of solids because of its adsorption at the solid/liquid interface. Adsorption profiles suggest that the hydrocarbon chains of biosurfactants attach to hydrophobic surfaces and that their polar groups project toward aqueous solutions. This combination of events makes a surface more hydrophilic. The orientation of surfactant molecules at the solid/liquid interface determines the ability of the surfactant to improve the wetting ability of the liquid. A phenomenon involving a change in orientation of the adsorbed molecules could also be important in the kinetics of the wetting process [20].

Total Gibbs Free Energy of Adhesion
The adhesion process was thermodynamically favorable (∆G adhesion < 0) for all tested strains on stainless steel surfaces conditioned with formulation B or a control formulation (Table 4). In contrast, the adhesion process was unfavorable (∆G adhesion > 0) on stainless steel surfaces conditioned with formulations C or D. According to thermodynamic theory, adhesion is favorable only if the process results in a decrease in total free energy. Therefore, the rhamnolipid biosurfactant may potentially be incorporated into detergent formulations to reduce or avoid microbial adhesion and subsequent biofilm formation.

Rheological Characteristics of the Detergent Formulations
A linear relationship between shear stress and shear rate was obtained across a range of experimental conditions for all surfactant mixtures, indicating that all formulations behaved as Newtonian fluids (Figs. 2 and  3). Penott-Chang et al. [21] observed similar As expected, an increase in temperature decreased the viscosity of all tested solutions. In an industrial context, a decreased viscosity can lead to more efficient cleaning procedures. More specifically, by decreasing the absolute viscosity (µ) at elevated temperatures, solvent flow across cleaning surfaces may be increased, thereby reducing pump power consumption and therefore energy costs.

Surfactant
formulations containing low concentrations of rhamnolipids achieved reductions in interfacial tension comparable to a formulation containing a higher concentration of a synthetic surfactant. The pre-conditioning with biosurfactant converted microbial adhesion to stainless steel into a thermodynamically unfavorable process. Moreover, at   room temperature, a detergent formulation containing a low rhamnolipid concentration exhibited a reduced viscosity relative to a control. This formulation potentially offers a great advantage for cleaning procedures within the food industry. Therefore, a biosurfactant can be superior to a synthetic surfactant for preventing or delaying microbial colonization of food contact surfaces.