Rational Design of Photosynthesis-Inspired Nanomedicines.

The sun is the most abundant source of energy on earth. Phototrophs have discovered clever strategies to harvest this light energy and convert it to chemical energy for biomass production. This is achieved in light-harvesting complexes, or antennas, that funnel the exciton energy into the reaction centers. Antennas contain an array of chlorophylls, linear tetrapyrroles, and carotenoid pigments spatially controlled by neighboring proteins. This fine-tuned regulation of protein-pigment arrangements is crucial for survival in the conditions of both excess and extreme light deficit. Photomedicine and photodiagnosis have long been utilizing naturally derived and synthetic monomer dyes for imaging, photodynamic and photothermal therapy; however, the precise regulation of damage inflicted by these therapies requires more complex architectures. In this Account, we discuss how two mechanisms found in photosynthetic systems, photoprotection and light harvesting, have inspired scientists to create nanomedicines for more effective and precise phototherapies. Researchers have been recapitulating natural photoprotection mechanisms by utilizing carotenoids and other quencher molecules toward the design of photodynamic molecular beacons (PDT beacons) for disease-specific photoactivation. We highlight the seminal studies describing peptide-linked porphyrin-carotenoid PDT beacons, which are locally activated by a disease-specific enzyme. Examples of more advanced constructs include tumor-specific mRNA-activatable and polyionic cell-penetrating PDT beacons. An alternative approach toward harnessing photosynthetic processes for biomedical applications includes the design of various nanostructures. This Account will primarily focus on organic lipid-based micro- and nanoparticles. The phenomenon of nonphotochemical quenching, or excess energy release in the form of heat, has been widely explored in the context of porphyrin-containing nanomedicines. These quenched nanostructures can be implemented toward photoacoustic imaging and photothermal therapy. Upon nanostructure disruption, as a result of tissue accumulation and subsequent cell uptake, activatable fluorescence imaging and photodynamic therapy can be achieved. Alternatively, processes found in nature for light harvesting under dim conditions, such as in the deep sea, can be harnessed to maximize light absorption within the tissue. Specifically, high-ordered dye aggregation that results in a bathochromic shift and increased absorption has been exploited for the collection of more light with longer wavelengths, characterized by maximum tissue penetration. Overall, the profound understanding of photosynthetic systems combined with rapid development of nanotechnology has yielded a unique field of nature-inspired photomedicine, which holds promise toward more precise and effective phototherapies.

UV-B radiation increases anthocyanin levels in cotyledons and inhibits the growth of common buckwheat seedlings.

The impact of short-term UV-B treatment on the content of individual flavonoids and photosynthetic pigments in cotyledons and the growth of common buckwheat (Fagopyrum esculentum Moench) seedlings was investigated. Seeds of four common buckwheat cultivars were germinated in darkness over a period of 4 days and acclimatized for 2 days under a 16/8 h light/dark photoperiod at 24/18 °C day/night, and exposure to 100-120 µmol ∙ m-2 ∙ s-1 of photosynthetically active radiation (PAR). Seedlings were divided into three batches, including two batches subjected to different doses of UV-B (5 W ∙ m-2 and 10 W ∙ m-2, one hour per day) for 5 days, and a control group exposed to PAR only. Exposure to UV-B increased anthocyanin levels in the cotyledons of all examined cultivars, it inhibited hypocotyl elongation, but did not affect the content of photosynthetic pigments. Flavone concentrations increased in cv. Red Corolla and Kora, remained constant in cv. Panda and decreased in cv. Hruszowska. Exposure to UV-B decreased rutin levels in cv. Hruszowska, but not in the remaining cultivars. Cultivars Hruszowska, Panda and Kora appeared to be less resistant to UV-B than Red Corolla. Higher resistance to UV-B radiation in Red Corolla can probably be attributed to its higher content of anthocyanins and rutin in comparison with the remaining cultivars.

Photo-excitation of carotenoids causes cytotoxicity via singlet oxygen production.

Carotenoids, natural pigments widely distributed in algae and plants, have a conjugated double bond system. Their excitation energies are correlated with conjugation length. We hypothesized that carotenoids whose energy states are above the singlet excited state of oxygen (singlet oxygen) would possess photosensitizing properties. Here, we demonstrated that human skin melanoma (A375) cells are damaged through the photo-excitation of several carotenoids (neoxanthin, fucoxanthin and siphonaxanthin). In contrast, photo-excitation of carotenoids that possess energy states below that of singlet oxygen, such as ß-carotene, lutein, loroxanthin and violaxanthin, did not enhance cell death. Production of reactive oxygen species (ROS) by photo-excited fucoxanthin or neoxanthin was confirmed using a reporter assay for ROS production with HeLa Hyper cells, which express a fluorescent indicator protein for intracellular ROS. Fucoxanthin and neoxanthin also showed high cellular penetration and retention. Electron spin resonance spectra using 2,2,6,6-tetramethil-4-piperidone as a singlet oxygen trapping agent demonstrated that singlet oxygen was produced via energy transfer from photo-excited fucoxanthin to oxygen molecules. These results suggest that carotenoids such as fucoxanthin, which are capable of singlet oxygen production through photo-excitation and show good penetration and retention in target cells, are useful as photosensitizers in photodynamic therapy for skin disease.

Carotenoids in human skin.

The interaction of free radicals with antioxidants is a topic of increasing interest in the development of prevention strategies against skin ageing. Carotenoids can serve as marker substances for the complete antioxidative network of human skin. Recently, it has become possible to measure the carotenoids non-invasively and online using resonance Raman spectroscopy. This method has been used in various studies to investigate the interaction of carotenoid antioxidants and free radicals in human skin. In this review, the results of the selected studies are summarized and compared. It could be demonstrated that the carotenoid concentration of the skin reflects the lifestyle of individuals. A high level of carotenoids can be achieved with a healthy diet rich, for instance, in fruit and vegetables. Stress factors such as illness, UV and IR radiation of the sun, and smoking and alcohol consumption reduce the concentration of the carotenoids in the skin. It could be demonstrated that premature skin ageing was less in people with a high level of antioxidants in their tissue. Consequently, the furrows and wrinkles were not so deep and dense as in the skin of individuals with a low antioxidant level. The measurements are highly suited for the development of anti-ageing strategies and can be efficiently used in the medical diagnostics and therapy control.

The gene for the heat-shock protein HSP100 is induced by blue light and heat-shock in the fungus Phycomyces blakesleeanus.

We cloned and sequenced the Phycomyces hspA gene. The hspA gene product is a 901-amino-acid protein member of the clpB/HSP100 family. HSP100 proteins are ATPases involved in high-temperature tolerance, proteolysis, and protein disaggregation. Phycomyces HSP100 is composed of a domain presumably involved in protein-protein interactions and two ATP-binding domains. The hspA promoter contains three heat-shock elements that are presumably involved in the activation of hspA after heat-shock. In addition, four short sequences are present in the hspA promoter and in the promoter of the photoinducible genes carB and carRA; and these are candidates as binding sites for light-regulated transcription factors. Blue light can increase transcription of the hspA gene 10-fold, with a threshold of 1 J/m2. The threshold for hspA photoactivation is 10(4) times higher than the thresholds for blue-light regulation of sporangiophore development and photocarotenogenesis, which suggests that there are differences in the photosensory systems for gene photoactivation and mycelial photoresponses. A heat-shock of 30 min at 34 degrees C or 42 degrees C increased hspA gene activity 160-fold. The differences in maximum hspA gene transcription by blue light and heatshock suggest the presence of different regulatory mechanisms.

Energy transfer pathways in the minor antenna complex CP29 of photosystem II: a femtosecond study of carotenoid to chlorophyll transfer on mutant and WT complexes.

The energy transfer processes between carotenoids and Chls have been studied by femtosecond transient absorption in the CP29-WT complex, which contains only two carotenoids per polypeptide located in the L1 and L2 sites, and in the CP29-E166V mutant in which only the L1 site is occupied. The comparison of these two samples allowed us to discriminate between the energy transfer pathways from the two carotenoid binding sites and thus to obtain detailed information on the Chl organization in CP29 and to assign the acceptor chlorophylls. For both samples, the main transfer occurs from the S(2) state of the carotenoid. In the case of the L1 site the energy acceptor is the Chl a 680 nm (A2), whereas the Chl a 675 nm (A4-A5) and the Chl b 652 nm (B6) are the acceptors from the xanthophyll in the L2 site. These transfers occur with lifetimes of 80-130 fs. Two additional transfers are observed with 700-fs and 8- to 20-ps lifetimes. Both these transfers originate from the carotenoid S(1) states. The faster lifetime is due to energy transfer from a vibrationally unrelaxed S(1) state, whereas the 8- to 20-ps component is due to a transfer from the S(1,0) state of violaxanthin and/or neoxanthin located in site L2. A comparison between the carotenoid to Chl energy transfer pathways in CP29 and LHCII is presented and differences in the structural organization in the two complexes are discussed.

The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants.

The npq1 mutant of Arabidopsis thaliana (L.) Heynh. has no xanthophyll cycle due to a lack of functional violaxanthin de-epoxidase. Short-term exposure (<2 days) of detached leaves or whole plants to the combination of high photon flux density (1,000 micromol m(-2) s(-1)) and low temperature (10 degrees C) resulted in PSII photoinhibition which was more acute in npq1 than in the wild type. This increased photosensitivity of npql at chilling temperature was attributable to the inhibition of nonphotochemical energy quenching (NPQ) and not to the absence of zeaxanthin itself. In contrast to PSII, PSI was found to be phototolerant to chilling stress in the light in both genotypes. In the long term (10-12 days), PSII activity recovered in both npql and wild type, indicating that A. thaliana is able to acclimate to chilling stress in the light independently of the xanthophyll cycle. In npql, photoacclimation involved a substantial reduction of the light-harvesting pigment antenna of PSII and an improvement of photosynthetic electron transport. Chilling stress also induced synthesis of early light-inducedproteins (ELIPs) which, in the long term, disappeared in npql and remained stable in the wild type. In both genotypes, photoacclimation at low temperature induced the accumulation of various antioxidants including carotenoids (except beta-carotene), vitamin E (alpha- and -gamma-tocopherol) and non-photosynthetic pigments (anthocyanins and other flavonoids). Analysis of flavonoid-deficient tt mutants revealed that UV/blue-light-absorbing flavonols have a strong protective function against excess visible radiations. In contrast to the defect in npq1, the absence of flavonoids could not be overcome in the long term by compensatory mechanisms, leading to extensive photooxidative and photoinhibitory damage to the chloroplasts. Depth profiling of the leaf pigments by phase-resolved photoacoustic spectroscopy showed that the flavonoid-related photoprotection was due to light trapping, which decreased chlorophyll excitation by blue light. In contrast to flavonoids, the xanthophyll cycle and the associated NPQ seem to be mainly relevant to the protection of photosynthesis against sudden increases in light intensity.

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments.

The one-electron reduction potentials of the radical cations of five dietary carotenoids (beta-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980-1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.

[The modification of a radiation lesion with 9a-homo-13-thiaprostanoids]. / Modifikatsiia luchevogo porazheniia 9a-gomo-13-tiaprostanoidami.

The radioprotective properties of 12 compounds of 9a-homo-13-thiaprostanoid series were investigated under gamma irradiation using the molecular model of beta-carotene radio-oxidation in oleic acid in vitro, erythrocyte radiomimetic model in ex vivo-in vitro system as well as in vivo radiation damage in mice. Most of these compounds stimulated the radio-oxidation of beta-carotene, however in this model two prostanoids with natural alpha-chain displayed radioprotective properties. Expressed membrane stabilizing effect of two 9a-homo-13-thiaprostanoid nor-analogues was revealed in radiomimetic model experiments. Two 10, 10-dimethyl-13-thiaprostanoids raised animal radioresistance during in vivo experiments.

[The effect of vitamin- and beta-carotene-enriched products on the vitamin A allowance and the concentration of different carotenoids of the blood serum in victims of the accident at the Chernobyl Atomic Electric Power Station]. / Vliianie produktov, obogashchennykh vitaminani i beta-karotinom, na obespechennost' vitaminom A i kontsentratsiiu razlichnykh karotinoidov v syvorotke krovi lits, postradavshikh ot avarii na ChAES.

The results of clinical trials of efficiency of foods enriched by vitamins and beta-carotene in people suffered from Chernobyl's accident are presented. The level of beta-carotene in clinical diets was the same during trial. Daily consumption of enriched food supplying ingestion of 4-5 mg of beta-carotene increased the level of beta-carotene in serum by 2-4 times. The concentration of total carotenoides in serum was increased by 1.6 times practically at the expense of beta-carotene.

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation.

Using dioleoylphosphatidylcholine liposomes incorporating various fatty acids and neutral lipids, we have examined the ability of such lipids to provide protection of Escherichia coli and vesicular stomatitis virus (VSV) against the lethal effect of ultraviolet (254 nm) radiation. While the presence of varying amounts of saturated (palmitic) or polyunsaturated (arachidonic) fatty acids or the lipid antioxidant, alpha-tocopherol, had little effect on killing by ultraviolet radiation, considerable radioprotection was observed with beta-carotene, retinal and vitamin K-1 at final concentrations of 1 mg/ml. In another approach, vesicular stomatitis virus grown under conditions in which its envelope fatty acid composition was substantially modified, showed little change in its sensitivity to inactivation by ultraviolet radiation. The results provide strong evidence for a radioprotective role of certain, relatively rare natural lipid components with conjugated polyene systems, but not of the more ubiquitous and abundant membrane fatty acids.

[Carotenoid pigments and the enhanced resistance of Pseudomonas methanolica to the action of ultraviolet radiation]. / Karotinoidnye pigmenty i povyshennaia ustoichivost' Pseudomonas methanolica k deistviiu ul'trafioletovogo izlucheniia.

The correlation was studied between the elevated resistance of Pseudomonas methanolica, a facultative methylotroph, to the bactericidal action of UV (254 nm) and the content of a pink pigment in this organism. Absorption spectra of cell extracts containing the pigment were recorded. The ethanol and acetone extracts had two absorption maxima at 420-430 and 500-510 nm, respectively, which was typical of carotenoids. The culture with the pigment and its variant without the pigment had nearly identical survival rates. Therefore, the elevated resistance of P. methanolica cells seems to stem from the activity of DNA reparation cellular systems rather than from the presence of carotenoid pigments in the cells.

[Effect of the cyclic nucleotide level on the degree of carotenoid pigment formation in the mycelial cells of Neurospora crassa]. / Vliianie urovnia tsiklicheskikh nukleotidov na intensivnost' obrazovaniia karotinoidnykh pigmentov v kletkakh mitseliia Neurospora crassa.

The relation between the content of cyclic nucleotides and the rate of formation of carotenoid pigments in the Neurospora crassa mycelium cells was investigated. Light derepression of the carotenoid synthesis during the photoinduction lag-period induced a transient decrease of the cAMP content. The intracellular cAMP content was in negative correlation with the constitutive level of carotenoid pigments. The cGMP content remained unchanged during the photoinduction lag-period and showed no correlation with the constitutive level of carotenoids in N. crassa cells.

Correlation between ATP synthesis and the decay of the arotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata.

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and Pi concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K+ and by ionophores. Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H+/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367-372). The ability of nucleotide diphosphates in the presence of Pi and Mg2+ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis. Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.

Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation.

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10-20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b/c2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.

The bleaching of the bacteriorhodopsin chromophore by ionizing radiation.

The visible chromophore of bacteriorhodopsin, BR(570), undergoes progressive bleaching when subjected to 60CO gamma-irradiation. The low G-value for bleaching confirms that the site of the chromophore is highly protected. Positive and negative circular dichroic (CD) bands associated with the chromosphone undergo concomitant decrease in a manner which is consistent with two independent chromophores rather than exciton coupling between neighbouring chromophoric site.