Hypericin Fluorescence Research Articles

Effect of pH on the Fluorescence and Absorption Spectra of Hypericin in Reverse Micelles¶

Effect of pH on the Fluorescence and Absorption Spectra of Hypericin in Reverse Micelles¶

Spectroscopic Study of Visible-light Effects on Hypericin-lens Proteins Systems†¶

Molecular interactions between hypericin and α-, β- and γ-crystallin proteins have been studied by means of absorption and steady-state fluorescence spectroscopy, aiming to clarify if and how the pigment binds to the proteins and to investigate the effects of visible-light irradiation on these molecular systems. Such a study is a prerequisite for assessing the possibility of using hypericin as a mild antidepressant and/or as a photodynamic agent for the treatment of eye tumors and eye viral and bacterial diseases without side injuries to the lens. We have shown that in dark-kept samples, with increasing α-crystallin concentration, both the fluorescence emission intensity and the ratio of the absorption maxima around 590 and 550 nm of hypericin increase. These effects have been attributed to the monomerization of nonfluorescent hypericin aggregates caused by the binding of the pigment to α-crystallin. The binding constant of hypericin has been evaluated to be of the order of 3.0 (mg/mL)−1, corresponding to a dissociation constant of the order of 0.3 mg/mL. Following irradiation with light of wavelengths over 400 nm, at an irradiance of 20 mW/cm2, both tryptophan and hypericin fluorescence emission intensities decrease. These effects are suggested to be the consequence of a spatial rearrangement of the protein framework which takes place following the α-crystallin photopolymerization sensitized by hypericin itself described in the literature. For the sake of comparison hypericin has been studied also in the presence of βH-, βL- and γ-crystallins at the same concentration.

Photodynamic therapy of malignant glioma with hypericin: Comprehensive in vitro study in human glioblastoma cell lines

The poor prognosis of patients suffering from malignant glioma requires further efforts. Photodynamic therapy (PDT) might be a therapeutic option to increase surgical radicality. Hypericin (HY) exhibit high phototoxicity to malignant cells and accumulates to a higher extent in glioblastoma cells as compared to neurons. Therefore, the impact of various experimental parameters on cytotoxicity, intracellular accumulation and phototoxicity of HY was quantitatively assessed in the three human glioblastoma cell lines U373 MG, LN229 and T98G. Additionally, intracellular location of HY was studied with fluorescence microscopic techniques. For all three cell lines, no cytotoxicity was found for incubation concentrations up to 5 microM. For short-time incubation (2 h), maximum HY fluorescence was achieved at an incubation concentration of about 5 microM. However, uptake kinetics of HY was dependent on its incubation concentration. Moreover, increase in HY fluorescence was negligible at 4 degrees C, which strongly indicates that the compound is taken up by an energy-dependent process. HY exhibited high phototoxicity (at 595 nm) in all three cell lines with ID50-values ranging from 0.15 J/cm(2) to 0.22 J/cm(2), but sensitivity decreased in the order U373 MG > LN229 > T98G. However, assessment of phototoxicity at different wavelengths revealed that highest cell inactivation was achieved at 600 nm. Fluorescence microscopy showed that HY fluorescence arose predominantly from the perinuclear region and the nuclear membrane. Fluorescence pattern of HY was significantly different from those observed for organelle markers staining lysosomes or mitochondria. Location of HY in the plasma membrane was proven by total internal reflection fluorescence microscopy. Thus, the present study demonstrates that glioblastoma cells can be effectively inactivated by HY-PDT after short-time incubation and exposure to low light doses. These results obtained in cell culture are encouraging and justify further evaluation HY-PDT for the treatment of malignant glioma in animal experiments.

Spectroscopic characterization and photobleaching kinetics of hypericin-N-methyl pyrrolidone formulations

Hypericin (HY) is a promising photosensitizer in photodynamic therapy (PDT). It was recently reported that appropriate use of N-methyl pyrrolidone (NMP) enhanced in vivo PDT efficacy of HY and enhanced in vivo delivery of HY. This present study further investigates the use of NMP and other known non-toxic pharmaceutical additives, polyvinylpyrrolidone (PVP, K29/32) and copolyvidonum (S630), for formulating HY to enhance its delivery with photodynamic activity as a goal in mind. Hence, the first objective of this study was to characterize the solubilization of HY by NMP, K29/32 and S630. Thermodynamic considerations were used to explain the solvation process. Photobleaching is another important property of photosensitizers. There is no report on the photostability of HY in pharmaceutical formulations used for PDT. Therefore, the second objective of this study was to investigate the photobleaching of HY in these formulations. The fluorescence of HY was found to increase significantly in higher concentrations of NMP or when 5% of polymer was co-mixed with 5% of NMP solution. The photobleaching of HY in these formulations followed first-order kinetics. The loss of fluorescence paralleled to the loss of absorption of HY. The formulation of HY with 40% NMP was found to be the most stable.

Evidence for Intracellular Aggregation of Hypericin and the Impact on its Photocytotoxicity in PAM 212 Murine Keratinocytes¶

AbstractWe have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 μM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 μM. However, there was a pronounced aggregation effect at 50 μM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 μM hypericin was comparable, whereas using 5 μM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.

A novel antioxidant role for ligandin behavior of glutathione S-transferases: attenuation of the photodynamic effects of hypericin.

Hypericin (HYP) is a major constituent of the herbal antidepressant St. John's wort with potential utility as an antitumor photodynamic sensitizer and antiviral agent. Upon irradiation at 540-600 nm, HYP generates reactive oxygen species (ROS) and induces oxidative stress. Here, human glutathione S-transferase (GST) isoforms GSTP1-1 (P1-1) and GSTA1-1 (A1-1) are shown to bind with high affinity to HYP and to differentially quench its photodynamic properties. In steady-state turnover studies, HYP inhibits A1-1 and P1-1 with IC(50) values of 160 and 190 nM, respectively. Fluorescence titration experiments and fitting of the data to the Hill equation yield apparent K(D)s for binding to A1-1 and P1-1 of 0.65 and 0.51 microM, respectively. The recovered Hill coefficients are 1.8 for both GSTA1-1 and GSTP1-1, indicating that multiple HYPs bind to each isoform. This behavior is reminiscent of classic "ligandin" activity of GSTs, wherein nonsubstrate planar aromatic anions are sequestered on, and inhibit, the enzyme. However, HYP complexed with P1-1 is photodynamically attenuated, with minimal protein oxidation. In contrast, light-dependent, oxygen-dependent, oxidation of A1-1 was modest and oxidation of human albumin was extensive in the presence of HYP, as monitored by electrospray mass spectrometry (ESI-MS). A peptide "trap" of diffusive ROS was oxidized extensively upon irradiation of HYP in the presence of albumin but very little in the presence of P1-1 or A1-1. Solute quenching studies were used to probe the accessibility of the bound HYP in each of the protein complexes. The fluorescence of HYP complexed with albumin, A1-1, or P1-1 was quenched by I(-) with quenching rate constants (k(q)) of 1.1 x 10(9), 2.4 x 10(9) and 0.5 x10(9) M(-1) s(-1), respectively, indicating that small molecules such as O(2) have similar diffusional access to the complexed HYP in each of the proteins, eliminating the possibility of differential accessibility of oxygen as the source of a different yield of ROS. This is the first demonstration of a possible antioxidant role for the ligandin activity of GSTs and a striking example of protein-specific effects on hypericin photodynamic activity. Even highly homologous protein isoforms can differentially promote or inhibit photosensitizer activity.

Evidence for Intracellular Aggregation of Hypericin and the Impact on its Photocytotoxicity in PAM 212 Murine Keratinocytes¶

We have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 microM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 microM. However, there was a pronounced aggregation effect at 50 microM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 microM hypericin was comparable, whereas using 5 microM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.

Novel photodynamic diagnosis of bladder cancer: Ex vivo fluorescence cytology using hypericin

In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non-invasive method for detecting early bladder cancers. To date this is the first study reported using this technique with hypericin. Urine samples from patients with early bladder cancers were processed for fluorescence cytology by incubation with hypericin, a novel photosensitizer. Normal urine samples incubated with hypericin served as normal controls. Laser confocal microscopy and spectroscopy was used to detect the fluorescence in the exfoliated low-grade urothelial tumor cells. Fluorescence cytology was considered positive if hypericin fluorescence of the low-grade urothelial tumor cells was detected to be stronger (>8.5 times) compared to the baseline fluorescence established for normal urine samples. Automated analysis for an objective reproducible outcome appears possible. The possibility of detection of malignant urothelial cells in early cancer makes ex vivo fluorescence cytology promising for routine diagnostic screening.

Microscopic quantification of hypercin fluorescence in an orthotopic rat bladder tumor model after intravesical instillation

We have previously investigated the possibility of using hypericin as a diagnostic tool for the fluorescence detection of flat bladder carcinoma. In these clinical studies, it was shown that following intravesical application in humans, hypericin becomes selectively localized in transitional papillary carcinoma and carcinoma in situ (CIS). In the present study, we characterized the biodistribution of hypericin in rat bladder tumor and normal bladder layers after intravesical administration. The biodistribution was evaluated using fluorescence microscopy with computerized image analysis to image and quantify the fluorescence of hypericin across the urothelial tumor and normal bladder wall. The results show that the photosensitizer intravesical administration route provides selective labelling of both the tumor and normal urothelium. A hypericin dose and instillation time-dependent increase in the hypericin fluorescence intensity in both the tumor and urothelium was observed, without significant hypericin fluorescence in the submucosa and muscle layers. The highest fluorescence ratios in hypericin accumulation in the tumor and normal urothelium to the muscle layer were achieved at 4 h with 30 micro M hypericin (20:1 for the urothelium to muscle layer and 30:1 for the tumor to muscle layer). The difference in fluorescence intensity in tumor tissue to the muscle layer following instillation of 8 micro M hypericin was 11:1, 25:1 and 28:1 at 1, 2 and 4 h, respectively. The difference in fluorescence intensity in tumor tissue to the muscle layer using 30 micro M hypericin was 17:1, 27:1 and 31:1 at 1, 2 and 4 h, respectively. The highest absolute fluorescence levels were observed in the tumor at 4 h with 30 micro M hypericin instillation. The results suggest that under these conditions, PDT with hypericin is likely to produce uniform urothelial tumor eradication, without causing damage to the underlying muscle layers.

Photosensitized structural modifications of the lens protein alpha-crystallin: do all modifications impair chaperone-like activity?

Among chaperone-like functioning proteins, the lens alpha-crystallins are of particular interest because they are not renewed, and even minor alterations can hurt their function of maintaining the proper refractive index and avoiding cataract formation in the lens. Several reports have suggested the occurrence of remarkable structural modifications in lens proteins in the presence of endogenous and exogenous sensitizers upon exposure to light. In particular, it has been shown in vitro that hypericin, the active ingredient of Hypericum, can bind to and, in the presence of light, cause the photopolymerization of alpha-crystallin. On the basis of these results it has also been suggested that a subsequent significant impairment of the protein function can occur. Using absorption and emission spectroscopic techniques, as well as circular dichroism, we have studied the structural modifications of alpha-crystallin resulting from its interaction with hypericin after irradiation with visible light. To investigate the chaperone-like function of alpha-crystallin, the heat-induced aggregation kinetics of another lens protein, betaLow-crystallin, was monitored by measuring the apparent absorption due to scattering at 360 nm as a function of time, and no apparent damage to its functional role was observed. Spectroscopic results, on the contrary, show a prominent reduction in both tryptophan and hypericin fluorescence emission intensity after light irradiation, suggesting an alteration in the tryptophan microenvironment and a high degree of packing of the chromophore due to photoinduced modification of the molecular framework. Control experiments on alpha-crystallin structurally modified by light in the presence of hypericin indicated that the protein still retains its ability to chaperone both lens crystallins and insulin.

Biodistribution of hypericin in orthotopic transitional cell carcinoma bladder tumors: implication for whole bladder wall photodynamic therapy.

In a recent clinical study, we reported a selective uptake of hypericin in superficial bladder tumors. The results suggested that hypericin, a potent photosensitizer, could be used not only for diagnosis but also for photodynamic therapy (PDT) of superficial bladder tumors. In the present study, we investigated the biodistribution of hypericin in an orthotopic rat bladder tumor model by assessing the extent of hypericin penetration and the kinetics of accumulation into rat bladder tumors and normal bladder wall. Hypericin (8 or 30 microM) was instilled into the bladder via the catheter for 1, 2 or 4 hr. The fluorescence of hypericin in the bladder tumors and normal bladder was documented using fluorescence microscopy. In situ quantification of hypericin fluorescence in the tumor or normal bladder was performed using the laser-induced fluorescence technique. There was much more hypericin fluorescence in the tumor than in the normal bladder, with the tumor-to-normal-bladder ratio mounting to 12:1 after 4 hr of hypericin (30 microM) instillation. Moreover, hypericin was retained in the tumor for at least 1 hr before it was gradually lost from the tissue. Microscopically, the fluorescence of hypericin was restricted to the urothelial tumor and normal urothelium without fluorescence in the submucosa and the muscle layers. Subsequently no hypericin was detected in plasma, indicating that under these conditions systemic side effects should not be expected. Because the conditions used in this study were similar to those used in our previous clinical study, it is therefore likely that whole bladder wall PDT in the clinic under these conditions will produce selective urothelial tumor destruction without causing damage to the underlying muscle layers.

Photochemical effects and hypericin photosensitized processes in collagen.

Emission and excitation spectra of collagen were recorded in the ultraviolet and visible regions. The existence of several types of chromophores absorbing and emitting throughout these spectral regions was observed. It was shown that laser irradiation at 355 and 532 nm caused collagen fluorescence photobleaching by 30%, when the delivered light doses were 9 and 18 J/cm2, respectively. This process of collagen fluorophores photodestruction was found to be a one-photon effect. The effect of hypericin (HYP), a polycyclic quinone, photosensitization on collagen was also studied. Addition of HYP aqueous solution to collagen produced quenching, redshift of the maximum, and broadening of the spectral form of its fluorescence. These effects became more prominent with increasing HYP concentration. The fluorescence of HYP sensitized collagen decreased in a spectrally nonproportional manner during laser irradiation at both 355 and 532 nm.

Spectroscopic study of visible-light effects on hypericin-lens proteins systems.

Molecular interactions between hypericin and alpha-, beta- and gamma-crystallin proteins have been studied by means of absorption and steady-state fluorescence spectroscopy, aiming to clarify if and how the pigment binds to the proteins and to investigate the effects of visible-light irradiation on these molecular systems. Such a study is a prerequisite for assessing the possibility of using hypericin as a mild antidepressant and/or as a photodynamic agent for the treatment of eye tumors and eye viral and bacterial diseases without side injuries to the lens. We have shown that in dark-kept samples, with increasing alpha-crystallin concentration, both the fluorescence emission intensity and the ratio of the absorption maxima around 590 and 550 nm of hypericin increase. These effects have been attributed to the monomerization of nonfluorescent hypericin aggregates caused by the binding of the pigment to alpha-crystallin. The binding constant of hypericin has been evaluated to be of the order of 3.0 (mg/mL)-1, corresponding to a dissociation constant of the order of 0.3 mg/mL. Following irradiation with light of wavelengths over 400 nm, at an irradiance of 20 mW/cm2, both tryptophan and hypericin fluorescence emission intensities decrease. These effects are suggested to be the consequence of a spatial rearrangement of the protein framework which takes place following the alpha-crystallin photopolymerization sensitized by hypericin itself described in the literature. For the sake of comparison hypericin has been studied also in the presence of beta H-, beta L- and gamma-crystallins at the same concentration.

Efficacy of antitumoral photodynamic therapy with hypericin: relationship between biodistribution and photodynamic effects in the RIF-1 mouse tumor model.

We investigated the hypericin-mediated PDT effects on the tumor and normal skin and in correlation with its biodistribution. These studies were carried out on C3H mice bearing RIF-1 tumors. The hypericin distribution and PDT effects were recorded at different intervals (0.5-24 hr) after intravenous injection of a 5-mg/kg dose of hypericin. After administration, rapid biphasic exponential decay was observed in the plasma drug concentration. It was found that hypericin was preferentially bound to the plasma lipoproteins. The tumor drug levels increased rapidly over the first few hours and reached a maximum around 6 hr after injection. In contrast, PDT efficacy was maximal when irradiation was performed at 0.5 hr after hypericin administration, which led to 100% cure. The PDT efficacy decreased rapidly as the administration-irradiation interval was prolonged. No tumor cure was obtained at the 6-hr interval, even though it was at this time that the tumor drug level peaked. Fluorescence microscopic studies showed that hypericin was mainly confined within the tumor vasculature at 0.5 hr after injection, whereupon it rapidly diffused to the surrounding tumor tissue. At 6 hr, a strong hypericin fluorescence was observed in the tumor tissue with only faint fluorescence within the vasculature, whereas at 24 hr the fluorescence in the tumor also decreased and became more diffused, and no fluorescence could be seen in the tumor vasculature. Like the tumor response, skin reactions were also found to be much more dramatic at short administration-irradiation intervals. Hypericin distribution and PDT response studies revealed a close correlation between the plasma drug level and the PDT effects, which suggests that vascular damage is the primary effect of hypericin-mediated PDT in this tumor model.

The uptake, location and fluorescence of hypericin in bovine intact lens

To determine the uptake, location and fluorescence of hypericin, the active ingredient in St. John's Wort, in situ in the isolated intact calf lens. The absorption and fluorescence spectra of hypericin 10(-5 ) M were measured in DMSO/phosphate buffer, pH 7.4) [PBS] (1/10 in volume) in the presence of alpha-crystallin (0.5 and 1.1 mg/ml). Bovine lenses were incubated in the dark for 24 hours in 10(-4) M hypericin in a DMSO/PBS (1/10 in volume) mixture. Diffused hypericin fluorescence emission was detected with a fluorescence stereomicroscope from the PBS washed lens surface. A lens-holder specially built for front-surface excitation-detection was used to measure fluorescence emission and excitation spectra of intact lenses incubated with hypericin solutions. As increasing concentrations of alpha-crystallin were added, the absorption and fluorescence spectra of hypericin in DMSO/PBS (1/10 in volume) changed, indicating a binding between the chromophore and the lens protein. Fluorescence emission spectra detected from the lens surface (lambda( em) = 601 and 651 nm; lambda(exc) = 550 nm) confirmed that hypericin does bind to the ocular tissues. The results we obtained in simplified model systems can provide clues to investigate the effects of hypericin on lens properties in physiological conditions. Hypericin could in fact bind to lens protein thus increasing the retention time of hypericin in the eye and possibly altering a-crystallin properties as a chaperone. Should therefore hypericin be taken up by the lens, this can be detected, non-invasively by its fluorescence. Therefore, ophthalmologists may use a slit-lamp or scanning fluorometry to monitor the uptake of hypericin in the eyes of patients using St. John's Wort or receiving high doses of hypericin while undergoing photodynamic therapy.

The uptake, location and fluorescence of hypericin in bovine intact lens

Purpose. To determine the uptake, location and fluorescence of hypericin, the active ingredient in St. John’s Wort, in situ in the isolated intact calf lens. Methods. The absorption and fluorescence spectra of hypericin 10 -5 M were measured in DMSO/phosphate buffer, pH 7.4) [PBS] (1/10 in volume) in the presence of a-crystallin (0.5 and 1.1 mg/ml). Bovine lenses were incubated in the dark for 24 hours in 10 -4 M hypericin in a DMSO/PBS (1/10 in volume) mixture. Diffused hypericin fluorescence emission was detected with a fluorescence stereomicroscope from the PBS washed lens surface. A lens-holder specially built for front-surface excitation-detection was used to measure fluorescence emission and excitation spectra of intact lenses incubated with hypericin solutions. Results. As increasing concentrations of a-crystallin were added, the absorption and fluorescence spectra of hypericin in DMSO/PBS (1/10 in volume) changed, indicating a binding between the chromophore and the lens protein. Fluorescence emission spectra detected from the lens surface (? em = 601 and 651 nm; ? exc = 550 nm) confirmed that hypericin does bind to the ocular tissues. Conclusions. The results we obtained in simplified model systems can provide clues to investigate the effects of hypericin on lens properties in physiological conditions. Hypericin could in fact bind to lens protein thus increasing the retention time of hypericin in the eye and possibly altering a-crystallin properties as a chaperone. Should therefore hypericin be taken up by the lens, this can be detected, non-invasively by its fluorescence. Therefore, ophthalmologists may use a slit-lamp or scanning fluorometry to monitor the uptake of hypericin in the eyes of patients using St. John’s Wort or receiving high doses of hypericin while undergoing photodynamic therapy.

Effect of pH on the fluorescence and absorption spectra of hypericin in reverse micelles.

The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa approximately 1.5 and approximately 12.5. These are assigned to the HypH+/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp-/Hyp2- equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH+, of Hyp and Hyp- and of Hyp2- are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2- or the HypH+ forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.

Fine fluorescence and absorption spectra of hypericin in a polymer matrix at a temperature of 4.2 K

Monochromatic photoexcitation and selective registration in bands of purely electronic S1↔S0 transitions resulted in fine fluorescence and absorption (fluorescence excitation) spectra of hypericin in polyvinylbutyral at a temperature of 4.2 K. Vibration analysis of the spectra is done and conclusions are made about the form of many normal vibrations. It is found that the formation of intramolecular hydrogen bonds in the hypericin structure has a substantial effect on the relative position of the electronic vibrational levels of the pigment molecule.

A study of photoinduced electron transfer and redox properties of hypericin

Hypericin (HYP), is a derivative of polyhydroxy substituted perylenequinones. In acetonitrile, the fluorescence of HYP can be quenched by electron donor: N,N-diethylaniline (DEA) and acceptors: methyl viologen (MV), anthraquinone (AN) (less than 1.2 × 10 −3 mol l −1), and the quenching rate constants are K q (DEA) = 3.52 × 10 10 l mol −1s −1 K q (MV) = 7.16 × 10 11 l mol −1 s −1, K q (AN) = 1.05 × 10 11 l mol −1s −1 respectively, indicating that hypericin can act either as an electron acceptor like quinones or as an electron donor like phenones. Hypericin in ground state maybe forms charge transfer complex with the higher concentrated anthraquinone which has a plane configuration. The stoichiometry is 1:2.6 (HYP:AN) determined by Job's photometric titration plots. The methyl viologen cation radical formed in the photoinduced interaction between hypericin and methyl viologen was also detected by the absorption spectrum.

Fluorescence Study of DNA–Dye Complexes Using One-Photon and Two-Photon Picosecond Excitation

Preliminary results of investigation of one-photon- and two-photon-induced fluorescence of acridine orange (AO), epirubicin (ER), hypericin (HYP), and ethidium bromide (EB) in complexes with DNA are presented. A spectrofluorometer based on a picosecond Nd:YAG laser was used for investigations of two-photon (1064-nm, 1-mJ, 40-ps) and one-photon (532- and 355-nm) dye excitation. The spectra of two-photon-induced fluorescence of dyes and their complexes with DNA as well as the kinetics of dyes' fluorescence intensification during their interactions with DNA in dependence on the biomacromolecule concentration were obtained. The intensities of AO, HYP, and EB fluorescence were increased 2.4, 3.2, and 8 times, respectively, after binding with DNA at two-photon excitation, while at one-photon excitation the corresponding values were 2.5, 3.7, and 10 times. The difference in fluorescence enhancement during DNA–dye complex formation at linear and nonlinear excitation may possibly be associated with the fact that the cross sections of one-photon and two-photon absorption, in general, change unequally during the binding of dyes to organic molecules and bathocromic shift of the electronic transitions. It was shown that the peak of AO fluorescence shifted to a longer wavelength on 10 nm after two-photon excitation at 1064 nm in comparison with one-photon excitation at 532 nm.