Photodynamic therapy (PDT) is a promising treatment for colorectal cancer owing to its high selectivity and limited systemic side effects. However, the true potential of PDT for therapeutic applications against tumors hasn’t been realized partly due to the complexity of PDT regimen. In the present study, we examine the efficacy of different fluence rates of hematoporphyrin derivative (HpD)-mediated PDT to predict long-term control rates of murine CT26 colorectal cancer. We further show how variation in the expression of immune checkpoints in the response of HpD-PDT at different fluence rates. Tumor-bearing mice were injected with 5 mg/kg HpD and subjected 48 h later to an 80 J/cm2 red light dose administered at fluence rates of 10, 50, and 100 mW/cm2. The expression of immune checkpoints(PD-1, LAG-3, and TIM-3)on tumor-infiltrating lymphocytes was measured 10 days after PDT. Mice treated with fluence rates of 10 and 50 mW/cm2 exhibited significantly longer survival than those treated at 100mW/cm2. Immune checkpoints on tumor-infiltrating CD8+ T cells were upregulated following PDT. Tumors treated with fluence rate of 10 mW/cm2 showed a significant increase of immune checkpoints on CD8+ T cells than those treated at 50 and 100 mW/cm2. Low light fluence rate results in significant tumor control and immune checkpoints upregulation. Improved tumor control could be expected by reducing the rate and combining checkpoint inhibitors with PDT using low fluence rate. Our data establish a correlation between activation of immune checkpoint and fluence rate and show the potential to combine PDT with checkpoint inhibitors.
Vascular-targeted photodynamic therapy (V-PDT) has been demonstrated to be an effective treatment modality for nonmalignant diseases such as capillary malformations port wine stains (PWS). V-PDT treatment of PWS involves a complex interaction of light, photosensitizer (PS), oxygen and biological tissue. A three-dimensional (3D) model of PWS with a Y-bifurcation blood vessels was proposed for mathematical simulation of the spatial and temporal distribution of singlet oxygen (1O2), which related to the distribution of light, PS and oxygen. A graphics processing units (GPU)-based method, Monte Carlo eXtreme (MCX), was applied to calculate the light deposition in the PWS, and a set of differential equations were utilized to describe the photochemical reactions involved in V-PDT. Using the obtained light energy deposition and the distributions of the PS and oxygen, a dynamic change model of the cumulative concentration of 1O2 ([1O2]) was estimated. The data show that the smaller vessels are more likely to acquire more [1O2].
Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new anti-gonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm wavelength), an innovative non-pharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no genotoxicity of aBL to the vaginal epithelial cells was observed at the radiant exposure for inactivating N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their co-cultures. No gonococcal resistance to aBL developed after 15 successive cycles of sub-therapeutic aBL inactivation. Taken together, aBL represents a potent potential treatment for antibiotic-resistant gonococcal infection.
KEYWORDS: In vivo imaging, Skin, In vitro testing, Raman spectroscopy, Tissues, Bacteria, Pathogens, Scanning electron microscopy, Laser therapeutics, Resistance
With the effectiveness of antimicrobials waning because of antimicrobial resistance, it is imperative that novel strategies are investigated for the treatment of infections. Antimicrobial blue light (aBL) is an innovative strategy that has proven efficacy against an array of pathogens, albeit, with different species having variable susceptibilities to the therapy. Quinine was discovered during the mid-17th century as a plant-derived potent antimalarial. More recently, its bactericidal properties were revealed, illustrating its potential as an antimicrobial adjuvant. Here we report a novel combination therapy, aBL+quinine hydrochloride (Q-HCL) for the treatment of multi-drug resistant infections. QHCL successfully potentiated the antimicrobial effects of aBL in numerous microbial pathogens of different etiologies, in vitro and in vivo. In addition, it synergistically improved the antimicrobial effects of aBL against bacterial biofilms. Raman spectroscopy revealed that concurrent exposure of aBL and Q-HCL improved uptake of Q-HCL into bacterial cells, when compared to the non aBL exposed sample. In addition, ultra-pure liquid chromatography (UPLC) revealed that Q-HCL increased the relative abundance of porphyrins in bacteria, suggesting the mechanism of this synergistic interaction is through increased production of intermediate photosensitizing porphyrins arising through perturbation of the heme biosynthesis pathway by Q-HCL. Genotoxic potential of the combination therapy against mouse skin tissue, was evaluated using the TUNEL assay, where it was revealed that a high dose exposure of aBL+Q-HCL (<3x the therapeutic dose) was not genotoxic to mouse skin tissue. In conclusion, the findings strongly suggest the potential of aBL+Q-HCL combination therapy as an alternative to traditional antibiotics for the treatment of localized infections.
Due to the growing global threat of antibiotic resistance, there is a critical need for the development of alternative therapeutics for infectious diseases. Antimicrobial blue light (aBL), as an innovative non-antibiotic approach, has attracted increasing attention. This paper discussed the basic concepts of aBL and recent findings in the studies of aBL. It is commonly hypothesized that the antimicrobial property of aBL is attributed to the presence of endogenous photosensitizing chromophores in microbial cells, which produce cytotoxic reactive oxygen species upon light irradiation. A wide range of important microbes are found to be susceptible to aBL inactivation. Studies have also shown there exist therapeutic windows where microbes are selectively inactivated by aBL while host cells are preserved. The combination of aBL with some other agents result in synergistically improved antimicrobial efficacy. Future efforts should be exerted on the standardization of study design for evaluating aBL efficacy, further elucidation of the mechanism of action, optimization of the technical parameters, and translation of this technique to clinic.
Neisseria gonorrhoeae is a human-adapted, gram-negative diplococcus that infects human reproductive tracts and causes gonorrhea, a sexually transmitted disease, resulting in discharge and inflammation at the urethra, cervix, pharynx, or rectum. Over the years, N. gonorrhoeae has developed resistance to nearly every drug ever used to treat it, including sulfonamides, penicillin, tetracycline, and fluoroquinolones. Drug-resistant N. gonorrhoeae is now considered by the Centers for Disease Control and Prevention (CDC) as an urgent threat. The present study aimed to evaluate the efficacy of antimicrobial blue light (aBL) at 405 and 470 nm for inactivating N. gonorrhoeae and reveal the mechanism of action. Our results showed that an exposure of 45 J/cm2 aBL at 405 nm reduced the bacterial CFU by 7.16-log10. When the aBL exposure was increased to 54 J/cm2, eradication of bacterial CFU was achieved. When the bacteria were exposed to aBL at 470 nm, 3-log10 reduction of CFU was observed at an aBL exposure of higher than 126 J/cm2. Absorption and fluorescence spectroscopic analyses revealed the presence of endogenous porphyrins and flavins in N. gonorrhoeae cells. The present study indicated that aBL is a potential strategy to control N. gonorrhoeae infections. Endogenous porphyrins play a vital role in the killing effects of aBL. In vivo experiments are ongoing in our laboratory to treat genital tract infections in mice using aBL and explore the potential clinical applications.
Background: An increasing prevalence of Candida infections has emerged with the wide use of immune-suppressants and antibiotics. Photodynamic inactivation (PDI) as a new approach to treat localized Candida infections is an emerging and promising field nowadays. This study evaluated the efficacy of photodynamic therapy using two new Cationic benzylidene cyclopentanone photosensitizers(P1 and P2) against strains of clinical fluconazole–resistant Candida albicans.
Methods: Suspensions and biofilms of Candida species were incubated with P1 and P2 concentrations (0.25~50 μM) for 30 min followed by 532nm laser irradiation. For planktonic suspensions, viability of cells was assayed by CFU counting. For biofilms, the metabolic activity was evaluated by XTT. Results: In PDI of a planktonic culture of clinical fluconazole–resistant Candida albicans, P2 showed the higher efficacy. After incubation with 25 μM of P2 for 30 min and irradiation with 532nm laser (36 J cm-2), the viability of C. albicans planktonic cells decreased by 3.84 log10. For biofilm cells, a higher light dose of 75 mW cm-2 was necessary to achieve 97.71% metabolic activity reduction.
Conclusions: The results of this investigation demonstrated that benzylidene cyclopentanone photosensitizer(P2)is an efficient photosensitizer to kill C. albicans. Moreover, single-species biofilms were less susceptible to PDT than their planktonic counterparts.
Vascular-targeted photodynamic therapy (V-PDT) is known to be an effective therapeutic modality for the treatment of port wine stains (PWS). Monitoring the PWS microvascular response to the V-PDT is crucial for improving the effectiveness of PWS treatment. The objective of this study was to use laser Doppler technique to directly assess the skin perfusion in PWS before and during V-PDT. In this study, 30 patients with PWS were treated with V-PDT. A commercially laser Doppler line scanner (LDLS) was used to record the skin perfusion of PWS immediately before; and at 1, 3, 5, 7, 10, 15 and 20 minutes during V-PDT treatment. Our results showed that there was substantial inter- and intra-patient perfusion heterogeneity in PWS lesion. Before V-PDT, the comparison of skin perfusion in PWS and contralateral healthy control normal skin indicated that PWS skin perfusion could be larger than, or occasionally equivalent to, that of control normal skin. During V-PDT, the skin perfusion in PWS significantly increased after the initiation of V-PDT treatment, then reached a peak within 10 minutes, followed by a slowly decrease to a relatively lower level. Furthermore, the time for reaching peak and the subsequent magnitude of decrease in skin perfusion varied with different patients, as well as different PWS lesion locations. In conclusion, the LDLS system is capable of assessing skin perfusion changes in PWS during V-PDT, and has potential for elucidating the mechanisms of PWS microvascular response to V-PDT.
Background: An increasing prevalence of Candida infections has emerged with the wide use of immune-suppressants and antibiotics. Current antifungal drugs exhibit low efficiency and high toxicity to the normal organs. Photodynamic inactivation (PDI) provides an alternative therapeutic strategy involving the use of photosensitizer (PS) and light irradiation. This study evaluated PDI effects against strains of C. albicans, C. parapsilosis, C. krusei and C. glabrata, using the PS of hematoporphyrin monomethyl ether (HMME), which is a second-generation PS clinically approved in China. Methods: Suspensions (~106 CFU/ml) were incubated with seven HMME concentrations (0.25~50 μM) for 30 min followed by 532-nm laser irradiation for 10 min at 40 mW/cm2. Viability of cells was assayed by CFU counting. Furthermore, fetal calf serum (10%) and singlet oxygen quencher sodium azide (100mM) were respectively added to the suspension of C. krusei to evaluate their roles in PDI process. Results: Among the four species, C. albicans was the most sensitive to PDI; 4 log10 killing was achieved at the concentration of 7.5 μM. C. glabrata was the most resistant; 3 log10 killing was not obtained even at PS concentration of 50 μM. PDI effects against C. krusei were inhibited by both serum and sodium azide. Conclusions: HMME-mediated PDI was able to effectively kill Candida in our experimental conditions, mainly through a Type Ⅱ photoprocess. However, the effects could be intensively reversed by the presence of serum. Thus, there might be a long way before HMME can be used in fighting against Candida in real infectious foci.
Response of port wine stain (PWS) to photodynamic therapy treatment (PDT) is variable and depends on treatment setting used and anatomic sites as well as on size and depth of ectatic vessels. Optical coherence tomography (OCT) is a non-destructive imaging modality which can reveal the layered structure of the upper part of the skin. The structural features of the PWS skin such as the diameter and depth of the blood vessels in different anatomic sites can be showed in the OCT images. In this study, the possible role of PWS skin structure in the response to PDT is assessed. 82 positions from 43 patients with PWS underwent OCT evaluation in cheek, zygomatic aera, preauricular and temporal region before and 3~4 months after the first PDT when treatment outcomes were also evaluated. After analyzing the structural features in different anatomic sites and the therapeutic effect of them, we found that the ectatic vessels diameter was obvious bigger in the cheek which had slightly poorer outcomes than other areas. Some typical structures had poor or good outcomes after first PDT. These can help the clinic doctors predict the response of some patients which have typical structures after PDT treatment. The OCT will be a potential tool for prediction of treatment response for port wine stain after photodynamic therapy.
We report the properties of two series of polyethylene glycol-functionalized bis(arylidene)cycloalkanone photosensitizers
designed for two-photon excited photodynamic therapy (PDT) with the aim to reveal the effect of the size of central ring
on the two-photon excited PDT efficiency. These photosensitizers are the derivatives of bis(arylidene) cyclopentanone
(B2, B3) and bis(arylidene) cyclobutanone (Q1-Q4). The bis(arylidene) cyclopentanone type photosensitizers were
found to have larger two-photon absorption cross sections than the bis(arylidene) cyclopentanone ones with the same
substituents. The singlet oxygen yields of the bis(arylidene) cyclobutanone derivatives are higher than the data of
bis(arylidene) cyclopentanone derivatives. All the studied photosensitizers showed no obvious toxicity under dark
situation. One- and two-photon excited PDT activities were successfully demonstrated by in vitro cell experiments.
Owing to the capability of destructing the cancerous cells under two-photon irradiation, bis(arylidene)cycloalkanone
based photosensitizers with proper substituents can be good candidates for two-photon excited PDT applications in the
future.
[Ru(bpy)2(dppn)]2+, one of Ru(II) polypyridyl complexes, present inner dicationic charge and high 1O2 quantum yield. In
this study, the synthetic compound was used as photosensitizer (PS) to photoinactivate a reference strain of
Staphylococcus aureus ATCC 25923. Bacterial suspensions consisting of 108 colony-forming units (CFU) per milliliter
were incubated with PS of different concentrations (0.025μM ~ 25μM). After a 30 minutes period, the suspensions
were exposed to 457nm laser light, determined by the absorption spectra of the PS in phosphate buffered saline (PBS),
with a power density of 40 mW/cm2 for 10 minutes (energy density of 24 J/cm2). PS group, light group and the blank
control were also concerned. Viability of bacteria was determined by pour plates. The Log10 reductions were calculated
and killing effects in photodynamic inactivation (PDI) group were analysed contrast to the blank control. We observed
that neither the laser light nor the PS per se had any inhibitory effect on the viability of the bacteria. PS at low dose
(0.025μM) followed by illumination yielded no significant decrease in the viable number. PS at 0.25μM and 2.5μM
with irradiation induced reductions of 1.69 Log10 and 5.97 Log10, respectively. PS at 10μM and 25μM combined with
light brought viable bacterial cells down to undetectable levels (reductions < 7 Log10). We concluded that with the PS of
appropriate doses, [Ru(bpy)2(dppn)]2+ mediated PDI inactivated S.aureus efficiently. At the concentration of 2.5μM,
bactericidal activity was reached where the viability of bacteria fell more than 3 Log10 based on previous researches.
To expand the optional laser wavelengths of photodynamic therapy (PDT) for port wine stain (PWS), the feasibility of applying a 457 nm laser to the PDT for infantile PWS was analyzed by mathematical simulation and was validated by clinical experiment. Singlet oxygen yield of 457 nm PDT or 532 nm PDT in an infantile PWS model and an adult PWS model was theoretically simulated. Fifteen PWS patients (14 infants and 1 adult) with 40 spots were treated with 457 nm (20 spots) and 532 nm (20 spots), respectively, in two PDT courses. Simulation results showed that under the same power density and irradiation time, singlet oxygen yield of 457 nm PDT and 532 nm PDT are similar in infantile PWS vessels. Yet, in adult PWS vessels, singlet oxygen yield of 457 nm PDT is lower than 532 nm PDT. Clinical outcomes showed that no statistic difference existed between 457 nm PDT and 532 nm PDT for infantile PWS. The result of this study suggested that 457 nm wavelength laser has the potential to be applied in PDT for infantile PWS.
Many laser wavelengths have been used in photodynamic therapy (PDT) for port wine stains (PWS). However, how these wavelengths result in different PDT outcomes has not been clearly illuminated. This study is designed to analyze which wavelengths would be the most advantageous for use in PDT for PWS. The singlet oxygen yield in PDT-treated PWS skin under different wavelengths at the same photosensitizer dosage was simulated and the following three situations were simulated and compared: 1. PDT efficiency of 488, 532, 510, 578, and 630 nm laser irradiation at clinical dosage (100 mW/cm2, 40 min); 2. PDT efficiency of different wavelength for PWS with hyperpigmentation after previous PDT; 3. PDT efficiency of different wavelengths for PWS, in which only deeply located ectatic vessels remained. The results showed that singlet oxygen yield is the highest at 510 nm, it is similar at 532 nm and 488 nm, and very low at 578 nm and 630 nm. This result is identical to the state in clinic. According to this theoretical study, the optimal wavelength for PDT in the treatment of PWS should near the absorption peaks of photosensitizer and where absorption from native chromophores (haemoglobin and melanin) is diminished.
Port wine stains (PWS) is a vascular malformation consisting of dilated capillaries in the superficial dermis.
Photodynamic therapy (PDT) is an effective approach in the treatment of PWS. However, the procedure of treatment is a
low efficient and hard work, as the doctor need to hold laser fiber to irradiate for 20 min to 50 min per lesion. So an
assisted novel robotic system was developed to instead part of doctor's work. The robotic system consisted of 7 degrees
of freedom, in which there were 5 passive joints and 2 active joints. Binocular surveillance system was used as guidance
for the robot. Clinical trial compared 20 patients (38 lesions) treated by the robotic system with another 20 patients (38
lesions) treated by a doctor. The patients in both groups were injected intravenously with photosensitizer (PSD-007,
4-5mg/kg) and irradiated with 532 nm laser (100mW/cm2, 120-300J/cm2) immediately. Both groups had same good
therapeutic results. The robotic system is helpful in the PWS-PDT and hopefully would become a part of PWS therapy
machine in the future.
To analyze the effect of concentration on the photobleaching of HMME, the loss of HMME absorption in simplex
solution (PBS) and complex solution (albumin buffer) was monitored using steady-state absorption spectra during
532nm laser irradiation. Three concentrations (4, 10, 80μmol/L) were set for each type solution. Photobleaching curves
at each concentration were drawn according to the concentration and time during irradiation. Photobleaching rate
coefficients (k) at each concentration were calculated with a mathematical model for singlet oxygen-mediated
photobleaching reaction. Then the photobleaching of HMME under different concentrations were compared using the
photobleaching rate coefficients (k) and the slope of photobleaching curves. Finally, the effect of concentration on
HMME photobleaching was analyzed combining the existence state of HMME at each concentration. The results showed
that in both PBS and albumin buffer, the photobleaching rate coefficient of HMME was the largest at 4μmol/L, smaller at
10μmol/L, and the smallest at 80μmol/L. In conclusion, there is a concentration-dependent relationship in the
photobleaching rate of HMME, photobleaching will be inhibited when the concentration of HMME increased above
certain level at which HMME aggregates formed.
Laser irradiation is the most acceptable therapy for PWS patient at present time. Its efficacy is highly dependent on the
energy deposition rules in skin. To achieve optimal PWS treatment parameters a better understanding of light
propagation in PWS skin is indispensable. Traditional Monte Carlo simulations using simple geometries such as planar
layer tissue model can not provide energy deposition in the skin with enlarged blood vessels. In this paper the structure
of normal skin and the pathological character of PWS skin was analyzed in detail and the true structure were simplified
into a hybrid layered mathematical model to character two most important aspects of PWS skin: layered structure and
overabundant dermal vessels. The basic laser-tissue interaction mechanisms in skin were investigated and the optical
parameters of PWS skin tissue at the therapeutic wavelength. Monte Carlo (MC) based techniques were choused to
calculate the energy deposition in the skin. Results can be used in choosing optical dosage. Further simulations can be
used to predict optimal laser parameters to achieve high-efficacy laser treatment of PWS.
To analyse the effect of oxygen on the photobleaching of HMME, photobleaching rates of HMME when bubbled with
oxygen or argon, added with singlet oxygen quenchers or hydroxyl radical quencher were compared. According to the
solvents in which HMME was dissolved, four groups were set, HMME-DMSO, HMME-PBS, HMME-Albumin buffer,
HMME-Cells suspension. Each group was divided into four subgroups, bubbled with oxygen or argon, added with
singlet oxygen quenchers or hydroxyl radical quencher respectively. Spectra measurement was performed at 10min
intervals for a period of 40 min before and after laser irradiation, photobleaching rates under different condition were
compared. Photobleaching was the fastest when bubbled with oxygen, apparentely inhibited when bubbled with argon or
added with singlet oxygen quenchers. Hydroxyl radical quencher can inhibit photopleaching of HMME slightly, but its
inhibition was gentler than that of singlet oxygen quenchers and argon. Oxygen plays an inportant role in thre
photobleaching of HMME, and the process is singlet oxygen mediated self-sensitized photooxidation.
KEYWORDS: Photodynamic therapy, Skin, Monte Carlo methods, Tissue optics, Absorption, Energy efficiency, Oxygen, Mass attenuation coefficient, Tissues, Blood vessels
Introduction: As it is always difficult to find the optimal combination of photosensitizer and of laser wavelength to achieve selective vascular damage in PWS-PDT, the selective vascular effects of HMME (Hematoporphyrin monomethyl ether) mediated PDT with 413 nm and with 532 nm were compared by mathematical simulation in this study.
Materials & Methods: Firstly, distribution of 413 nm, 532 nm light in PWS tissue was simulated by Monte Carlo model. Two energy density groups were set, one is 80mW/cm2x40min for both 413 nm and 532 nm, the other is 80mW/cm2x40min for 532 nm while 80mW/cm2x20min in for 413 nm. Secondly, the productivity of reactive oxygen species (ROS) in target vessels and normal tissue were simulated using a simulation system for PDT of PWS established in our lab, which considering the amount of light and photosensitizer in tissue, the molar extinction coefficient of photosensitizer, and quantum yield of ROS. Concentration of HMME for each wavelength were same. Finally, the productivity of ROS n in target vessels and normal tissue were compared between 413 nm PDT and 532 nm PDT under different energy density.
Result: Under the same energy density, ROS productivity in target vessels of 413 nm PDT was significantly higher than that of 532 nm PDT. Moreover, it was still higher at low energy density than that of 532nm PDT with high energy density.
Conclusion: HMME mediated PDT using 413 nm has the potential to increase the selective vascular effect of PDT for PWS by shortening treatment time.
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