Immune responses triggered by nanobody-targeted photodynamic therapy

Nanobodies are single domain antibodies derived from heavy chain antibodies that exist in Camelids1. The relatively small dimensions of nanobodies combined with high binding affinities to their targets, lead to rapid tumor accumulation, homogenous distribution, and rapid clearance of unbound fractions. We took advantage of these properties and have developed an alternative approach for targeted photodynamic therapy (PDT) by conjugating photosensitizers to nanobodies2. Several of our studies are targeting the epidermal growth factor receptor (EGFR) for head and neck cancer. As photosensitizer, we employ the IRDye700DX, that is a silicon phthalocyanine derivative, currently being tested in clinical trials conjugated to the monoclonal antibody cetuximab, and already approved for clinical use in Japan. Notably, with nanobody-targeted PDT, cytotoxicity is selectively induced in cells which have high target expression level. Preclinical studies have shown selective tumor necrosis3 and significant tumor regression after a single treatment session4.Similar to conventional PDT, we have shown that nanobody-targeted PDT can stimulate the immune system5. This was documented by the release of damage-associated molecular patterns (DAMPs), such as HSP70 and ATP, as well as the pro-inflammatory cytokines IL- 1β and IL-6. Conditioned medium from EGFR-expressing tumor cells treated with nanobody-targeted PDT led to the maturation of human monocyte-derived dendritic cells, and subsequently, these dendritic cells induced CD4+ T cell proliferation. After our initial studies exploring the immunomodulatory potential of nanobody-targeted PDT in vitro, we are currently conducting studies in vivo to further investigate the systemic reach of this selective and localized treatment. This presentation will summarize these studies and discuss preliminary data obtained from testing this treatment in cats that are diagnosed with oral squamous cell carcinoma.Acknowledgements: This work was supported by the European Research Council (ERC, Starting Grant 677582) and is supported by the Dutch Cancer Foundation (KWF, High Risk Grant 12736) and the Morris Animal Foundation (MAF, Established Investigator Grant D21FE-021).     

The photodynamic/photothermal synergistic therapeutic effect of BODIPY-I-35 liposomes with urea

Photodynamic therapy (PDT) has a successful track record in cancer. . Urea is a naturally occurring metabolite in the human body. Some studies have shown that it can inhibit the proliferation of tumor cells and cause oxidative stress. In order to explore the application of urea in enhancing the PDT effect, we synthesized a new photosensitizer (BODIPY-I-35) with good phototherapeutic effect and encapsulated it in liposomes. Compared with free BODIPY-I-35, water-soluble nanoliposomes (LipoBOD) produced a huge redshift (> 122 nm) of fluorescence emission in solution. When LipoBOD was irradiated with 808 nm laser (1 W/cm²) for 10 min, the temperature contrast increased by 20 °C, which was 4 times higher than free BODIPY-I-35. Confocal microscopy showed appreciable accumulation of LipoBOD in HeLa cells. In addition, when LipoBOD was incubated with urea in HeLa cells, we found that urea not only obviously enhanced the production of ROS, but also increased the apoptosis of HeLa cells. The synergistic effect of LipoBOD (20 μg/mL, at BODIPY-I-35-eq) with urea (250 mM) showed significantly higher phototoxicity than LipoBOD alone. Low dose can reduce the cell viability to 10%. Therefore, we have obtained an effective method of using urea to enhance the PDT effect.     

Multifunctional nanoplatform based on g-C3N4, loaded with MnO2 and CuS nanoparticals for oxygen self-generation photodynamic/photothermal synergistic therapy

Photodynamic therapy (PDT) and photothermal therapy (PTT) are both promising therapeutic approaches for cancer. Unfortunately, the anticancer efficiency of PDT is restricted by the hypoxic tumor microenvironment and the performance of the photosensitizer (PS) while the efficiency of PTT is limited by the penetration depth of NIR light, making it difficult to further improve the efficiency of the treatment. In this paper, we strategically proposed a multifunctional nano-platform based on g-C₃N₄ and loaded with CuS and MnO₂ nanoparticals. Interestingly, the obtained F127@CNs-CuS/MnO₂ nano-platform with high singlet oxygen quantum yield and excellent photothermal performance were used in synergistic PTT and PDT therapy to cope with the limitation of single mode cancer treatment under irradiation and has greatly improved the treatment effect. Additionally, MnO₂ nanoparticles loaded on the CNs surface could not only generate oxygen to ameliorate hypoxia in the tumor environment by reacting with H₂O₂ in tumor cells, but also react with the over-expressed reduced glutathione (GSH) in cancer cells to further improve the synergistic therapeutic effect. In the in vitro hepatocarcinoma cell inactivation experiment, the maximum cell inactivation efficiency of the PDT, PTT and PDT/PTT synergistic treatment group reached at 65% (F127@CNs-MnO₂), 69.2% (CNs-MnO₂) and 88.6% (F127@CNs-MnO₂) respectively, which means that the F127@CNs-CuS/MnO₂-mediated PTT/PDT synergy anticancer treatment was more effective than single mode therapy. In summary, the innovative multifunctional nanoplatform F127@CNs-CuS/MnO₂ used for synergistic PTT and PDT treatment has greatly improved the inactivation efficiency of cancer cells and has provided a new scheme for the treatment of hypoxic tumors.     

Recent advances and trends in nanoparticles based photothermal and photodynamic therapy

Light-mediated therapies, including photodynamic therapy (PDT) and photothermal therapy (PTT) have been exploited as minimally invasive techniques for ablation of various tumors., Both modalities may eradicate tumors with minimal side effects to normal tissues and organs. Moreover, developments of light-mediated approaches using nanoparticles (NPs) and photosensitizer (PS) as diagnostic and therapeutic agents may have a crucial role in achieving successful cancer treatment. In recent years, novel nanoplatforms and strategies have been investigated to boost the therapeutic effect.. In this regard, gold, iron oxide, graphene oxide nanoparticles and hybrid nanocomposites have attracted attention.. Moreover, the combination of these materials with PS, in the form of hybrid NPs, reduces in vitro and in vivo normal tissue cytotoxicity, improves their solubility property in the biological environment and enhances the therapeutic effects. In this review, we look into the basic principles of PTT and PDT with their strengths and limitations to treat cancers. We also will discuss light-based nanoparticles and their PTT and PDT applications in the preclinical and clinical translation. Also, recent advances and trends in this field will be discussed along with the clinical challenges of PTT and PDT.     

Rose bengal-modified gold nanorods for PTT/PDT antibacterial synergistic therapy

In this study, Rose Bengal (RB) was loaded onto mesoporous silica coated gold nanorods (AuNR@SiO₂-NH₂) to form a novel multifunctional platform for antimicrobial therapy (AuNR@SiO₂-NH₂-RB). The platform combines the photothermal functions of AuNR and the photodynamic functions of RB to effectively inactivate bacteria under irradiation. Moreover, AuNR@SiO₂-NH₂-RB showed negligible cytotoxicity and good blood compatibility. Therefore, this work has potential significance for the development of new antibacterial agents.     

Antibacterial photodynamic therapy for dental caries: evaluation of the photosensitizers used and light source properties

Photodynamic therapy studies have shown promising results for inactivation of microorganisms related to dental caries. A large number of studies have used a variety of protocols, but few studies have analyzed photosensitizers and light source properties to obtain the best PDT dose response for dental caries. This study aims to discuss the photosensitizers and light source properties employed in PDT studies of dental caries. Three questions were formulated to discuss these aspects. The first involves the photosensitizer properties and their performance against Gram positive and Gram negative bacteria. The second discusses the use of light sources in accordance with the dye maximum absorbance to obtain optimal results. The third looks at the relevance of photosensitizer concentration, the possible formation of self-aggregates, and light source effectiveness. This review demonstrated that some groups of photosensitizers may be more effective against either Gram positive or negative bacteria, that the light source must be appropriate for dye maximum absorbance, and that some photosensitizers may have their absorbance modified with their concentration. For the best results of PDT against the main cariogenic bacteria (Streptococcus mutans), a variety of aspects should be taken into account, and among the analyzed photosensitizer, erythrosin seems to be the most appropriate since it acts against this Gram positive bacteria, has a hydrophilic tendency and even at low concentrations may have photodynamic effects. Considering erythrosin, the most appropriate light source should have a maximum emission intensity at a wavelength close to 530 nm, which may be achieved with low cost LEDs.     

Melanoma-targeted photodynamic therapy based on hypericin-loaded multifunctional P123-spermine/folate micelles

Multifunctional P123 micelle linked covalently with spermine (SM) and folic acid (FA) was developed as a drug delivery system of hypericin (HYP). The chemical structures of the modified copolymers were confirmed by spectroscopy and spectrophotometric techniques (UV–vis, FTIR, and ¹H NMR). The copolymeric micelles loading HYP were prepared by solid dispersion and characterized by UV–vis, fluorescence, dynamic light scattering (DLS), ζ potential, and transmission electron microscopy (TEM). The results provided a good level of stability for HYP-loaded P123-SM, P123-FA, and P123-SM/P123-FA in the aqueous medium. The morphology analysis showed that all copolymeric micelles are spherical. Well-defined regions of different contrast allow us to infer that SM and FA were localized on the surface of micelles, and the HYP molecules are located in the core region of micelles. The uptake potential of multifunctional P123 micelle was accessed by exposing the micellar systems loading HYP to two cell lines, B16-F10 and HaCaT. HYP-loaded P123 micelles reveal a low selectivity for melanoma cells, showing significant photodamage for HaCat cells. However, the exposition of B16-F10 cells to Hyp-loaded SM- and FA-functionalized P123 micelles under light irradiation revealed the lowest CC₅₀ values. The interpretation of these results suggested that the combination of SM and FA on P123 micelles is the main factor in enhancing the HYP uptake by melanoma cells, consequently leading to its photoinactivation.     

Light source is critical to induce glioblastoma cell death by photodynamic therapy using chloro-aluminiumphtalocyanine albumin-based nanoparticles

Selection of an efficient light source is fundamental in the development of photodynamic therapy (PDT) protocols. However, few studies provide a comparison of different light sources with regard to phototoxic effects. Here, we compared the cell death induced by photoactivation of chloro-aluminiumphtalocyanine (AlClPc)-loaded human serum albumin nanoparticles under irradiation with different light sources: continuous laser (CL), pulsed laser (PL), and light-emitting diode (LED). Cells were exposed to three different AlClPc concentrations (1, 3, and 5 μM) and three different light doses (200, 500, and 700 mJ/cm²) for each light source. Cell death and differentiation of apoptosis and necrosis pathway were measured by flow cytometry. CL was the best light source for improving the photodynamic action of AlClPc-loaded albumin nanoparticles in glioblastoma cells and avoiding undesirable side effects, especially at low photosensitizer doses (200 mJ/cm²). In addition, apoptosis was the main cell death pathway in all evaluated cases (70% for CL, and greater than 50% for PL and LED). In conclusion, the search for optimal light sources and light/photosensitizer doses is a crucial step in improving PDT outcomes and enhancing the clinical translation of PDT.     

Clinical evaluation of a short illumination duration (1 hour) when performing photodynamic therapy of actinic keratosis using the Dermaris light source

Photodynamic therapy (PDT) using daylight as the photoactivating light source (DL-PDT) is an effective treatment for actinic keratosis (AK). Among the artificial daylight sources, the Dermaris (Surgiris, Croix, France) is specially designed for SDL-PDT (simulated daylight PDT) of AK. To perform the PDT session, a long duration (2.5 h) and low-intensity light exposure (2.9 mw/cm²) is used. This long duration is considered as a major limitation by both the patient and the dermatologist. The study aims to evaluate the clinical outcomes of SDL-PDT using the Dermaris in patients treated for AK lesions of the scalp at our medical dermatology center using only one hour low-intensity light exposure.Thirty patients (19 males, 11 females), mean age: 72.8 ± 9.3, with phototype 1 (11 patients), with phototype 2(17 patients) and phototype 3 (2 patients), with grade I-II AK of the scalp were treated with a drug-light interval (DLI) of 10 min and a light exposure of 1 h. The cure rate of AK lesions at six months after the treatment was determined. Erythema, crusts, discomfort and during or/and post the treatment were also evaluated. In total, 293 AK were treated. Six months following treatment, the cure rate of patients was 93%. Pain score reported after the first PDT session was from 0 to 1 on a visual analogue scale (VAS) ranging from 0 to 10. Erythema was observed in 28 patients and lasted 3 days, crusts were seen in 19 patients. Discomfort was as mild or less in more than 97% of patients.The shortening of the exposure time to one hour does not modify the efficacy of the SDL-PDT using the Dermaris. This observation is in agreement with recent published data demonstrating that PDT can be performed successfully with half the illumination time used in daylight PDT today. Besides, this clinical study confirmed that SDL-PDT is an effective and nearly painless treatment with minimal side effects for patients with AK lesions of the scalp.     

The synergistic effect of Nano-propolis and curcumin-based photodynamic therapy on remineralization of white spot lesions: An ex vivo study

ObjectiveOne of the major problems in fixed orthodontic treatments is the control of enamel demineralization, white spots lesions (WSL), and dental caries around the brackets, which can be stopped by strengthening the remineralization process. The aim of this study was to investigate the synergistic effect of propolis nanoparticles (NPro) and nano-curcumin-based photodynamic therapy (NCur-PDT) in the remineralization of WSL ex vivo.Materials and MethodsExperimental period was 5.5 months. After synthesis and characterization of NPro, the extracted bovine teeth were demineralized using a demineralization solution. They were divided into 7 groups (n=10) and treated in the following groups: 1) Negative control (artificial saliva), 2) Positive control or control of treatment (2% neutral sodium fluoride gel; NSF), 3) Nano-curcumin (NCur), 4) NPro, 5) Diode laser irradiation (light), 6) NCur with irradiation (NCur-PDT) and 7) NPro plus NCur-PDT (NPro+NCur-PDT). The treatment duration was 3 months and each treatment was conducted on T1 (the end of the third week), T2 (the end of the sixth week), T3 (the end of the ninth week), and T4 (the end of the twelfth week). Then, the microhardness, surface changes, and surface topography of the enamel were examined using digital hardness tester, DIAGNOdent Pen Reading, and scanning electron microscope (SEM), respectively.ResultsIn all groups, after demineralization, the microhardness of tooth enamel decreased and after each of the treatments, the amount of microhardness significantly increased over time except light and artificial saliva groups. The earliest significant change in microhardness improvement was observed in the NPro+NCur-PDT group at T2 (P<0.05). Also, at T2, DIAGNODent Pen revealed the earliest significant improved change in the level of mineralization degree in the NPro+NCur-PDT group. NCur and light irradiation groups alone, like the control group (artificial saliva), did not lead to remineralization (P>0.05). Also, the NPro+NCur-PDT treatment results obtained from SEM after the last treatment in the twelfth week showed that remineralization in that group has significantly improved compared to other groups.ConclusionThe results of this study showed that combined use of NPro and NCur-PDT had more enamel remineralization efficacy in a shorter period. Simultaneous application of NPro and NCur-PDT had also a stronger therapeutic effect after 3 months.     

Development of a light emitting device for the treatment of peritoneal carcinomatosis of ovarian origin by intracavitary photodynamic therapy

IntroductionPeritoneal carcinomatosis of ovarian origin (PCO) is an evolution of ovarian cancer, which is the fourth leading cause of death by cancer in women in France. PCO is defined by dissemination of cancerous cells from ovarian cancer to the peritoneal cavity. Photodynamic therapy (PDT) has been proposed in complement to the standard of care, consisting of surgery and chemotherapy. However, litterature has highlighted the lack of selectivity of available photosensitizers (PS) leading to inconclusive results [1, 2]. In this context, PRODYNOV project, initiated by our research team INSERM U1189 ONCOTHAI, has enabled the development of a patented PS. The high selectivity of this PS makes possible to carry out relevant PDT for PCO provided that an adequate illumination device would exist. In this study, we developed and assessed such a device.MethodsFirst, a test bench aiming to evaluate quantity and homogeneity of the delivered illumination has been developed. It is composed of a fantom of peritoneal cavity in which seven optical probes connected to a power-meter were placed in strategic zones characterised by high recurrence risk.Then, three illumination devices were implemented and assessed. The first one consisted of six fixed light emitting fabrics (LEF), the second one was a moving luminous wand, and the last one, a hybrid one, combined a fixed luminous wand and six fixed LEF. Light doses received by the probes have been calculated by integrating measured powers over illumination time.ResultsEach of the seven optical probes received a mean light dose of 0.68 mJ with the first illumination device (minimum: 18.37 10⁻³ mJ, maximum: 2.66 mJ), 0.11 mJ with the second one (minimum: 5.25 10⁻³ mJ, maximum: 0.35 mJ) and 0.65 mJ with the third one (minimum: 48.19 10⁻³ mJ, maximum: 1.27 mJ). With a variation coefficient of 77.1% (versus 93.1% for the first device and 119.5% for the second one), the hybrid device enabled to homogeneously illuminate the largest part of the cavity. For these reasons, the hybrid method has been selected as illumination process for PDT of PCO.ConclusionIllumination solutions for PDT of PCO have been proposed and tested. One of these solutions has been approved.The light dose necessary for an effective treatment remains to be determined and first feasability tests will be led on mini-pig by the end of the year.     

Efficacy of erythrosine and cyanidin-3-glucoside mediated photodynamic therapy on Porphyromonas gingivalis biofilms using green light laser

BackgroundThe purpose of this in vitro study was to evaluate the efficacy of erythrosine and cyanidin-3-glucoside as photosensitizers in PDT for the elimination of Porphyromonas gingivalis (P. gingivalis) biofilms.MethodsP. gingivalis biofilms were prepared from a chronic periodontitis subject. Erythrosine and cyanidin-3-glucoside were prepared and randomly allocated as follows: 110, 220, 330, and 440 μM erythrosine; 101, 202, 303, and 404 μM anthocyanin; and 440 μM erythrosine + 404 μM cyanidin-3-glucoside. There were 18 PDT experimental groups (non-irradiated/irradiated with a 532-nm green light diode laser at 1.29 J/cm² for 60 s). The 3 controls were grouped as follows: biofilms exposed to the photosensitizers alone, biofilms exposed to the laser alone, and biofilms exposed to 0.12% chlorhexidine. All sample groups were cultured at 1, 3 and 6 h after PDT and incubated in an anaerobic chamber at 37 °C for 4 days. The surviving fraction was calculated from the log₁₀ CFU/ml. The 330 and 440 μM erythrosine and the 440 μM erythrosine + 404 μM cyanidin-3-glucoside were mixed with spin traps (TEMPO, DMPO), and the electron spin resonance spectra were evaluated.ResultsThe log₁₀ CFU/ml measurements showed that the PDT groups with 330 μM or 440 μM erythrosine and 440 μM erythrosine + 404 μM cyanidin-3-glucoside had statistically significant differences from the other groups (one-way ANOVA and Bonferroni’s multiple comparison test, p- value ≤ 0.05).ConclusionsPDT using 330 μM erythrosine, 440 μM erythrosine or 440 μM erythrosine + 404 μM cyanidin-3-glucoside irradiated with the laser more effectively inhibited P. gingivalis in biofilms.     

Evaluation of antimicrobial photodynamic therapy on wounds infected by Staphylococcus aureus in animal models

BackgroundAntibiotic-resistant Staphylococcus aureus bacteria are one of the expanding challenges. The purpose of current study is to evaluate the antimicrobial effect of photodynamic therapy (aPDT) on wounds infected to Staphylococcus aureus.MethodsIn this study, 40 six-month-old rats were divided into 4 groups: control, photosensitizer (PS), laser, and aPDT. A full-thickness wound was created on their skin and it was infected by Staphylococcus aureus. For aPDT, the Indocyanine Green (Germany, Nürnberg, A.R.C. Laser, EmunDo) photosensitive agent and laser diod 810 nm (Germany, Nürnberg, A.R.C. Laser) was utilized. The wound healing procedure was monitored every 24 h until the 12th day with photography. The number of the bacteria was counted on the 12th day also. All results were compared using ANOVA and Tukey post hoc tests. Significance level was considered P-Value < 0.05.ResultsThe average area of wound reduced in days 5–11th in photosensitizer, laser, and aPDT, respectively. The absolute colonization rate of bacteria in the wounds showed a significant decrease in two groups laser and aPDT compared to the control group. However, the lowest value was for the aPDT.ConclusionIn the conditions of this study, it emerged that aPDT and laser have an antimicrobial effect against antibiotic-resistant bacteria (particularly Staphylococcus aureus) and improve wound healing.     

In vitro evaluation of adhesion/proliferation of human gingival fibroblasts on demineralized root surfaces by toluidine blue O in antimicrobial photodynamic therapy

BackgroundAntimicrobial photodynamic therapy (aPDT) in Dentistry has important effects as bacterial destruction in areas with periodontal disease. Some dyes applied in aPDT could present low pH and, consequently, result in tooth demineralization. This study evaluated demineralization produced by aPDT with toluidine blue O (TBO) at low pH and analyzed adhesion/proliferation of human gingival fibroblasts (HGF).MethodsIn the 1st phase, bovine enamel and root dentin fragments received 2 treatments: PDT4 group (TBO–100 μg/ml—pH 4–60 s) plus laser (660 nm, 45 J/cm², 1.08 J, 30 mW, 30 s, spot 0.024 cm², 1.25 W/cm², sweeping, non-contact) and CA group (citric acid plus tetracycline—pH 1–180 s). Surface hardness loss and tooth wear were statistically analyzed (Student’s t test, ANOVA/Tukey, p < 0.05). In the 2nd phase, human dentin fragments were divided in C (control group—scaling and root planing), PDT4 and CA. HGF (10⁴, 5th passage) were cultured on these fragments for 24, 48 and 72 h and counted in scanning electron microscopy photographs. Number of HGF was analyzed using repeated-measures ANOVA and Tukey (p < 0.05).ResultsPercentage of surface hardness loss was similar in dentin for PDT4 (71.5%) and CA (76.1%) (p > 0.05) and higher in enamel for CA (68.0%) compared to PDT4 (34.1%) (p < 0.05). In respect to wear, no difference was found between PDT4 (dentin: 12.58 μm, enamel: 12.19 μm respectively) and CA (dentin: 11.74 μm and enamel: 11.03 μm) (p > 0.05). Number of HGF was higher after 72 h in CA group (2.66, p < 0.05) compared to PDT4 (2.2) and C (1.33).ConclusionPDT4 is not as aggressive as CA for enamel. However, dentin demineralized promoted by PDT4 does not stimulate HGF adhesion and proliferation as CA.     

Emerging trends in photodynamic therapy for head and neck cancer: A 10-year bibliometric analysis based on CiteSpace

BackgroundHead and neck cancer (HNC) was the seventh most common cancer worldwide. Photodynamic therapy (PDT) is a clinically approved, minimally invasive treatment, which was shown to be effective in the treatment of head and neck cancer and potentially malignant disorders. We used a bibliometric analysis to analyze the publications of radiomics in oncology to clearly illustrate the current situation and future trends and encourage more researchers to participate in radiomics research in oncology.MethodsPublications for Photodynamic therapy in for head and neck cancer and potentially malignant disorders were downloaded from the Web of Science Core Collection (WoSCC). CiteSpace was used for a bibliometric analysis of countries, institutions, journals, authors, keywords, and references pertaining to this field. The state of research and areas of focus were analyzed through burst detection.ResultsA total of 1002 studies were used for analysis on CiteSpace. The USA is in first place by number of publications. Hopper C, was the most prolific author, and the author with the most citations was Chen XY. Among the journals and the co-cited journals, “Photodiagnosis and Photodynamic Therapy” was the first. “Nanoparticle” showed the highest burst strength level and materials research is major area of focus in this field.ConclusionsThis bibliometric analysis of photodynamic therapy in head and neck cancer, provides a visual analysis of publications in this field. The conclusion of the current research in this field was that it focused on the research of photosensitizers, particularly nanomaterials and targeted therapies.