Gelatin hydrogel cross-linked with glutaraldehyde loaded with methylene blue for photodynamic action in gliosarcoma strain 9L/lacZ.

Synthesis and characterization of photosensitive gelatin-based hydrogels for photodynamic therapy in HeLa-CCL2 cell line

Methylene blue (MB) is a commonly used dye that can be used for near-infrared (NIR) imaging and photodynamic therapy (PDT) by producing reactive oxygen species after light exposure, inducing apoptosis. The limiting factor of MB is its poor penetration through cell membranes. Its decreased cellular uptake can be prevented by encapsulation in drug delivery systems such as liposomes. Additionally, the enhanced permeability and retention effect of tumors enables enhanced accumulation of nanocarriers at the target site. Nanosized, MB encapsulated, Tc-99m radiolabeled Lipoid S PC:PEG2000-PE:Chol: DTPA-PE and DPPC:PEG2000-PE:Chol:DTPA-PE liposomes were formulated to design multifunctional theranostic nanocarriers for: 1) NIR imaging, 2) gamma probe detection of sentinel lymph nodes (SLNs), and 3) PDT, which can provide accurate imaging and therapy helping surgery with a single liposomal system. The characterization of liposomes was performed by measuring particle size, zeta potential, phospholipid content, and encapsulation efficiency. Additionally, the in vitro release profile of MB and physical stability were also evaluated over 6 months at determined time intervals by measuring the mean particle size, zeta potential, encapsulation efficiency, and phospholipid content of liposomes kept at room temperature (25°C) and 4°C. Tc-99m radiolabeled, nanosized Lipoid S PC:PEG2000-PE:Chol:DTPA-PE and DPPC:PEG2000-PE:Chol:DTPA-PE liposomes showed suitable particle size (around 100 nm), zeta potential (-9 to -13 mV), encapsulation efficiency (around 10%), phospholipid efficiency (around 85-90%), and release profiles. Additionally, the liposomes found stable for 3 months especially when kept at 4°C. MB encapsulated, Tc-99m radiolabeled, nanosized Lipoid S PC:PEG2000-PE:Chol:DTPA-PE and DPPC:PEG2000-PE:Chol:DTPA-PE liposomes were found to have potential for SLN imaging by gamma probe detection, NIR imaging, and PDT. In vitro and in vivo imaging and therapeutic efficiency should be definitely evaluated to enable a final decision and our studies on this research topic are continuing.

Open AccessCCS ChemistryRESEARCH ARTICLE1 Mar 2022Building Block Symmetry Relegation Induces Mesopore and Abundant Open-Metal Sites in Metal–Organic Frameworks for Cancer Therapy Jing Sun†, Xuan Zhang†, Dong Zhang, Ying-Pin Chen, Fei Wang, Lan Li, Tian-Fu Liu, Huanghao Yang, Jibin Song and Rong Cao Jing Sun† State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002 School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210 , Xuan Zhang† MOE Key Laboratory for Analytical Science of Food Safety and Biology, College of Chemistry, Fuzhou University, Fuzhou 350116 , Dong Zhang State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002 , Ying-Pin Chen NSF's ChemMatCARS, The University of Chicago, Argonne, IL 60439 , Fei Wang State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002 University of Chinese Academy of Sciences, Beijing 100049 , Lan Li State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002 University of Chinese Academy of Sciences, Beijing 100049 , Tian-Fu Liu State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002 University of Chinese Academy of Sciences, Beijing 100049 , Huanghao Yang MOE Key Laboratory for Analytical Science of Food Safety and Biology, College of Chemistry, Fuzhou University, Fuzhou 350116 , Jibin Song *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] MOE Key Laboratory for Analytical Science of Food Safety and Biology, College of Chemistry, Fuzhou University, Fuzhou 350116 and Rong Cao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002 School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210 University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.021.202000634 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The judicious choice of metal clusters and organic building blocks leads to a wide variety of structures for metal–organic frameworks (MOFs). In this work, we demonstrated that relegating the symmetry of a building block can also lead to the proliferation of new MOF structures. Herein, a triangle building block was elongated with reduced symmetry for MOF construction, which gave rise to a novel (3,4,6)-connected idp (based on the definition of Reticular Chemistry Structure Resource, http://rcsr.net/) network ( PFC-16) with mesopores and abundant open-metal sites. The framework is composed of the rarely observed tetrakis hexahedral cages, surrounding which are small cages arranged in sodalite topology. The relegated symmetry was required for this novel self-assembly. The obtained MOF with mesopores, a robust backbone, and abundant open-metal sites can incorporate functional species in the structure, which is representatively demonstrated by internalizing the photosensitizer zinc(II) phthalocyanine (ZnPc) and the modifying tumor-targeting molecule folic acid (FA) in PFC-16. The obtained composite [email protected] shows excellent photodynamic therapy (PDT) efficiency for both in vitro and in vivo experiments, representing a promising candidate for cancer therapy. Download figure Download PowerPoint Introduction Metal–organic frameworks (MOFs), self-assembled through metal clusters and organic ligands, are a class of long-range ordered crystalline materials attracting much attention because of their high surface area, diverse structures, and wide applications.1–11 Among them, a great deal of research interest has been focused on Zr-based MOFs since a series of Zr-MOFs were initially reported in 2008.12 Zr-MOFs show not only excellent chemical stability but also highly predictable structure, which offers this class of materials large design space for potential applications. Although a variety of structures have been reported,13–15 there remains a pressing need to explore new Zr-MOFs combining high chemical stability, large pore size, and abundant open-metal sites for specific application demands. Elongation of organic building blocks is a traditional paradigm for synthesis of MOFs with large cavities.16,17 However, this strategy usually causes the obtained MOFs have interpenetrated structures instead of large cavities.18,19 Moreover, attempts of linker extension are very likely to yield the same topologies as the underlying frameworks, which misses out on new structures for MOF library.20–22 Previous studies show that using asymmetric organic ligands can result in highly porous materials, new topologies, and sometimes multiple metal clusters within a framework.23 These findings inspired us to introduce symmetry inequivalence into a multitopic building block for Zr-MOF design, which would bring an opportunity to create new types of structures unprecedented in Zr-MOFs. Moreover, relegated symmetry may induce the absence of connections in certain directions and therefore generate abundant open-metal sites, which would be beneficial to the catalytic performance of bulky MOFs. Taking triangular carboxylate linkers as examples, self-assembly of trimesic acid (H3BTC) with Zr6 clusters results in six-connected MOF-808 ( spn topology; spn = spinel).24 Symmetrically extending H3BTC to H3TATB and H3BTE for Zr-MOF construction gave rise to 6-connected PCN-777 (β-cristobalite topology) and 12-connected MOF-1004 [ sky (based on the definition of Reticular Chemistry Structure Resource) topology], respectively.25,26 If trimesic acid was elongated with a reduced symmetry, would it induce a new structure different from MOF-808, PCN-777, and MOF-1004? This scenario was supported in this work by deliberately designing a building block with reduced symmetry where two of the carboxylic moieties of H3BTC were replaced by ethynylbenzoic acid, and the other one was replaced by the relatively short benzoic acid (named as H3BDBA). Moreover, a methyl group was intentionally introduced on the benzoate group to force the connected carboxylate group perpendicular to the plane of two ethynylbenzoate arms (Figure 1, detail synthetic routine as shown in Supporting Information Scheme S1). With this modification, a triangle planer building block was constructed into a tetrahedral geometry. Self-assembly of H3BDBA and Zr ions generated a (3,4,6)-connected network PFC-16 [PFC = porous materials from FJIRSM (Fujian Institute of Research on the Structure of Matter), Chinese Academy of Science] with an idp network combining both six- and four-connected Zr6 clusters in one structure, which is unprecedented in Zr-MOFs. The entire framework is composed of the sodalite (SOD) cages and rarely observed tetrakis hexahedral cages (Catalan solids), and the twisted direction of three carboxylate groups is the prerequisite for this Catalan solid self-assembly. The obtained structure with mesopore, robust backbone, and abundant open-metal sites allows the incorporation of functional species in the structure, which is representatively demonstrated by internalizing photosensitizer zinc(II) phthalocyanine (ZnPc) and modifying tumor-targeting molecule folic acid (FA) in PFC-16. The obtained composite [email protected] shows excellent photodynamic therapy (PDT) efficiency for both in vitro and in vivo experiments, representing a promising candidate for bio-related applications.27,28 Figure 1 | Conceptual design of organic linker with relegated symmetry. Download figure Download PowerPoint Experimental Procedures Preparation of PFC-16 for single-crystal X-ray diffraction analysis H3BDBA (10 mg, 0.02 mmol) was dissolved in 1 mL of N,N-dimethylformamide (DMF) in a 10-mL uncapped vial, to which ZrCl4 (14 mg, 0.06 mmol) and trifluoroacetic acid (TFA; 100 μL, 1.35 mmol) were added. The mixture was ultrasonicated for 5 min and kept at 120 °C for 3 days. Light yellow cubic crystalline products were attained with 71.9% (12 mg) yield. Preparation of nano-PFC-16 H3BDBA (10 mg, 0.02 mmol) was dissolved in 10 mL DMF in a 20-mL vial, to which ZrCl4 (14 mg, 0.06 mmol) and TFA (10 μL, 0.135 mmol) were added, and then the mixture was kept at 90 °C for 24 h. A light yellow precipitate was attained with 83.9% (14 mg) yield. Preparation of [email protected] ZnPc (3.5 mg, 6 μmol) was added to the reaction mixture of nano-PFC-16, and after the reaction, a blue-green precipitate was attained with 80.7% (16.3 mg) yield. Preparation of [email protected] 10 mg of dried [email protected] powder was suspended in 1 mL DI, 4.4 mg FA was dissolved in 1 mL dimethyl sulfoxide (DMSO). The mixture was kept in a 90 °C oven for 2 days. Then the mixture was centrifuged and washed three times with DMSO/DI (v/v, 1/1) to remove excessive free folic acid. Cell culture MCF-7 (human breast adenocarcinoma cell line) cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and cultured at 37 °C containing 5% CO2 in the air with RPMI 1640 (purchased from HyClone, South Logan, UT) supplemented with 1% penicillin-streptomycin solution and 10% fetal bovine serum (FBS; HyClone). In vitro cytotoxicity of [email protected] The cell cytotoxicity was examined by standard CCK-8 assay. In brief, MCF-7 cells were seeded in a 96-well cell-culture plate (104 cells per well, 100 μL) and incubated in a humidified incubator at 37 °C with 5% CO2 for 24 h. Then the cells were treated with [email protected] at concentrations of 0, 10, 20, 40, 60, 100, and 200 μg mL−1 for another 12 h. Each cell well was washed with phosphate-buffered saline (PBS) twice after removing the culture medium. Finally, 100 μL mixture containing 10 μL CCK-8 solution and 90 μL culture medium was added into each well and incubated for 2 h. The OD450 (absorbance at 450 nm) value was measured by microplate reader. In vitro photodynamic performance of [email protected] Briefly, MCF-7 cells were seeded in 96-well plates (104 cells per well) for 24 h. Cells were refreshed with fresh medium containing different concentrations of [email protected] for another 12 h. Next, cells of each concentration group were irradiated with a 660 nm laser (0.8 W cm−2) for 10 min, and then incubated for another 2 h. Ultimately, 10 μL CCK-8 solution and 90 μL culture medium were added to determine the cell viability. Intracellular PDT performance of [email protected] MCF-7 cells were first plated onto confocal dishes (NEST, Scientific USA) for 12 h and incubated with [email protected] for another 12 h. The irradiating experimental group was treated with 660 nm laser for 10 min independently. Then, after washing twice with PBS, 1 μL calcein-AM and 1 μL propidium iodide (PI) were added and the cells were incubated for 30 min to be observed by fluorescence microscope. Intracellular reactive oxygen species generation of [email protected] The cancer cells were seeded in a glass bottom cell culture dish and incubated with [email protected] at 50 μg mL−1 for 24 h. After sequential 10 min of 660 nm irradiation, 10 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which is a reactive oxygen species (ROS) detector, was added into each plate and incubated for another 30 min. After washing with PBS twice, the generated ROS from each group was observed by the Nikon A1 confocal laser scanning microscope (CLSM). In vivo PDT efficacy All the animal experiments abided by the guide for the care and use of laboratory animals (Ministry of Science and Technology of China, 2006) and were approved by the Institutional Animal Care and Use Committee of Fujian Medical University. All the female BALB/c nude mice (6 weeks) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The subcutaneously implanted MCF-7 tumor models were constructed by hypodermic inoculation of MCF-7 cells (100 μL, 1 × 106) into the hind limbs of nude mice. When the tumor grew to 40 mm3, all the models were randomly divided into four groups (three mice in each group): PBS group, [email protected] group, laser only group, and [email protected] + laser group. All doses of [email protected] were 20 mg kg−1. A 100 μL dose was injected into each mouse in all groups through tail vein. The laser only and [email protected] + laser groups were irradiated at 660 nm for 15 min. Every 2 days, body weight was measured, and tumor size was calculated as follows: V = L × W2, in which L and W represent the length and width of the tumor. Results and Discussion Crystal structure Slightly yellow cubic crystals of PFC-16 were synthesized by solvothermal reaction of H3BDBA (H3L) and ZrCl4 in DMF at 120 °C for 3 days. Single-crystal X-ray diffraction revealed that PFC-16 crystallizes in space group Im 3 ¯ , and the structure can be formulated as Zr6O4(OH)4(BDBA)2(OH)6 ( Supporting Information Table S1). There are two kinds of Zr-oxo clusters in the structure. One is a disordered cluster connecting with six ethynylbenzoate groups of H3L, and the other one is an ordered cluster connecting with four methylbenzoate groups of H3L, resulting in a (3,4,6)-c net with idp topology (Figure 2 and Supporting Information Figures S1–S3).29 Moreover, the Im 3 ¯ space group is the maximum symmetric embedding of this net, further verifying the conclusion drawn from crystallography analysis.30 Of special note, this is the first observation of Zr-oxo clusters with different connectivity in one MOF structure ( Supporting Information Figure S4). Previous studies show that the Zr6 clusters can exhibit diverse geometries compatible to various organic building blocks. For example, in fcu-a or ftw networks, each Zr6 cluster is fully occupied by 12 carboxylates adopting cuboctahedron geometry (Oh symmetry).12,31 In an eight-connected bcu-a or flu network, the symmetry of Zr6 is downgraded to D4h.24,32 Further reducing the connectivity of the network, for example, in the six-connected β-cristobalite network, the Zr6 cluster acts as an antiprismatic node with D3d symmetry.25 The aforementioned MOFs are all based on highly symmetric organic building blocks. However, with relegating the symmetry of organic linkers, the connectivity and symmetry of Zr6 cluster in PFC-16 can be further reduced to six-connected (octahedron with Oh symmetry) and four-connected (rectangle with C4v symmetry) nodes. The four-connected Zr6 nodes, although having been reported, are still rare in Zr-MOF structure.33–36 In a search for MOFs with idp topology, we found that most reported idp networks are constructed by Zn4O(COO)6 (six-connected node in octahedral geometry) and Cu(COO)4 (four-connected node in rectangular geometry) clusters.29,37 Therefore, this work demonstrates that degrading the symmetry of organic linkers caused downgraded connectivity for Zr6 clusters and therefore gave rise to some topologies which are usually observed in Cu-, Mn-, Zn-MOF, and so forth, presenting an effective strategy for expanding the Zr-MOF library.37–39 Figure 2 | The view of organic building block, Zr6 cluster, and two types of cavities in crystal structure (top). Irregular tiles with SOD topology surrounding the tetrakis hexahedron tile (bottom). Download figure Download PowerPoint Accompanied with the downgraded Zr6 clusters of PFC-16 are the features of novel architecture, abundant open-metal sites, and mesoporosity. The dihedral angles between the central benzene ring and the methylbenzoate fragments in BDBA3− ligand are 90° because of the steric hindrance of the –CH3 group, and therefore the carboxylate group on methylbenzoate is perpendicular to the other two carboxylate groups on the ethynylbenzoate fragments. Further investigation of the detailed structure revealed, 12 BDBA3− ligands connected with 6 four-connected Zr6-SBUs and 8 six-connected Zr6-SBUs generating a tetrakis hexahedron cage (Oh symmetry), which can be considered as a Catalan solid by putting a square pyramid onto each of the faces of a cube ( Supporting Information Figure S2).40–42 All the six-connected Zr6 clusters were located at the eight vertices of the cube, and the four-connected Zr6 clusters were located at the 6 vertices of the square pyramid ( Supporting Information Figure S2). It is known that Catalan solids are a class of polyhedrons composed of irregular polygons. In PFC-16, the symmetry inequivalent BDBA3− ligands served as some of the triangular faces of the pyramid, and we found that this structure was also obtained in other MOF structures based on symmetry inequivalent ligands.29 Moreover, in the tetrakis hexahedron, the pyramids' lateral faces have to be at an angle of strictly <45° to the base plane so that the lateral faces of adjacent pyramids do not lie in the same plane to form a rhombic face. This also required that the building blocks sat on the face of the pyramid must be non equilateral triangles such as H3BDBA. Therefore, besides the appropriate direction of each carboxylate group, the inequivalent extension of the ligand into irregular triangular building blocks is a prerequisite for the construction of this Catalan solid. We can further speculate that this type of ligand has the possibility of generating other Catalan solids unexplored in the MOF field. The tetrakis hexahedron cages exhibit pentagonal windows with an opening of ∼16 Å and an internal diameter of approximately 36 Å ( Supporting Information Figure S1). Such a tetrakis hexahedron was further connected with six adjacent tetrakis hexahedra by a vertex-transitive manner causing a pcu stacking (Figure 2 and Supporting Information Figures S2 and S3), between which are 24 irregular void tiles forming a SOD network with a tetrakis hexahedron embedded inside (Figure 2). This novel architecture gave rise to a highly porous framework with a calculated solvent-accessible surface area of 3510.47 m2 g−1. The experimental powder X-ray diffraction (PXRD) pattern of PFC-16 matched well with the simulated one, indicative of the attainment of the pure phase (Figure 3b). Experimental N2 adsorption isotherm exhibits type IV behavior without hysteresis upon desorption, further supporting the existence of permanent micro- and mesopores in the structure (Figure 3c). The deduced Brunauuer–Emmett–Teller (BET) surface area is 3231 m2 g−1 based on the N2 adsorption isotherms, consistent with the calculated results.43 Despite the high porosity, PFC-16 exhibits good acid and base stability in various pH aqueous solutions (pH 3–12) as indicated by the intact PXRD patterns and N2 uptakes (Figure 3c and Supporting Information Figure S5). A lower synthetic temperature (decreasing from 120 to 90 °C) gave rise to a smaller particle size of approximately 100 nm (denoted as nano-PFC-16) as confirmed by scanning electron microscopy (SEM; Supporting Information Figure S6), suitable for the subsequent in vitro and in vivo experiments. Figure 3 | Incorporation of photosensitizer and targeting molecule in nano-PFC-16. (a) Incorporation of ZnPc and FA into PFC-16 for PDT. (b) PXRD patterns of simulated-PFC-16, bulk-PFC-16, [email protected], and [email protected]. (c) N2 isotherms of PFC-16 treated with different pH aqueous solutions. (d) Digital photographs of nano-PFC-16 (left), [email protected] (middle), and [email protected] (right) suspended in DMF. (e) Solid luminescent spectra of ZnPc, nano-PFC-16, [email protected], and [email protected]. (f) Solid UV spectra of ZnPc, FA, nano-PFC-16, [email protected], and [email protected]. (g) IR spectra of nano-PFC-16, [email protected], FA, and [email protected]. Download figure Download PowerPoint Incorporation of photosensitizer and targeting molecule in nano-PFC-16 The addition of ZnPc or phthalocyanine nickel (NiPc) to the reaction mixtures yields composite material with ZnPc (7.03% mol·mol−1) or NiPc (16.22% mol·mol−1) being successfully encapsulated in nano-PFC-16 (denoted as [email protected] and [email protected], respectively) as proved by inductively coupled plasma (ICP) analysis ( Supporting Information Scheme S2).44 Solid-state fluorescence and UV–vis spectra show the characteristic peaks from the ZnPc component (Figures 3e and 3f), again confirming the successful incorporation of ZnPc in nano-PFC-16. The successful of ZnPc can be to mesopores large for and abundant open-metal sites within the structure or Moreover, the existence of Zr-oxo clusters in the structure allows for some functional through In the carboxylate of FA ( Supporting Information Scheme can with Zr6 clusters to form nano-PFC-16, which is in Zr-MOFs the of we further FA on metal This caused an from to yellow (Figure The successful incorporation of FA was confirmed by the of [email protected], where new peaks at and were which can be to the and groups of FA, (Figure from the UV–vis (Figure and as shown in Supporting Information Figure can with to the generation of [email protected], nano-PFC-16, and [email protected] was by the of at = UV–vis spectra an excellent generation of [email protected], nano-PFC-16 and [email protected] exhibit much lower or for ( Supporting Information Figures and analysis of PFC-16, [email protected], [email protected], and after are revealed in Supporting Information Figure all of the materials can their frameworks 450 The N2 adsorption of [email protected], and [email protected] been at exhibit type IV behavior without hysteresis upon ( Supporting Information Figure The CO2 adsorption of PFC-16 been and the were calculated based on adsorption isotherms at and by using the ( Supporting Information Figure In vitro photodynamic performance of [email protected] in vitro the generation of [email protected] in aqueous solution was by oxygen The fluorescence of a at nm in the of [email protected] and 660 nm laser (Figure the efficiency was also The solutions with different concentrations of [email protected] were irradiated at 660 and the UV–vis spectra were to determine the of The concentrations of with the of [email protected] Moreover, the efficiency is in a concentration as as 20 μg it is a promising for PDT in vivo (Figure The group without [email protected] not show in concentration the 660 nm laser The of [email protected] was then through the cell after MCF-7 cells with [email protected] in various shown in Figure [email protected] cancer cells to 200 μg the PDT the cancer cells were irradiated with 660 nm laser for 10 min. The cell upon with that of the group without irradiation, confirming the photodynamic of [email protected] (Figure of MCF-7 cells generated by calcein-AM and a much cell in the group treated with [email protected] and laser (Figure that was by photodynamic a which can be to by was as an ROS shown in Figure only MCF-7 cells treated with [email protected] and laser These results indicated that upon the at 660 [email protected] ROS and leads to cell potential for in vivo photodynamic Figure | In vitro photodynamic performance of [email protected]. (a) spectra of solution for generated (b) UV–vis spectra and of aqueous solution treated with various concentrations of [email protected] 20, 40, 60, and 100 μg with the same (c) of MCF-7 cells incubated with various concentration of [email protected] 10, 20, 40, 60, 100, and 200 μg for 12 h. (d) of MCF-7 cells after with [email protected] treated with or without 660 nm laser (e) cells of calcein-AM and 100 (f) confocal laser scanning fluorescence microscope of ROS with the in MCF-7 cells treated with [email protected] at μg mL−1 660 nm laser for 10 min. 100 Download figure Download PowerPoint In vivo PDT efficacy by the in vitro results we constructed groups of mice with of MCF-7 cells to further explore the photodynamic efficacy of [email protected] in in Figure the 40 in measured [email protected] or PBS was injected into four various groups of mice tail ( [email 20 mg 100 μL, and 660 Figure 5 | In vivo PDT (a) In vivo PDT with [email protected] by 660 nm laser MCF-7 mice were randomly divided into four PBS group, [email protected] only group, laser only group, and [email protected] + laser group. (b) tumor and (c) body weight of MCF-7 mice after being treated with different (d) tumor from mice in four (e) treated with after PDT 100 Download figure Download PowerPoint shown in Figure the tumor were other for a of days. of laser only and [email protected] only groups grew at a in the first which was the PBS group without After the PDT on the the of both was different and a tumor the [email protected] + laser group efficacy that with the PBS group. In in body weight was observed for all caused by such a (Figure The photographs of tumor from groups (Figure also revealed the analysis a to the photodynamic performance of in and that the of [email protected] and 660 nm laser where the cancer cells of tumor a

Objective: To evaluate 11.5% polyacrylic acid (PA) containing 0.3% methylene blue (MB) dye as a photosensitizer for photodynamic therapy (PDT) of carious dentin. Methods: One hundred twenty molars were selected and the dentin was exposed for cariogenic challenge, where the molars were placed in brain heart infusion medium containing a standard strain of Streptococcus mutans (ATCC). Samples were randomly divided into eight groups (n = 15): S: saline, PA, MB: MB 0.3%, PA+MB: PA containing 0.3% MB + LLL: irradiation with low-level laser, PDT (MB): MB 0.3% + laser, PDT (PA): PA + laser, and PDT (PA+MB): PA containing 0.3% MB + laser. Carious dentin was collected before and after exposure to S. mutans. All samples of carious dentin were homogenized, diluted, and seeded in mitis salivarius bacitracin medium, and the cultures were incubated at 37°C for 15 days in anaerobic jars. The Wilcoxon test was used for analysis. Results: The percent microbial reduction achieved with each treatment was as follows: PDT (MB), 53.62%; PDT (PA+MB), 50.47%; PDT (PA), 46.73%; PA, 38.51%; MB, 19.75%; PA+MB, 17.18%; LLL, 12.83%; S, 5.99%. The greatest reductions in S. mutans growth occurred with PDT (MB), PDT (PA+MB), and PDT (PA) when compared to the S group (p = 0.0002, 0.0023, and 0.0232, respectively). Conclusions: PA containing 0.3% MB can be used as a photosensitizer for PDT to reduce S. mutans burden in carious dentin.

Background: Methylene blue has a long history of clinical application. Thanks to phenothiazine chromophore, it has potential in photodynamic anticancer therapy. In spite of the growing body of literature that has evaluated the action of this dye on different types of cancer, the systematic understanding of this problem is still lacking. Therefore, this systematic review was performed to study the efficacy of methylene blue in photodynamic anticancer therapy.Methods: This systematic review was carried out in accordance with the PRISMA guidelines, and the study protocol was registered in PROSPERO (CRD42022368738). Articles for the systematic review were identified through the PubMed database. SYRCLE’s risk of bias tool was used to assess the studies. The results of systematic analysis are presented as narrative synthesis.Results: Ten studies met the inclusion criteria and these full texts were reviewed. In the selected articles, the dosage of dye infusion ranged from 0.04 to 24.12 mg/kg. The effectiveness of photodynamic therapy with methylene blue against different types of cancer was confirmed by a decrease in tumor sizes in seven articles.Conclusion: The results of the systematic review support the suggestions that photodynamic therapy with methylene blue helps against different types of cancer, including colorectal tumor, carcinoma, and melanoma. In cases of nanopharmaceutics use, a considerable increase of anticancer therapy effectiveness was observed. The further research into methylene blue in photodynamic anticancer therapy is needed.Systematic Review Registration: (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=368738), identifier (CRD42022368738).

Antimicrobial effects of photodynamic therapy on Staphylococcus aureus biofilm grown on a specific acrylic resin surface for ocular prostheses.

Enterococcus faecalis biofilm is a major contributor to persistent root canal infections due to its high resistance to conventional irrigants. This study aimed to evaluate and compare different disinfection protocols, including sodium hypochlorite (NaOCl), photodynamic therapy (PDT) with methylene blue (MB), and MB functionalized with reduced graphene oxide (MB-rGO) nanoparticles with or without sonic activation, in disrupting E. faecalis biofilm on root canal dentin using SEM and quantitative FIJI-based image analysis. Eighty root dentin specimens were obtained from forty extracted single-rooted human teeth. Following canal preparation and instrumentation, thirty-five teeth were inoculated with Enterococcus faecalis and incubated for 21days to develop mature biofilms, while five teeth served as negative controls. Each tooth was longitudinally sectioned to yield two standardized dentin specimens, which were randomly allocated to six experimental groups (n = 10 specimens per group) according to the disinfection protocol. Groups 1-2 received 5% sodium hypochlorite (NaOCl), Groups 3-4 underwent photodynamic therapy (PDT) using methylene blue (MB), and Groups 5-6 received PDT with methylene blue functionalized with reduced graphene oxide (MB-rGO). Sonic activation was applied in Groups 2, 4, and 6. Biofilm removal was assessed using scanning electron microscopy (SEM) and quantified with FIJI software employing the Trainable Weka Segmentation plugin. A 6-point visual scoring scale was additionally applied. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test and the Wilcoxon signed-rank test (p < 0.05). The positive control showed extensive biofilm coverage (73.36 ± 6.36%). NaOCl-treated groups showed moderate reduction (Group 1: 35.57 ± 3.11%; Group 2: 31.67 ± 2.09%). PDT with MB exhibited greater reduction (Group 3: 21.68 ± 2.65%; Group 4: 19.11 ± 1.78%). The MB-rGO groups presented the highest efficacy (Group 5: 17.71 ± 2.66%; Group 6: 10.46 ± 1.92%), with Group 6 significantly outperforming all others (p < 0.001). Semi-quantitative scores reflected similar trends, with Group 6 showing the lowest score (1.20 ± 0.63, p < 0.001). MB-rGO combined with sonic and laser activation demonstrated the highest efficacy against E. faecalis biofilm, indicating a promising approach for enhanced endodontic disinfection.

Photodynamic therapy (PDT) is a cutting-edge cancer treatment that utilizes both light and photosensitizers (PSs) to attack cancer cells. Methylene blue (MB) has emerged as a highly promising photosensitizer (PS) in PDT therapy due to its exceptional ability to produce singlet oxygen, which is attributed to its high quantum yield. However, the main challenge in utilizing MB in photodynamic therapy is its effective delivery to the target tissue. This challenge can be addressed by utilizing silica nanoparticles (SiNPs) as a drug delivery agent. Silica nanoparticles encapsulate MB and prevent its leakage, offering a novel approach to improving PDT therapy by reducing the toxicity of MB and increasing its bioavailability at the target cell. In this study, an extensive analysis of the size and shape evolution of the synthesized silica nanoparticles loaded with MB was conducted using TEM. Various encapsulated and bare MB concentrations were tested for cytotoxicity against osteosarcoma cells. Moreover, the optimal concentration and exposure time under light (with an intensity of approximately 8.9 mW/cm2 in the visible range) were determined to achieve maximum cell elimination. The results revealed that encapsulated MB in SiNPs exhibited a higher efficacy compared to naked MB, with a 50% increase in concentration effectiveness and a 90% increase in exposure time efficacy. This confirms that encapsulated MB in SiNPs is more effective in killing osteosarcoma cells than bare MB, thereby enhancing photodynamic therapy through increased bioavailability of MB in target cells. The enhanced bioavailability of MB in target cells as a result of its encapsulation in SiNPs makes it a highly promising drug delivery candidate for significantly enhancing the efficacy of photodynamic therapy against osteosarcomas.

This study was performed to compare the use of methylene blue (MB) and rose bengal (RB) in antimicrobial photodynamic therapy (PDT) targeting Enterococcus faecalis (E. faecalis) bacteria in planktonic and biofilm forms with potassium iodide (KI) potentiation. E. faecalis bacteria in planktonic form were exposed to antimicrobial PDT protocols activating MB and RB, with or without KI potentiation, following laser irradiation with different exposure times, 60 mW/cm2 laser power, and different photosensitizer agent (PS)/potentiator concentrations to observe relationships among the variables. Two continuous-wave diode lasers were used for irradiation (red light: λ = 660 nm and green light: λ = 565 nm). The pre-irradiation time was 10 minutes. The vitality of E. faecalis biofilm was assessed by confocal laser scanning microscopy, and the morphology was determined by scanning electron microscopy. The effects on the proliferation of stem cells from the apical papilla (SCAPs) were analyzed by cell counting kit-8 assay. The staining effect of antimicrobial PDT on dentin slices was investigated. Statistical analysis using a one-way analysis of variance was done. KI-potentiated RB and MB antimicrobial PDT both effectively eradicated E. faecalis bacteria in planktonic and biofilm forms. The minimum bactericidal concentrations of PSs (±100 mM KI) were obtained through PDT on planktonic E. faecalis, and the optimal light parameters were 60 mW/cm2 , 6 J/cm2 for 100 seconds. KI-potentiated PDT effectively strengthened the ability to inhibit E. faecalis biofilm with 86.50 ± 5.78% for MB (P = 0.0015 < 0.01) and 91.50 ± 1.75% for RB (P = 0.0418 < 0.05) of bactericidal rate, with less toxicity for SCAPs (P < 0.001) and less staining. KI could reduce the staining induced by antimicrobial PDT on dentin slices. A combination of KI and antimicrobial PDT may be a useful alternative to conventional disinfection methods in endodontic treatment. MB and RB antimicrobial PDT at much lower concentrations with KI could hopefully achieve disinfection effects comparable with those of 1.5% NaClO while causing few adverse effects on SCAPs. KI helps to avoid staining problems associated with high concentrations of photosensitizer agents. Lasers Surg. Med. © 2020 Wiley Periodicals, LLC.

Photodynamic therapy (PDT) based on photosensitizers (PSs) constructed with nanomaterials has been widely applied to treat cancer. This therapy is characterized by an improved PS accumulation in tumor regions. However, challenges, such as short penetration depth of light and low extinction coefficient of PSs, limit PDT applications. In this study, a nanocomposite consisting of NaYF4:Yb/Er upconversion nanoparticles (UCPs) conjugated with gold nanorods (Au NRs) was developed to improve the therapeutic efficiency of PDT. Methylene blue (MB) was embedded in a silica shell for plasmon-enhanced PDT. UCPs served as a light converter from near-infrared (NIR) to visible light to excite MB to generate reactive oxygen species (ROS). Au NRs could effectively enhance upconversion efficiency and ROS content through a localized surface plasmon resonance (SPR) effect. Silica shell thickness was adjusted to investigate the optimized MB loading amount, ROS production capability, and efficient distance for plasmon-enhanced ROS production. The mechanism of plasmon-enhanced PDT was verified by enhancing UC luminescence intensity through the plasmonic field and by increasing the light-harvesting capability and absorption cross section of the system. This process improved the ROS generation by comparing the exchange of Au NRs to Au nanoparticles with different SPR bands. NIR-triggered nanocomposites of UCP@SiO2:MB-NRs were significantly confirmed by improving ROS generation and further modifying folic acid (FA) to develop an active component targeting OECM-1 oral cancer cells. Consequently, UCP@SiO2:MB-NRs-FA could highly produce ROS and undergo efficient PDT in vitro and in vivo. The mechanism of PDT treatment by UCP@SiO2:MB-NRs-FA was evaluated via the cell apoptosis pathway. The proposed process is a promising strategy to enhance ROS production through plasmonic field enhancement and thus achieve high PDT therapeutic efficacy.

Methylene blue-enhanced photodynamic and sonodynamic therapy againstStaphylococcus aureus: From laboratory research to clinical evaluation.

Background: Unwanted hair is one of the most common medical problems affecting women of reproductive age inducing a lot of psychological stress and threatening their femininity and self-esteem. Old methods of removing unwanted hair include shaving, waxing, chemical epilation, and electrolysis, all of which have temporary results. However laser-assisted hair removal is the most efficient method of long-term hair removal currently available. It is desirable to develop a reduced-cost photodynamic therapy (PDT) system whose properties should include high efficiency and low side-effects. Objectives: The aim is to develop an adequate PDT system including helium-neon (He-Ne) laser and its effect on the photodynamic activity of methylene blue (Figs. 1 and 2) in biological experiments. Method: Mice skin tissues were used in this study and divided into the following six groups: controls (Fig.3), free methylene blue (MB) incubation, liposomal MB incubation, laser without MB, free MB for 3 and 4 hrs and laser, liposome MB for 3 hrs and laser. MB was applied to wax epilated areas. The areas were irradiated with CW He-Ne laser system that emits orange-red light with wavelength 632.8 nm and 10 mW at energy density of 5 J/cm2 for 10 minutes. The UV-visible spectrum was collected by Cary spectrophotometer. Results: MB is selectively absorbed by actively growing hair follicles due to its cationic property (Fig.4). MB untreated sections showed that hair follicles and sebaceous glands are intact and there is no change due to the laser exposure (Fig.5). Free MB sections incubated for 3 hrs showed that He:Ne laser induced destruction in hair follicles, leaving an intact epidermis (Fig. 6). Treated section with free MB for 4 hrs showed degeneration and necrosis in hair follicles, leaving an intact epidermis (Fig. 7). Liposomal MB sections incubated for 3 hrs showed He:Ne laser induced destruction in hair follicles with intradermal leukocytic infiltration (Fig. 8). Conclusion: Low power He:Ne laser and MB offers a successful PDT system in selectively damaging hair follicles, leaving an intact epidermis. The current PDT system provides better outcome than hair destruction through laser heat transfer procedures and laser-mediated hair removal, due to complete destruction of hair follicles.

The authors investigated the antimicrobial effect of methylene blue (MB)-mediated photodynamic therapy (PDT) on Mycobacterium fortuitum keratitis. In the in vitro study, the mycobacterial suspension and colonies were treated with the following: no MB, no light (normal control); MB and no light (dye control); light and no MB (light control); MB and light (PDT). Morphologic characteristics were examined by transmission electron microscopy. The bactericidal effects of combined PDT and antibiotic therapy (ciprofloxacin, moxifloxacin, and amikacin) were determined using the broth microdilution technique. Twenty-one rabbits with Mycobacterium keratitis were randomly divided into three groups (no treatment, topical amikacin treatment, and PDT combined with amikacin treatment). The clinical features of keratitis were scored and graded before treatment and before euthanatization. The diseased corneas were trephined for quantitative bacteriologic analysis to determine the antibacterial efficacy of the treatment. In the in vitro tests, the bacterial count had a 2-log reduction immediately after PDT treatment at 100 J/cm(2) with 10(-3)% MB. After PDT at 100 J/cm(2) with 10(-2)% MB, almost no viable bacteria were detected. PDT had a synergistic antimicrobial effect in combination with antibiotics. The phototoxicity occurred in the cytoplasm first and then disrupted the mycobacterial cell walls by lysis. In the rabbit keratitis model, combined PDT resulted in significantly less bacterial burden (P < 0.01) than in the amikacin group. This study demonstrated the effectiveness of MB-mediated PDT against Mycobacterium fortuitum. PDT could be a potential alternative treatment for nontuberculous mycobacterial corneal infections.

Photodynamic therapy mediated by chlorin-type photosensitizers against Streptococcus mutans biofilms.

Improved photodynamic efficiency for methylene blue from silica-methylene blue@tannic acid-Fe(III) ions complexes in aqueous solutions