Opto-microfluidic assisted synthesis of photo-protoporphyrin (pPP) conjugated to hollow gold-albumin hybrid nanoshells to enhance the efficiency of photodynamic therapy of triple negative breast cancer cells.

INTRODUCTION: Protoporphyrin-IX (PpIX), a photosensitizer used in photodynamic therapy, has limitations due to its hydrophobicity, rapid photobleaching, and low absorption peak in the red region. These limitations make the use of PpIX less effective for photodynamic therapy treatments. In this study, we harnessed the power of microfluidic technology to manipulate the properties of PpIX and quickly synthesize albumin-based hybrid nanoshells with high reproducibility. METHODS AND MATERIAL: To begin with, we designed a microfluidic chip with SolidWorksⓇ software; then the chip was fabricated in Poly(methyl methacrylate) (PMMA) material using micromilling and thermal bonding. We synthesized PpIX-loaded CTAB micelles and subsequently transformed the PpIX structure into photo-protoporphyrin (PPP,) by opto-microfluidic chip (Integrating a microfluidic chip with a light source). Simultaneously with CTAB-PPP synthesis complex, we trapped it in binding sites of bovine serum albumin (BSA). Afterward, we used the same method (without irradiating) to generate a hybrid nanostructure consisting of hollow gold nanoshells (HGN) and BSACTAB-PPP. Then, after physical characterization of nanostructures, the photodynamic effects of the agents (HGNs, CTAB-PpIX, BSA-CTABPpIX, HGN-BSA-CTAB-PpIX, CTAB-PPP, BSA-CTAB-PPP, and HGNs-BSA-CTAB-PPP) were evaluated on MDA-MB-231 and 4T1 cells and the cytotoxic properties of the therapeutic agents after treatment for 24, 48, and 72 hours were investigated using MTT assay. Finally, we analyzed the findings using GraphPad Prism 9.0 software. RESULTS: Results revealed that the opto-microfluidic assisted synthesis of HGN-BSA-CTAB-PPP is highly efficient and reproducible, with a size of 120 nm, a zeta potential of -16 mV, and a PDI index of 0.357. Furthermore, the cell survival analysis demonstrated that the HGNBSA-CTAB-PPP hybrid nanostructure can significantly reduce the survival of MDA-MB-231 and 4T1 cancer cells at low radiation doses (< 10 J/cm2) when exposed to an incoherent light source due to its strong absorption peak at a wavelength of 670 nm. CONCLUSION: This research indicates that developing albumin-based multidrug hybrid nanostructures using microfluidic technology could be a promising approach to design more efficient photodynamic therapy studies.

In vivo evaluation of Sono-chemo therapy via hollow gold nanoshells conjugated to mitoxantrone on breast cancer.

Objectives: Conventional methods of cancer treatment include surgery, chemotherapy, radiation therapy, and immunotherapy. Chemotherapy, as one of the main methods of cancer treatment, due to the lack of targeted distribution of the drug in tumor tissues, is not able to destroy cancer cells and also affects healthy tissues and causes serious side effects in patients. Sonodynamic therapy (SDT) is a promising strategy for non-invasive treatment of deep solid cancer tumors. In this study, for the first time, the sono-sensitive activity of mitoxantrone was investigated and then mitoxantrone (MTX) was conjugated to hollow gold nanostructure (HGN) to improve the efficiency of in vivo SDT. Materials and Methods: Firstly, after the synthesis of hollow gold nanoshells and the PEGylation process, conjugation of MTX was performed. Then, after evaluating the toxicity of the treatment groups in vitro, in order to perform an in vivo study, 56 male Balb/c mice that had been tumorized by subcutaneous injection of 4T1 cells were divided into eight groups of breast tumor model. Ultrasonic irradiation (US) conditions including intensity of 1.5 W/cm2 (with a frequency of 800 kHz, 5 min), MTX concentration of 2 µM, and HGN dose of 2.5 mg/kg (unit of animal weight) were used. Results: The results show that administration of PEG-HGN-MTX caused a slight reduction in tumor size and growth compared with free MTX. Ultrasound also improved the therapeutic effect of the gold nanoshell in treated groups, and the HGN-PEG-MTX-US treated groups were able to significantly reduce and control tumor size and growth. Conclusion: The findings also show that MTX and HGN can be used as sonosensitizers in SDT. Also, HGN-PEG-MTX can act as a sono-chemotherapy agent for the combination of sonodynamic therapy and chemotherapy for in vivo breast tumors.

The multimodal effect of Photothermal/Photodynamic/Chemo therapies mediated by Au-CoFe2O4 @Spiky nanostructure adjacent to mitoxantrone on breast cancer cells.

BACKGROUND: Conventional cancer treatments are associated with a number of limitations, including non-selectivity, toxicity and multidrug resistance, so new nanotechnologies are being developed forcancer diagnosis and therapy. Phototherapy approach based on nanotechnology is a hopeful strategy to overcome these problems. Photothermal (PTT) and photodynamic therapies (PDT), in addition to having non-invasive properties, are known as promising methods for treatment of tumors. In this study, CoFe2O4 theranostic magnetic nanoparticles coated with spiky gold nanoparticles were designed and synthesized and its photothermal effects were evaluated in combination with the photodynamic and chemotherapeutic effects of mitoxantrone (MTX) under in vitro conditions. METHODS AND MATERIALS: At first, CoFe2O4 @Spiky Au nanostructure was synthesized and after its characterization, cytotoxicity of MTX, CoFe2O4 @ Spiky Au (MGNS) and CoFe2O4 @ Au were determined on MDA-MB-231 cell line. Then, the concentrations required for inducing 50% cell death (IC50) and appropriate concentration for this study was obtained. Cells were irradiated by an 808 nm laser and a non-synchronous light source at 670 nm at the separate groups. The viability of treated cells was determined via MTT test 48 h after treatment. RESULTS: In the groups receiving energy density (5-40) J/cm2, at the lower laser dose an increase in cell survival was observed (P < 0.05) and then cell survival was decreased (P < 0.05). In the groups receiving non-coherent light (2-18 J/cm2) from the beginning, a decreasing trend in cell survival is observed. CONCLUSION: The overlap of the emission spectrum of the light source and the absorption spectrum of the nanostructure amplified the cell death. Similar to the Hormesis model reported for ionizing radiation effects, at low light doses with the bio-phasic response dose model, increased cell survival and proliferation can be expected.

Surface modification of gold nanoparticles with 6-mercapto-1-hexanol to facilitate dual conjugation of protoporphyrin IX and folic acid for improving the targeted photochemical internalization.

Objectives: Photochemical internalization (PCI) is an important type of photodynamic therapy for delivering macromolecules into the cytosol by the endocytosis process. In this study, 6-mercapto-1-hexanol (MH) was used to functionalize the gold nanostructure as a primer for surface modification to improve conjugation of multi-agents such as protoporphyrin IX (Pp-IX) and folic acid with gold nanoparticles (PpIX/FA-MH-AuNP) to facilitate the photochemical internalization. Materials and Methods: After surface modification of AuNPs with MH, PpIX and FA are bonded to the surface of the MH-AuNPs through the coupling reaction to produce the desired conjugated AuNPs. In the next step, the synthesized nanostructures were characterized by different methods. Finally, after selecting specific concentrations, light treatments were applied and cell survival was measured based on MTT analysis. Also, in order to better study the morphology of the cells, they were stained by the Giemsa method. The SPSS 16 software was used for data analysis. Results: By surface modification of the nanostructure with MH and then conjugation of FA to it, the incubation time of the drug in PpIX/FA-MH-AuNP was reduced from 3 hr to 30 min. Also, at each light dose, cell death in the presence of PpIX/FA-MH-AuNP was significantly reduced compared with unconjugated conditions (P<0.001). Under these conditions, the ED50 for PpIX and PpIX-MH-AuNP and PpIX/FA-MH-AuNP at a concentration of 2.5 µg/ml is 8.9, 9.1, and 6.17 min, respectively. Conclusion: The results show that the PCI of PpIX/FA-MH-AuNP increases the selective phototoxicity efficiency on cancer cells compared with the conventional process of photodynamic therapy.

A whole-thermoplastic microfluidic chip with integrated on-chip micropump, bioreactor and oxygenator for cell culture applications.

Microfluidics provides enabling platforms for various cell culture, drug testing and synthesis of drug carriers using chip-based microsystems. In this study, we present a novel integrated whole-thermoplastic microfluidic chip to provide a platform for on-chip cell culture at static and dynamic conditions. The whole chip was made of polymethyl methacrylate (PMMA) and thermoplastic polyurethane (TPU) using high precision micromilling and laser micromachining, assembled by thermal fusion bonding. Prior to fabricate the integrated microchip, a pneumatic solo diffuser-nozzle micropump was fabricated and characterized to evaluate its functionality for on-chip pumping. Then the micropump was integrated with a microbioreactor and an oxygenator in a microchip for flow pumping required for on-chip cell culture. Oxygenator, made of a thin TPU membrane and a reservoir, was implemented in the microchip because of low oxygen permeability of PMMA. To design the oxygenator for sufficient oxygen delivery to the chip, numerical simulation was performed using COMSOL Multiphysics® to evaluate oxygen concentration distribution inside the microchip. Finally, the diffuser-nozzle micropump was integrated with the oxygenator and a bioreactor on the microchip for cell culture with on-chip pumping. Culture of DFW cells was performed on the integrated chip for three days, and cell survival was evaluated with Trypan Blue assay. The findings reveal that the proposed integrated chip with on-chip pumping could be employed for conducting various cell culture studies.

In vitro evaluation of the intensifying photodynamic effect due to the presence of plasmonic hollow gold nanoshells loaded with methylene blue on breast and melanoma cancer cells.

BACKGROUND: Hypoxia is one of the most important limiting factors in photodynamic therapy that can reduce the effectiveness of this treatment. By designing a nanocomplex of plasmonic nanoparticles and photosensitizers with similar optical properties, the rate of free oxygen radical production can be increased and the efficiency of photodynamic therapy can be improved. in this study, we tried to use the outstanding capacities of hollow gold nanoshells (HGNSs) as a plasmonic nanocarrier of methylene blue (MB) to improve the performance of photodynamic therapy. METHODS AND MATERIAL: After synthesis and optimization of hollow gold nanoshells loaded with Methylene blue (HGNSs-PEG-MB), the characteristics of MB, HGNSs, HGNSs-PEG, HGNSs-PEG-MB, and their toxicity at different concentrations on the cell lines was determined. After determining of optimum concentration of nano agents, irradiation of cell was performed with non-coherent of light source with 670 nm wavelength and an intensity of 14.9 mW/cm2. Twenty-four hours after irradiation, an MTT assay was used to determine cell survival percentage. To compare the results, we defined different indexes such as treatment efficiency (TE), synergism ratio (SYN), and the amount of exposure required for 50% cell death (ED50). All the tests were repeated at least four times on the DFW and MCF-7 cancer cell lines. RESULTS: For combination therapies with Lumacare irradiated HGNSs-PEG-MB, the UC index was less than one for all concentrations (P < 0.05). Also, the IC50 index for this nanostructure in non-irradiated conditions and less than 9 min irradiation time was lower than other treatment groups (P < 0.05). ED50 amounts for HGNSs-PEG-MB in all concentrations were greater than the other groups. TE Index was also reported to be greater than 1 in all irradiation conditions and concentrations. CONCLUSION: In this study, HGNSs-PEG in the role of nanocarriers for methylene Blue was used. The results showed that irradiated HGNSs-PEG-MB by 670 nm light severely induced cell death and greatly improved the efficiency of photodynamic therapy in melanoma and breast cancer cells.

In-vitro investigation of cold atmospheric plasma induced photodynamic effect by Indocyanine green and Protoporphyrin IX.

BACKGROUND: Photodynamic therapy (PDT) is one of the non-invasive methods for the treatment of superficial malignant cancers. One of the limiting challenges of PDT is the hypoxic conditions during treatment that reduces PDT Efficiency. Because of ROS and free radicals in plasma flame output, Cold atmospheric plasma (CAP) may improve treatment efficiency. In this study, the effect of plasma-induced photodynamic effect of two Photosensitizers (PSs) include Indocyanine green (ICG) and Protoporphyrin IX (PPIX) on two cell lines (MCF-7 and HT-29) was investigated. METHODS: First, toxicity of different concentrations of PSs (5-50 µM) were examined on cell lines. After that, we surveyed low toxicity of PSs concentrations with different plasma radiation doses. To quantitative of cell survival, MTT assay was performed after 48 h. Finally, in order to statistical analysis of data, we used SPSS software (version 20) and also in order to better comparison the results, we used indexes such as Plasma sensitivity index and Synergism index. RESULTS: The results indicate that in most irradiant conditions; for ICG + CAP group, PSI > 1 and SI < 1 in the both of cell line (P < 0.05). Also for PPIX + CAP group in most irradiant conditions, only in the HT-29 cell line can it be said with certainty that both indexes (PSI and SI) are higher than 1 (P < 0-05). CONCLUSION: The results show that the plasma-induced photodynamic therapy with ICG and PPIX has more effective results on MCF-7 (breast cancer) and HT-29 (colon cancer) cell line, respectively. Also, the synergistic effect was observed only for PPIX in the HT-29 cell line.

A novel nano-superparamagnetic agent for photodynamic and photothermal therapies: An in-vitro study.

BACKGROUND: In this study, iron oxide nanoparticles (SPIONs) were synthesized and coated by GA (SG) and then SG was encapsulated by ICG (SGI). After identifying specifications and cytotoxicity of the agents, the potential of SGI for photodynamic therapy (PDT) and photothermal therapy (PTT) was studied. METHODS: An SGI size of 12-13 nm was determined by TEM images and its zeta potential was measured at -23.8 ± 5.8 mV. MCF-7 and HT-29 cells were exposed to a non-coherent light source at a wavelength of 730 nm and a range of 3.9-124.8 J/cm2 under two different concentrations of agents. The viability of treated cells was determined via MTT assay. To analyze the effects of different irradiation conditions, some indices such as Coefficient of Light Effect, Synergism Index, Addition Ratio, Treatment Efficacy and ED50 were defined. RESULTS: Cell survival at the highest power of irradiation in the absence of any agent was decreased to 93% and 73% for HT-29 and MCF-7, respectively. In both cell lines, the cellular survival dropped by increasing the light source intensity. The maximum cell death recorded for SG, ICG and SGI was 63 ± 2%, 63 ± 2% and 21 ± 2% for MCF-7 cells and 67 ± 2%, 78 ± 1% and 53 ± 1% for HT-29 cells, respectively. CONCLUSION: SGI had a significant photodynamic and photothermal effect on cells. This is a promising outcome, which can help enhance the effectiveness of a minimally invasive treatment. Moreover, SPIONs can be used to apply magnetic hyperthermia or act as a contrast agent in MRI images.

Pegylated hollow gold-mitoxantrone nanoparticles combining photodynamic therapy and chemotherapy of cancer cells.

BACKGROUND: In recent years, Mitoxantrone (MTX) has been introduced as a chemotherapy drug which also serves as a photosensitizer and radiosensitizer. Due to its serious side effects, there are limitations to the application of MTX so scientists are looking for solutions to overcome this problem. Hollow gold nanoparticles (HAuNP) have attracted growing attention due to their unique physical-chemical properties, such as biocompatibility, tunable plasmonic absorption peak ranging from visible to near infrared, high stability and various medical applications in imaging, drug delivery and combinational cancer treatments. In this paper, the combinational effect of photodynamic therapy (PDT) and chemotherapy of MTX conjugated to HAuNP is studied. METHOD: After optimizing the synthesis of PEGylated HAuNP and preparing nanostructures conjugated with MTX, the characteristics of pharmacological agents including MTX, HAuNP, mPEG-HAuNP,and MTX-mPEG-HAuNP and their toxicity were determined at different concentrations on two cell lines of DFW and MCF7 derived from human melanoma and breast cancer, respectively. To select the optimal concentration for PDT, the cytotoxicity of agents was investigated at concentrations of 3, 6, 9 and 12 µM. Moreover, a LEDs system at 630 nm and power output of 3 W was used to apply PDT process. MTT test was used to determine cell survival 24 h after treatment. Several indexes were utilized for data comparison, such as therapeutic efficacy (TE), necessary concentration to kill 50% of cells (IC50), and necessary light exposure to induce 50% cell death (ED50). RESULTS: LED exposure alone did not cause significant cell death. For MTX-mPEG-HAuNP, at both cell lines, IC50 had the least exposure to dark condition with an exposure time of less than 9 min and this nanostructure had the smallest ED50 in each cell line at all concentrations. TE of MTX-mPEG-HAuNP at different exposures and concentrations was greater than 1 for the DFW cells. It was also true for concentrations greater than 6 µM with irradiation times longer than 3 min for MCF7 cells. CONCLUSION: This is the first paper to use PEGylated hollow gold nanoparticles as the nanocarrier for MTX. The results indicated that MTX-mPEG-HAuNP improved the efficacy of PDT with Light Emission diode.