Tuberculosis (TB) remains a significant global health concern, necessitating the development of sensitive and efficient diagnostic methods. This study compares the performance of the custom-built Surface Plasmon Resonance (SPR) phase difference with conventional SPR in detecting the tuberculosis antigen MPT64. Our objective is to evaluate whether the SPR phase shift offers improved sensitivity compared to the conventional SPR technique. MPT64 antigen samples with varying concentrations were prepared and passed over the sensor chip. The resulting binding events were monitored using SPR phase difference and conventional SPR measurement techniques. Our findings demonstrate that SPR phase difference exhibits enhanced sensitivity compared to conventional SPR in the detection of MPT64 antigen. The SPR phase shift technique effectively amplifies the signal response and offers a greater dynamic range, enabling the detection of lower antigen concentrations. The experimental results were analyzed statistically, comparing the limits of detection between SPR phase difference and conventional SPR. The data indicate that the SPR phase difference outperforms the conventional SPR method in terms of sensitivity, highlighting its potential as a superior tool for TB antigen detection. The results obtained in this study may contribute to the advancement of TB diagnosis. The improved sensitivity offered by the SPR phase difference technique has promising implications for early and accurate TB diagnosis, aiding in the timely initiation of treatment and reducing disease transmission.
Tuberculosis (TB) is a major global health issue. To address this, there is a need for rapid and highly sensitive diagnostic methods. Surface plasmon resonance (SPR) is a promising optical sensing platform for various medical applications due to its high sensitivity and label-free detection. In this study, we introduce and evaluate surface plasmon resonance phase difference (SPR-PD) as a novel approach for TB detection. Our method uses the optical properties of SPR-PD to monitor the interaction between MPT64 biomarkers and immobilized MPT64 antibodies on a sensor surface. MPT64 antibodies were immobilized on a gold-coated biosensor chip and the MPT64 TB antigen was added to the biosensing surface. To enhance the detection signal for biomolecular binding events, gold nanoparticles (AuNPs) were conjugated to the MPT64 secondary antibodies. Ultraviolet-visible (UV-vis) spectroscopy was used to characterize the conjugation. The optimized biosensor chip was then analyzed using the custom-built SPR-PD system. Our preliminary results demonstrate successful immobilization of MPT64 antibodies on the biosensing surface, facilitating the capture of MPT64 TB antigen. Furthermore, SPR-PD provides real-time insights into the binding events and enables the sensitive and specific detection of MPT64 TB antigen. Our findings highlight the successful detection of TB biomarkers.
Globally, multidrug resistance (MDR) in breast cancer has become the major cause of morbidity and mortality among women. This study was designed to overcome resistance, reduce dose-dependence in photodynamic therapy (PDT) and evaluate cell death mechanisms induced by green synthesized silver nanoparticles (AgNPs) in combination with pheophorbide-a mediated PDT on superlative, and most architectured three-dimensional (3-D) doxorubicin (DOX) resistant MCF-7 breast cancer cells with overexpressed p-glycoproteins in vitro. In addition to the aforementioned scope, the combination of green NPs with PDT has been reported to yield a good disease prognosis which in most cases is accompanied with manageable adverse effects. Briefly, MDR MCF-7 breast cancer cells were cultured in a 96 well plate to form 3D tumor spheroids and later treated with optimized concentrations of AgNPs and pheophorbide-a in monotherapy. After 24 h treatment, 3-[4,5-dimethylthiazole-2- yl]-2,5- diphenyl tetrazolium bromide (MTT) assay was performed to determine the 50% inhibitory concentration (IC50) for both experimental models. Morphological changes were observed by using an inverted light microscope, viability by MTT assay, and cell death analysis by Annexin VFITC-PI staining. Taken together, the results from this study displayed a dose-dependent decrease in cell viability which was accompanied by significant morphological changes. Furthermore, Annexin V-FITC-PI assay showed apoptosis as the most prominent cell death mechanism induced by PPBa-mediated PDT and AgNPs. Taken together, the findings from the present study highlight the advantages of green nanotechnology in cancer therapy.
Melanoma is a dreadful skin malignancy caused by genetic mutations in melanocytes. The inherent unresponsiveness of melanoma cells to conventional therapies leads to uncontrollable tumour growth and alarming fatalities rates. Photodynamic therapy (PDT) is a novel therapeutic option for the eradication of malignant tumours and, compared to traditional therapies, is minimally invasive and exhibits increased efficacy. Currently, most PDT experiments are still conducted on two-dimensional (2-D) monoculture, which may not sufficiently mimic the physiological conditions and the three-dimensional (3-D) architecture of native tumours. Therefore, 3-D cell cultures serve as excellent models to replicate tumour tissue in terms of structural and functional properties. Commercially available A375 melanoma cells were cultivated as monolayers and 3-D tumour spheroids for this study. A375 cells were treated with zinc phthalocyanine tetrasulfonic acid (ZnPcS4) at varying doses (0.125-20 μM) and photoactivation was achieved using a 673 nm diode laser at a fluency of 10 J/cm2. Photoactivated ZnPcS4 resulted in a dose-dependent reduction in cell proliferation, and increased cytotoxicity as determined by adenosine 5′-triphosphate (ATP), and lactate dehydrogenase (LDH) assay respectively. Morphological changes also confirmed the phototoxic effect of ZnPcS4, while cell death pathways were detected via annexin V-FITC-PI. The half-maximal inhibitory concentration (IC50) of ZnPcS4-mediated PDT on 3-D tumour spheroids was higher than that of monolayers. In conclusion, 3-D cell cultures are unresponsive to PDT compared to traditional monolayer cell cultures and can therefore provide more realistic data and reduce significant discrepancies between in vitro and in vivo studies for better prediction in clinical responses.
The development of effective cancer therapies is required to improve outcomes in colorectal cancer (CRC) treatment. Photodynamic therapy (PDT) has been proposed as more beneficial over common therapeutic approaches and is currently used to treat numerous cancer indications. PDT relies on a selective photosensitizer (PS) that gets activated by laser light in the presence of molecular oxygen, thereby inducing tumour death. To date, zinc phthalocyanine tetrasulfonate acid (ZnPcS4) - mediated photodynamic therapy (PDT) has shown strong cytotoxic effects in twodimensional (2D) cell culture. Three-dimensional (3D) structures such as multicellular cancer tumour spheroids (MCTS) are being recognised to accurately mimic similarities to the in vivo tumour tissue architecture. In this study, the cytotoxicity assessment of ZnPcS4 against MCTS established from human colorectal carcinoma (Caco-2 cell line), under irradiation at 673nm at a fluence of 10 J/cm2 was evaluated. Spheroids were cultured in 96-well non adherent plates, using low attachment technique. Post treatment, MCTS morphology was characterized. Cytotoxic responses on spheroids were assessed post ZnPcS4-based PDT using lactase dehydrogenase-cytotoxicity assay and quantitative assessment of cell death mechanism using Annexin V/PI Staining protocol was conducted. ZnPcS4 in combination with laser irradiation exerted a dose dependent increase in LDH level patterns. In addition, an induction of apoptotic cell death mechanism was observed. These findings suggest that ZnPcS4 exhibits considerable potential as a photosensitizer for photodynamic therapy against multicellular cancer tumour spheroids
In the presence of diabetes mellitus (DM), the wound healing process is interrupted, and chronic wounds develop. DM affects the wound healing response and leads to early apoptosis and reduced number/function of fibroblast cells. In this study we investigated the hypothesis that photobiomodulation (PBM) initiates healing through reduced apoptosis and increased cell survival in diabetic wounded fibroblast (WS1) cells. Cells were irradiated using a diode laser at a wavelength of 660 nm, power output of 100 mW/cm2 and a fluence of 5 J/cm2 . Non-irradiated cells were used as controls. Irradiated cells were incubated for 48 h, after which assays for cell viability (Trypan blue exclusion assay), proliferation (5-Bromo-2′-deoxyuridine-BrdU), apoptosis (Annexin V/PI) and the expression of B-Cell Lymphoma 2- Bcl2 (ELISA) were done. PBM at a wavelength of 660 nm, power output of 100 mW/cm2 and a fluence of 5 J/cm2 significantly increased cell survival through reduced apoptosis in diabetic wounded fibroblast cells. The PBM effects observed in this study are critical in the wound healing process.
Lung cancer has high incidence and mortality rates worldwide and so there is strong need for the development of novel therapies. Photodynamic therapy (PDT) is a photochemotherapeutic cancer treatment that utilizes a photosensitizer (PS) drug that, when activated by laser light at a specific wavelength, yields reactive oxygen species, which in turn induces cell death. However, due to the passive diffusion of PSs, normal surrounding cells are sometimes affected and their targeted concentrations in cancer cells tends to be minimal, thus limiting the effectiveness of this treatment. Therefore, a multicomponent drug targeting strategy is often applied to improve PS specific delivery and concentration in cancer cells, which in turn can improve the effectiveness of PDT. The intention of this study was to improve the PS drug delivery of Zn(II) Phthalocyanine tetrasulfonic acid (ZnPcS4) in lung cancer cells, by enhancing its chemical structure. ZnPcS4 was successfully conjugated to pegylated gold nanoparticles in order to maximize its solubility and stability, as well as bound to specific active tumour-associated antibody-antigens (Cetuximab: Anti-EGFR1 Ab) to aid specific targeted PS delivery. Within in vitro cultured lung cancer cells, this molecular drug delivery system noted improved and specific sub-cellular location of ZnPcS4. Furthermore, after conducting in vitro PDT experiments, a significant amount of cell death and cytotoxicity was found. Overall, this nano immunotherapy drug conjugation combination of ZnPcS4 with AuNP and Cetuximab, proved to enhance concentrated PS uptake in lung cancer cells and so improve PDT treatment outcomes for this form of cancer.
Studies have shown that using high fluences of Low Intensity Laser Irradiation (HF-LILI) produce apoptotic effects on
normal and neoplastic cells. This study aimed to determine whether HF-LILI induce cell death in lung CSCs. Lung CSCs
were isolated using the stem cell marker CD 133, characterized using flow cytometry, and applied in experiments which
included treatment with LILI at wavelengths of 636, 825 and 1060 nm with fluences ranging from 5 J/cm2 to 40 J/cm2.
Viability and proliferation studies, using Alamar blue assay and adenosine triphosphate luminescence (ATP), indicated
an increase when treating lung CSCs with low fluences of 5 - 20 J/cm2 and a decrease in viability and proliferation as
well as an increase in apoptosis when applying a fluence of 40 J/cm2 indicated by flow cytometry using Annexin V and
propidium iodide (PI) dyes. Results indicate that LILI, when treating lung CSCs, can induce either a bio-stimulatory or
bio-inhibitory effect depending on the wavelength and fluence used. This study indicated successful apoptotic induction
of lung CSCs. Future experiments should be able to conclude the exact mechanism behind HF-LILI, which can be used
in the targeted treatments of CSC elimination, implementing HF-LILI in the same manner as PDT in the absence of a
photosensitizer.
Photodynamic cancer therapy (PDT) had already proved its effectiveness in vitro but the search for more effective photosensitizers is encouraged and ongoing. Gold nanoparticles (AuNPs), have been shown to be good drug delivery agents, but in this work AuNPs were investigated as photodynamic agents for PDT. AuNPs were synthesized and characterized by means UV-Vis spectroscopy and transmission electron microscopy (TEM). Photodynamic effects of AuNPs in MCF-7 cells were evaluated using trypan blue, adenosine triphosphate (ATP) luminescence and lactate dehydrogenase (LDH) membrane integrity for cell viability, proliferation and cytotoxicity, respectively. AuNPs had a peak absorption at 540 nm, spherical in shape and were successfully taken up by MCF-7 cells. As a result of light activation or not, cell damage was observed and AuNPs modified into dendrimer-entrapped gold nanoparticles (AuDENPs), which only yielded effects in a light dependent manner. AuNPs is not a suitable photodynamic agent but its modified AuDENPs can be essential in improving the efficiency of PDT.
Mesenchymal stem cells (MSCs) have the capacity to differentiate into a variety of cell types that could potentially be
used in tissue engineering and regenerative medicine. Low intensity laser irradiation (LILI) has been shown to induce a
significant increase in cell viability and proliferation. Growth factors such as retinoic acid (RA) and transforming growth
factor β1 (TGF-β1) play important roles in the differentiation of cells. The aim of this study was to investigate whether LILI in combination with growth factors could induce the differentiation of adipose derived stem cells (ADSCs) cocultured with smooth muscle cells (SMCs). The study used primary and continuous ADSC cell lines and a SMC line
(SKUT-1) as control. Cells were co-cultured directly at a ratio of 1:1 using established methods, with and without growth
factors and then exposed to LILI at 5 J/cm2 using a 636 nm diode laser. The cellular morphology, viability and
proliferation of the co-cultures were assessed over a period of one week. The study also monitored the expression of cell
specific markers over the same period of time. Genetic expression of the markers for both adipose derived stem cells (β1
Integrin and Thymidine 1) and smooth muscle cells (Heavy Myosin Chain) was monitored using flow cytometry. Cell
viability and proliferation increased significantly in the co-cultured groups that were exposed to laser alone, as well as in
combination with growth factors. Furthermore, there was a significant decrease in the expression of stem cell markers in
the ADSCs over time. The results indicate that LILI in combination with growth factors not only increases the viability
and proliferation of co-cultured cells but also decreases the expression of ADSC stem cell markers. This could indicate
the possible differentiation of ADSCs into SMCs.
Background/purpose: Stress induced premature senescence (SIPS) is defined as the long-term effect of subcytotoxic
stress on proliferative cell types. Cells in SIPS display differences at the level of protein expression which
affect energy metabolism, defense systems, redox potential, cell morphology and transduction pathways. This study
aimed to determine the effect of laser irradiation on second messengers in senescent cells and to establish if that
effect can be directly linked to changes in cellular function such as cell viability or proliferation. Materials and
Methods: Human keratinocyte cell cultures were modified to induce premature senescence using repeated sub-lethal
stresses of 200 uM H2O2 or 5% OH every day for four days with two days recovery. SIPS was confirmed by
senescence-associated β-galactosidase staining. Control conditions included normal, repeated stress of 500 uM H2O2
to induce apoptosis and 200 uM PBN as an anti-oxidant or free radical scavenger. Cells were irradiated with
1.5 J/cm2 on day 1 and 4 using a 648 nm diode laser (3.3 mW/cm2) and cellular responses were measured 1 h post
irradiation. The affect on second messengers was assessed by measuring cAMP, cGMP, nitric oxide and intracellular
calcium (Ca2+) while functional changes were assessed using cell morphology, ATP cell viability, LDH membrane
integrity and WST-1 cell proliferation. Results: Results indicate an increase in NO and a decrease in cGMP and
Ca2+ in 200 uM H2O2 irradiated cells while PBN irradiated cells showed a decrease in cAMP and an increase in ATP
viability and cell proliferation. Conclusion: Laser irradiation influences cell signaling which ultimately changes the
biological function of senescent cells. If laser therapy can stimulate the biological function of senescent cells it may
be beneficial to conditions such as immune senescence, skin ageing, muscle atrophy, premature ageing of arteries in
patients with advanced heart disease, neurodegenerative disorders and chronic renal failure.
Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To
ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when
determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures
simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to
determine the effect of laser irradiation on the genetic integrity of the cell.
Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to
induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy
may help the medical condition initially but damage DNA at the same time leaving undetected damage that may
result in late onset, more severe, induced medical conditions including cancer.
Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by
incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions.
Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a
fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after
irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase
(FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2.
A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when
compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model
showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular
DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2, there was a
significant increase in DNA damage in irradiated cells with and without the addition of FPG. These results are
indicative of the importance of both cell injury model as well as fluence when assessing the effect of phototherapy
on DNA integrity.
Background/purpose: In vivo studies have demonstrated that phototherapy accelerates wound healing in the clinical
environment; however the exact mechanism is still not completely understood. The main focus of this study was to use
in vitro laboratory results to establish an effective treatment regimen that may be practical and applicable to the clinical
environment. This in vitro study aimed to compare the cellular responses of wounded fibroblasts following a single
exposure of 5 J/cm2 or multiple exposures of low doses (2.5 J/cm2 or 5 J/cm2) on one day of the week to a single
application of a higher dose (16 J/cm2) on day 1 and day 4. Methodology: Cellular responses to Helium-Neon
(632.8 nm) laser irradiation were evaluated by measuring changes in cell morphology, cell viability, cell proliferation,
membrane integrity and DNA damage. Results: Wounded cells exposed to 5 J/cm2 on day 1 and day 4 showed an
increase in cell viability, increase in the release of bFGF, increase in cell density, decrease in ALP enzyme activity and
decrease in caspase 3/7 activity indicating a stimulatory effect. Wounded cells exposed to three doses of 5 J/cm2 on day
1 showed a decrease in cell viability and cell proliferation and an increase in LDH cytotoxicity and DNA damage
indicating an inhibitory effect. Conclusion: Results indicate that cellular responses are influenced by the combination of
dose administered, number of exposures and time between exposures. Single doses administered with sufficient time
between exposures is more beneficial to restoring cell function than multiple doses within a short period. Although this
work confirms previous reports on the cumulative effect of laser irradiation it provides essential information for the
initiation of in vivo clinical studies.
Photodynamic therapy (PDT) involves the use of a photosensitizer, which, when activated by light becomes toxic to the cancer cells. Lasers provide light at a specific wavelength required to activate the photosensitizer while the monochromaticity of the lasers at specific wavelengths results in maximum effectiveness of the photosensitizer during treatment. An important property of photosensitizers is that they should absorb light at a long wavelength as the light has to be able to penetrate tissue, and low energy light is able to travel further through tissue than light which absorbs at a shorter wavelength. This study aimed at evaluating the effects of 2 different photosensitizers, Al (AlPcSmix), commercially known as Photosens(R) and Ge (GePcSmix), both from the Phthalocyanine family of sensitizers, on oesophageal (SNO) and breast cancer (MCF-7) cells. Cells were irradiated at 660nm with a power output of 100 mW and a fluence of 10 J/cm2. Cell viability and proliferation were assessed using adenosine triphosphate (ATP) luminescence and alamarBlueTM staining. Lactate dehydrogenase (LDH) activity was used as a measure of cytotoxicity while the Comet assay was used to evaluate DNA damage. Heat shock protein 70 (Hsp70) induction acted as a measure of cellular stress. Both photosensitizers used during the course of this study are effective in targeting malignant cells, and have a cytotoxic effect on these cells when activated using laser irradiation. However, cytotoxic effects were also measured in the absence of laser irradiation, indicating the importance of photosensitizer concentration. Lower concentrations of photosensitizer in the presence of laser irradiation showed greater apoptotic inducing ability than with high concentrations. Morphologically, cells were affected to the detriment despite viability tests indicating the contrary.
An alternative treatment modality for diabetic wound healing includes low level laser therapy (LLLT). Biostimulation of such wounds may be of benefit to patients by reducing healing time. Structural, cellular and genetic events in diabetic wounded human skin fibroblasts (WS1) were evaluated after exposing cells in culture to a Helium-Neon (632.8nm), a Diode laser (830nm) and a Nd:YAG (Neodynium:Yttrium-Allumina-Gallium) laser (1064nm) at either 5J/cm2 or 16J/cm2. Cells were exposed twice a week and left 24 hours post-irradiation prior to measuring effects. Structural changes were evaluated by assessing colony formation, haptotaxis and chemotaxis. Cellular changes were evaluated using cell viability, (adenosine-triphosphate, ATP production), and proliferation, (alkaline phosphatase, ALP and basic fibroblast growth factor, bFGF expression), while the Comet assay evaluated DNA damage and cytotoxicity was determined assessing membrane permeability for lactate dehydrogenase (LDH). Caspase 3/7 activity was used as an estimate of apoptosis as a result of irradiation. The irradiated diabetic wounded cells showed structural, cellular as well as molecular resilience comparable to that of unwounded normal skin fibroblast cells. With regards to fluence, 5J/cm2 elicit positive cellular and structural responses while 16J/cm2 increases cellular and genetic damage and cellular morphology is altered. Different wavelengths of LLLT influences the beneficial outcomes of diabetic wounded cells and although all three wavelengths elicit cellular effects, the penetration depth of 830nm plays a significant role in the healing of diabetic wounded human fibroblast cells. Results from this study validate the contribution of LLLT to wound healing and elucidate the biochemical effects at a cellular level while highlighting the role of different dosages and wavelengths in LLLT.
A variety of strategies have been utilised for prevention and treatment of chronic wounds such as leg ulcers, diabetic foot ulcers and pressure sores1. Low Level Laser Therapy (LLLT) has been reported to be an invaluable tool in the enhancement of wound healing through stimulating cell proliferation, accelerating collagen synthesis and increasing ATP synthesis in mitochondria to name but a few2. This study focused on an in-vitro analysis of the cellular responses induced by treatment with three different laser beam profiles namely, the Gaussian (G), Super Gaussian (SG) and
Truncated Gaussian (TG), on normal wounded irradiated (WI) and wounded non-irradiated (WNI) human skin fibroblast cells (WS1), to test their influence in wound healing at 632.8 nm using a helium neon (HeNe) laser. For each beam profile, measurements were made using average energy densities over the sample ranging from 0.2 to 1 J, with
single exposures on normal wounded cells. The cells were subjected to different post irradiation incubation periods, ranging from 0 to 24 hours to evaluate the duration (time) dependent effects resulting from laser irradiation. The promoted cellular alterations were measured by increase in cell viability, cell proliferation and cytotoxicity. The results obtained showed that treatment with the G compared to the SG and TG beams resulted in a marked increase in cell
viability and proliferation. The data also showed that when cells undergo laser irradiation some cellular processes are driven by the peak energy density rather than the energy of the laser beam. We show that there exist threshold values for damage, and suggest optimal operating regimes for laser based wound healing.
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