Patience Mthunzi-Kufa

The effects of low level laser therapy on both HIV-1 infected and uninfected TZM-bl cells

Human immunodeficiency virus (HIV-1) infection remains a major health problem despite the use of highly active antiretroviral therapy (HAART), which has greatly reduced mortality rates. Due to the unavailability of an effective vaccine and treatment that would completely eradicate the virus in infected individuals, the quest for new therapies continues. Low level laser therapy (LLLT) involves the exposure of cells to low levels of red or infrared light. LLLT has been widely used in different medical conditions, but not in HIV-1 infection. This study aimed to determine the effects of LLLT on HIV-1 infected and uninfected TZM-bl cells. Both infected and uninfected cells were irradiated at a wavelength of 660xa0nm with different fluences from 2xa0J/cm2 to 10xa0J/cm2 . Changes in cellular responses were assessed using cell morphology, viability, proliferation, cytotoxicity and luciferase activity assays. Upon data analysis, uninfected irradiated cells showed no changes in cell morphology, viability, proliferation and cytotoxicity, while the infected irradiated cells did. In addition, laser irradiation reduced luciferase activity in infected cells. Finally, laser irradiation had no inhibitory effect in uninfected cells, whereas it induced cell damage in a dose dependent manner in infected cells.

Point of Care Diagnostics for HIV in Resource Limited Settings: An Overview

Human immunodeficiency virus (HIV) is a global health problem. Early diagnosis, rapid antiretroviral therapy (ART) initiation and monitoring of viral load are the key strategies for effective HIV management. Many people in resource limited settings where timely access to medical care is a challenge and healthcare infrastructure is poor have no access to laboratory facilities and diagnosis is dependent on the presence of point of care (POC) devices. POC instruments have shown to be easy to operate, maintain and transport and can easily be operated by less skilled health workers. Additionally, POC tests do not require laboratory technicians to operate. POC devices have resulted in a growing number of people testing for HIV and thereby receiving treatment early. In recent years, there has been great improvement in the development of POC technologies for early HIV diagnosis, HIV viral load and cluster of differentiation 4 (CD4) measurement. This review discusses POC technologies that are currently available and in the pipeline for diagnosing and monitoring HIV. We also give an overview of the technical and commercialization challenges in POC diagnostics for HIV.

Femtosecond laser assisted photo-transfection and differentiation of mouse embryonic stem cells

In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.

Pros and cons of characterising an optical translocation setup

The delivery of genetic material and drugs into mammalian cells using femtosecond (fs) laser pulses is escalating rapidly. This novel light based technique achieved through a precise focusing of a laser beam on the plasma membrane is called photoporation. This technique is attained using ultrashort laser pulses to irradiate plasma membrane of mammalian cells, thus resulting in the accumulation of a vast amount of free electrons. These generated electrons react photochemically with the cell membrane, resulting in the generation of sub-microscopic pores on the cell membrane enabling a variety of extracellular media to diffuse into the cell. This study is aimed at critically analysing the “do’s and don’ts” of designing, assembling, and characterising an optical translocation setup using a femtosecond legend titanium sapphire regenerative amplifier pulsed laser (Gaussian beam, 800 nm, 1 kHz, 113 fs, and an output power of 850 mW). The main objective in our study is to determine optical phototranslocation parameters which are compatible to the plasma membrane and cell viability. Such parameters included beam profiling, testing a range of laser fluencies suitable for photoporation, assessment of the beam quality and laser-cell interaction time. In our study, Chinese Hamster Ovary-K1 (CHO-K1) cells were photoporated in the presence of trypan blue to determine optimal parameters for photoporation experiment. An average power of 4.5 μW, exposure time of 7 ms, with a laser beam spot of ~1.1 μm diameter at the focus worked optimally without any sign of cell stress and cytoplasmic bleeding. Cellular responses post laser treatment were analysed using cell morphology studies.

In-vitro photo-translocation of antiretroviral drug delivery into TZMbl cells

The current human immunodeficiency virus (HIV) treatment regime possesses the ability to diminish the viral capacity to unnoticeable levels; however complete eradication of the virus cannot be achieved while latent HIV-1 reservoirs go unchallenged. Therapeutic targeting of HIV therefore requires further investigation and current therapies need modification in order to address HIV eradication. This deflects research towards investigating potential novel antiretroviral drug delivery systems. The use of femtosecond (fs) laser pulses in promoting targeted optical drug delivery of antiretroviral drugs (ARVs) into TZMbl cells revolves around using ultrafast laser pulses that have high peak powers, which precisely disrupt the cell plasma membrane in order to allow immediate transportation and expression of exogenous material into the live mammalian cells. A photo-translocation optical setup was built and validated by characterisation of the accurate parameters such as wavelength (800 nm) and pulse duration (115 fs). Optimisation of drug translocation parameters were done by performing trypan blue translocation studies. Cellular responses were determined via cell viability (Adenosine Triphosphate activity) and cell cytotoxicity (Lactate Dehydrogenase) assays which were done to study the influence of the drugs and laser exposure on the cells. After laser irradiation, high cell viability was observed and low toxicity levels were observed after exposure of the cells to both the ARVs and the laser. Our results confirmed that, with minimal damage and high therapeutic levels of ARVs, the fs laser assisted drug delivery system is efficient with benefits of non-invasive and non-toxic treatment to the cells.

Phototodynamic activity of zinc monocarboxyphenoxy phthalocyane (ZnMCPPc) conjugated to gold silver (AuAg) nanoparticles in melanoma cancer cells

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality for the treatment of neoplastic and non-neoplastic diseases. In PDT of cancer, irradiation with light of a specific wavelength leads to activation of a photosensitizer which results in generation of reactive oxygen species (ROS) which induces cell death. Many phthalocyanine photosensitizers are hydrophobic and insoluble in water, which limits their therapeutic efficiency. Consequently, advanced delivery systems and strategies are needed to improve the effectiveness of these photosensitizers. Nanoparticles have shown promising results in increasing aqueous solubility, bioavailability, stability and delivery of photosensitizers to their target. This study investigated the photodynamic activity of zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc) conjugated to gold silver (AuAg) nanoparticles in melanoma cancer cells. The photodynamic activity of ZnMCPPc conjugated to AuAg nanoparticles were evaluated using cellular morphology, viability, proliferation and cytotoxicity. Untreated cells showed no changes in cellular morphology, proliferation and cytotoxicity. However, photoactivated ZnMCPPc conjugated to AuAg nanoparticles showed changes in cell morphology and a dose dependent decrease in cellular viability, proliferation and an increase in cell membrane damage. The ZnMCPPc conjugated to AuAg nanoparticles used in this study was highly effective in inducing cell death of melanoma cancer cells.

Phototoxic effects of free phthalocyanine and phthalocyanine conjugated to gold nanoparticles for targeted photodynamic therapy of melanoma cancer

Photodynamic therapy (PDT) has emerged as an effective treatment modality for various malignant neoplasia and diseases. In PDT, the photochemical interaction of photosensitizer (PS), light and molecular oxygen produces singlet oxygen which can lead to tumour cell apoptosis, necrosis or autophagy. The success of PDT is limited by the hydrophobic characteristic of the PS which hinders treatment administration and efficiency. To circumvent this limitation, PS can be incorporated in nanostructured drug delivery systems such as gold nanoparticles (AuNPs). In this study, we investigated the effectiveness of free zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc) and ZnMCPPc conjugated to AuNPs. Commercially purchased melanoma cancer cells cultured as cell monolayers were used in this study. Changes in cellular response were evaluated using cellular morphology, viability, proliferation and cytotoxicity. Untreated cells showed no changes in cellular morphology, proliferation and cytotoxicity. However, photoactivated free ZnMCPPc and ZnMCPPc conjugated to AuNPs showed changes in cellular morphology and a dose dependent decrease in cellular viability and proliferation as well as an increase in cell membrane. ZnMCPPc conjugated to AuNPs showed an improved efficiency in PDT as compared to free ZnMCPPc, which might be as a result of the vehicle effect of AuNPs. Both PSs used in this study were effective in inducing cell death with ZnMCPPc conjugated to AuNPs showing great potential as an effective PS for PDT.

Investigation of HIV-1 infected and uninfected cells using the optical trapping technique

Optical trapping has emerged as an essential tool for manipulating single biological material and performing sophisticated spectroscopy analysis on individual cell. The optical trapping technique has been used to grab and immobilize cells from a tightly focused laser beam emitted through a high numerical aperture objective lens. Coupling optical trapping with other technologies is possible and allows stable sample trapping, while also facilitating molecular, chemical and spectroscopic analysis. For this reason, we are exploring laser trapping combined with laser spectroscopy as a potential non-invasive method of interrogating individual cells with a high degree of specificity in terms of information generated. Thus, for the delivery of as much pathological information as possible, we use a home-build optical trapping and spectroscopy system for real time probing human immunodeficiency virus (HIV-1) infected and uninfected single cells. Briefly, our experimental rig comprises an infrared continuous wave laser at 1064 nm with power output of 1.5 W, a 100X high numerical aperture oil-immersion microscope objective used to capture and immobilise individual cell samples as well as an excitation source. Spectroscopy spectral patterns obtained by the 1064 nm laser beam excitation provide information on HIV-1 infected and uninfected cells. We present these preliminary findings which may be valuable for the development of an HIV-1 point of care detection system.

Targeted femtosecond laser driven drug delivery within HIV-1 infected cells: in-vitro studies

Human immunodeficiency virus (HIV-1) infection still remains one amongst the world’s most challenging infections since its discovery. Antiretroviral therapy is the recommended treatment of choice for HIV-1 infection taken by patients orally. The highly active antiretroviral therapy (HAART) prevents the replication of HIV-1 and further destruction of the immune system, therefore enabling the body to fight opportunistic life-threatening infections, cancers, and also arrest HIV infection from advancing to AIDS. The major challenge with HAART is the inability to reach the viral reservoirs where the HIV-1 remains latent and persistent, leading to inability to fully eradicate the virus. This study is aimed at initially designing and assembling a fully functional optical translocation setup to optically deliver antiretroviral drugs into HIV-1 infected cells in a targeted manner using Gaussian beam mode femtosecond laser pulses in-vitro. The main objective of our study is to define the in-vitro drug photo-translocation parameters to allow future design of an efficient drug delivery device with potential in-vivo drug delivery applications. In our experiments, HEK 293T cells were used to produce HIV-1 enveloped pseudovirus (ZM53) to infect TZM-bl cells which were later treated with laser pulses emitted by a titanium sapphire laser (800 nm, 1KHz, 113 fs, ~ 6.5 μW) to create sub-microscopic pores on the cell membrane enabling influx of extracellular media. Following laser treatment, changes in cellular responses were analysed using cell morphology studies, cytotoxicity, and luciferase assay studies. Controls included laser untreated cells incubated with the drug for 72 hours. The data in this study was statistically analysed using the SigmaPlot software version 13.

Could low level laser therapy and highly active antiretroviral therapy lead to complete eradication of HIV-1 in vitro?

Human immunodeficiency virus (HIV-1) infection remains a major health problem despite the use of highly active antiretroviral therapy (HAART), which has greatly reduced mortality rates. Due to the unavailability of an effective vaccine or a treatment that would completely eradicate the virus, the quest for new and combination therapies continues. In this study we explored the influence of Low Level Laser Therapy (LLLT) in HIV-1 infected and uninfected cells. Literature reports LLLT as widely used to treat different medical conditions such as diabetic wounds, sports injuries and others. The technique involves exposure of cells or tissue to low levels of red and near infrared laser light. Both HIV infected and uninfected cells were laser irradiated at a wavelength of 640 nm with fluencies ranging from 2 to 10 J/cm2 and cellular responses were assessed 24 hours post laser treatment. In our studies, laser therapy had no inhibitory effects in HIV-1 uninfected cells as was indicated by the cell morphology and proliferation results. However, laser irradiation enhanced cell apoptosis in HIV-1 infected cells as the laser fluencies increased. This led to further studies in which laser irradiation would be conducted in the presence of HAART to determine whether HAART would minimise the detrimental effects of laser irradiation in infected cells.