Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation.

Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here, an advanced ultrasound-responsive GAM is described containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 coexpression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immunohistochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies. Copyright © 2017 John Wiley & Sons, Ltd.

Pt-based electro-catalytic materials derived from biosorption processes and their exploitation in fuel cell technology.

Yeast-based biomass, immobilised in polyvinyl alcohol (PVA) cryogels, was used as a biosorbant material for the recovery of platinum (PtCl (6) (2-) ) from aqueous solutions. The resulting biomass-Pt matrices were then employed directly as an electro-catalytic anode in a fuel cell configuration to generate electrical energy from renewable sources such as glucose and ethanol. We suggest an integrated strategy incorporating the derivation of a high-value product from a bioremediative process with a view towards producing energy from renewable fuels such as glucose and ethanol.

A versatile, stimulus-responsive nanoparticle-based platform for use in both sonodynamic and photodynamic cancer therapy.

A PLGA-based multifunctional biodegradable nanoparticle platform co-harboring hematoporphyrin and indocyanine green has been developed. In vitro studies demonstrate ultrasound and light stimulated generation of cytotoxic reactive oxygen species. In vivo studies show that the ICG component facilitates nIR fluorescence imaging that demonstrates accumulation of IV- administered nanoparticles in tumours. In vivo studies also demonstrate ultrasound- and light-mediated inhibition of tumour growth in animals treated with the platform. Since the platform consists entirely of clinically-approved agents it could find use in sonodynamic- and photodynamic-based therapies for cancer. STATEMENT OF SIGNIFICANCE: We describe a biocompatible and biodegradable nanoparticle-based platform for use in sonodynamic and photodynamic therapeutic approaches for the treatment of cancer. The non-toxic nanoparticles produce cytotoxic reactive oxygen species when exposed to ultrasound and/or light at levels that have no impact on tissues. The system is unique in that it is accumulated by tumours within six hours and has the ability to release its sensitising capability while retaining its imaging capability within a therapeutic time frame. The former could enhance dispersion and sensitising capabilities in less permeable tumour tissues and the latter permits the design of therapeutic approaches that minimize collateral damage to normal tissues.

Studies on N-acetylglucosaminidase activity produced by Streptomyces hygroscopicus.

Following growth on chitin-containing media the actinomycete, Streptomyces hygroscopicus produces N-acetylglucosaminidase. When the organism was grown in submerged culture on chitin-containing media, at 30 degrees C, production of the N-acetylglucosaminidase activity has been shown to increase to a maximum at around day 18. Following electrophoretic analysis of culture filtrates and subsequent zymogram staining with fluorescent substrate analogues, at least three general pools of N-acetylglucosaminidase activity were detected. These were named NA1, NA2 and NA3. In addition, a potential chitinase was also identified. The N-acetylglucosaminidases, referred to above as NA1 and NA2, were separated and their enzymatic properties were investigated.

cDNA cloning and expression of a Talaromyces emersonii beta-glucosidase determinant in Escherichia coli.

Intact mRNA has been isolated from the thermophilic fungus Talaromyces emersonii following growth on lactose-containing media and this has been used as template to produce cDNA. This cDNA has been cloned into the Escherichia coli expression system, pUC18 and this DNA was used to transform E. coli. A 2.1 kb fragment was isolated and shown to encode functional beta-glucosidase activity in E. coli. When the fragment was sub-cloned into a leaky strain of E. coli K-12, beta-glucosidase activity was detected in culture supernatants. The fragment was characterised further using restriction enzyme analysis and was also used as a probe in Northern and Southern blotting analyses of T. emersonii mRNA and genomic DNA, respectively. Results obtained from these analyses verified that the cloned insert DNA was of T. emersonii origin.

Combined electric field and ultrasound therapy as a novel anti-tumour treatment.

The permeabilising effects of electric pulses on cell membranes and the use of ultrasound energy of various intensities, for both thermal effects and enhancement of drug and gene delivery, have led to extensive research into the potential applications of these systems in the development of novel anti-cancer treatments. In the present study we have demonstrated for the first time that the application of brief electric pulses 'sensitises' tumour cells to the effects of low intensity ultrasound. The studies were conducted in human tumours established in athymic nude mice and in many instances resulted in the reduction of tumour mass. The combined electric field and ultrasound approach (CEFUS) was applied in vivo to a murine colon adenocarcinoma (C26) and a human oesophageal adenocarcinoma (OE19). The experiments performed demonstrated the anti-tumour effects of the combined therapy. Varying the electrosensitisation parameters used (voltage, waveform, electrode type) contributed to optimise the procedure. Exponential electric pulses with a peak of 1000 V/cm were initially used, but square wave pulses (1000 V/cm, 1 ms, x2, 1 Hz) were found to be just as effective. All ultrasound application parameters were kept constant during the study. The growth rate of C26 tumours treated with CEFUS was significantly reduced with respect to untreated controls at day 7 (96% of average initial tumour volume in CEFUS group versus 615% for controls, P < 0.05). Similar reduction was observed in OE19 tumours treated with CEFUS by day 4 (82% versus 232%, P < 0.032). Our preliminary data suggest that this novel technology could potentially be of wide application in clinical practice for the treatment of solid tumours and is worth further investigation.

Electro-sensitisation of mammalian cells and tissues to ultrasound: a novel tumour treatment modality.

This study demonstrates that mammalian cell targets (erythrocytes and tumour cells) may be sensitised to ultrasound using electric pulses and this combination treatment results in destruction of those cells in vitro. It further demonstrates that when a tumour mass is treated in vivo using combined electric field and ultrasound therapy, significant retardation of tumour growth has been observed using a mouse tumour model. We suggest that combined electric field and ultrasound (CEFUS) therapy may provide a novel, drug-free treatment modality for cancer.

Microbial biosorption of metals: potential in the treatment of metal pollution.

The phenomenon of metal biosorption by microorganisms has been thoroughly documented. Although this phenomenon is exhibited by both living and non-living forms of biomass, the purpose of this chapter will be to review biosorption by the latter. In addition, the application of various technological processes required for exploitation of this phenomenon in waste treatment will be examined.

Differential response of photosensitized young and old human erythrocytes to photodynamic activation.

It has recently been proposed that photosensitized erythrocytes may play an important role in the delivery and targeting of agents such as photosensitizers and chemotherapeutics for use in cancer treatment. It has been suggested that loading of photosensitized erythrocytes with chemotherapeutic agents would provide an ideal means of combining both treatment modalities. The recent application of real-time confocal laser scanning microscopy to the study of immediate effects of photodynamic activation on photosensitized erythrocytes has enabled us, in this study, to distinguish between the differential susceptibility of age-density resolved sub-populations of human erythrocytes to photodynamic activation. In this study we demonstrate that younger (low age-density) sub-populations of photosensitized erythrocytes are less susceptible than older (high age-density) sub-populations to photodynamic activation. We also demonstrate that this phenomenon is exhibited by cells photosensitized using hematoporphyrin derivative and rose bengal as photosensitizers. In both cases no significant difference in uptake of photosensitizer by both populations could be observed using absorbance spectrophotometry. The study suggests that age-density resolution of erythrocytes prior to loading and photosensitization might provide a means of enhancing the release of loaded components from the photosensitized system and this would, in turn, enhance the potential use of photosensitized erythrocytes as delivery or targeting systems for use in combination cancer therapies.

Use of a tetrazolium based colorimetric assay in assessing photoradiation therapy in vitro.

The suitability of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay to the determination of cell viability following photoradiation therapy (PRT) of human breast and melanoma cell lines has been examined. Results have been shown to correlate with those obtained using a clonogenic assay system. Using the MTT assay system it was shown that differences occur in the susceptibility of both lines to PDT. In addition it has been demonstrated that both lines differ with respect to their ability to develop photosensitivity in the presence of hematoporphyrin derivative (HpD). In the absence of serum this difference is not as obvious. This MTT assay provides a valid, simple and semi-automatable system for assessment of PRT in vitro.

Methotrexate-loaded, photosensitized erythrocytes: a photo-activatable carrier/delivery system for use in cancer therapy.

With a view towards the design of a system incorporating both the use of chemotherapeutics and photodynamic therapy for use in cancer treatment modalities, erythrocytes have been loaded with methotrexate and subsequently photosensitized by exposure to hematoporphyrin derivative. Loading of methotrexate into erythrocytes has been optimized by examining variations in electroporation conditions. Maximum loading indices observed were in the region of 64%. In order to obtain rapid pre-defined release of chemotherapeutic from the system, the erythrocytes were photosensitized. Light-dependent release of methotrexate from the system was examined. In addition, studies measuring the cytotoxic effects of light-activated release from the system using Hela cells as a target, suggested that decreases in cell viability following exposure to light resulted from the combined effects of chemotherapy and photoradiation therapy. Potential applications and advantages associated with this novel system are discussed.

Magnetically responsive photosensitizing reagents for possible use in photoradiation therapy.

The ability of a magnetically responsive material to function as a carrier for photosensitizing agents for use in photoradiation therapy (PRT) has been examined in vitro. The photosensitizer has been attached to the magnetically responsive matrix (Dynabeads) by non-specific adsorption, Intralipid-mediated adsorption and poly-L-lysine mediated adsorption. In these studies, it has been demonstrated that conditions of attachment of photosensitizer to the matrix may be adapted in order to facilitate a diffuse or highly localized photo-toxic effect on target cells in vitro. The authors believe that this system may represent a novel approach to targeting photosensitizing agents to specific areas, thereby circumventing some of the problems associated with conventional photoradiation therapy (PRT), particularly in hollow organs.

The effects of electric fields on photosensitized erythrocytes: possible enhancement of photodynamic activation.

In this study it has been found that exposure of photosensitized erythrocytes to short, intense electric pulses, resulted in cell lysis. When erythrocytes were photosensitized with increasing concentrations of the photosensitizer, hematoporphyrin derivative (HPD), and subjected to electric pulses in the absence of light, cell lysis increased with increasing photosensitizer concentration. In addition, it has been shown that exposure of photosensitized erythrocytes to electric field pulses of increasing field strength resulted in increased cell lysis. Light activation of photosensitized erythrocytes, pre-treated with electric pulses, also resulted in increased cell lysis. The results presented here suggest that HPD may be activated in the absence of light using electric pulses. We suggest that enhancement of activation by electric field stimulation may find application in increasing the overall efficiency of photodynamic therapy.

Electric field-enhanced activation of hematoporphyrin derivative: effects on a human tumour cell line.

In a recent report we described the effects of combined electroactivation and photoactivation of hematoporphyrin derivative (HPD) on human erythrocytes and established that activation-induced cell lysis was more pronounced when both modes of activation were sequentially applied to the system. Here we demonstrate that electric field-induced activation of HPD-treated HeLa cells results in cell death. This effect is shown to be dependant on both electric field strength and on HPD concentration. In addition, we demonstrate that exposure of HPD-treated cells to short and intense electric pulses prior to photoactivation, results in increased cell mortality. The results confirm our earlier suggestion that HPD may be activated in the presence of an applied electric field. The results further suggest that activation of photosensitizers using combined exposure to electric fields and light may play an important role in increasing the efficiency of photodynamic therapy (PDT) in the treatment of cancer.

Use of real-time confocal laser scanning microscopy to study immediate effects of photodynamic activation on photosensitized erythrocytes.

With a view towards the design of systems capable of combining the use of chemotherapy and photodynamic therapy in the treatment of cancer and other disorders, it has been proposed that photosensitized erythrocytes might be employed as carriers/vehicles for agents such as cancer chemotherapeutics. In studying the light dependent release of entrapped agents from such a system, the efficacy of light induced release is usually studied by measuring release of an entrapped component into centrifugation supernatants following photoactivation. It has hitherto been extremely difficult to examine what occurs upon immediate irradiation at the microscopic level in real-time. In this study we demonstrate that, using real-time confocal laser scanning microscopy, it is possible to directly observe immediate short-term events occurring during direct irradiation with the visualizing beam. Following irradiation of photosensitized erythrocytes with the visualizing beam form the confocal scanning system, it was noticed that some from of cell-disruptive event occurred. In this study we demonstrate a dose dependent response between this relatively immediate, light induced disruptive event with respect to both irradiation exposure and photosensitizer concentration. We suggest that this system may provide a novel means of observing, at a microscopic level, events occurring in real-time during photodynamic therapy.

The tumoricidal potential of extracorporeal shock wave therapy.

A potential role of extracorporeal shock wave lithotripsy (E.S.W.L.), in the destruction of tumours has been proposed in recent literature. To examine further this potential, we have studied the effects of E.S.W.L. on the sarcoma-derived osteoblast-like cell line MG-63. An in-situ assay of cell viability was used to establish the cellular response to high energy shock wave therapy. A significant tumoricidal effect was confirmed when the cells were grown and tested in conventional monolayer phase. However cells grown in the three-dimensional matrix of alginate beads were significantly less vulnerable to extracorporeal shock wave therapy as earlier studies have suggested.

Encapsulation of the thrombolytic enzyme, brinase, in photosensitized erythrocytes: a novel thrombolytic system based on photodynamic activation.

In order to circumvent many of the problems associated with the systemic administration of agents used in thrombolytic therapy, it was decided to investigate the possibility of using erythrocytes as carriers and delivery vehicles for these agents. The enzyme brinase, a fibrinolytic enzyme produced by Aspergillus oryzae, was loaded into rabbit erythrocytes using electroporation. The loading index for this enzyme was found to be 60% and incorporation appeared to be relatively stable over a period of 4 h. In order to facilitate the predetermined release of the loaded component from the erythrocytes, they were photosensitized using haematoporphyrin derivative (HPD) and release was demonstrated within 5 min of photoactivation. Inclusion of the loaded, photosensitized system into clotting blood and subsequent exposure to light demonstrated almost complete lysis of the clot. We believe that this system exhibits potential for use in thrombolytic therapy.

Real time confocal laser scanning microscopy: potential applications in space medicine and cell biology.

Photodynamic therapy (PDT), in which tissues may be rendered fatally light-sensitive represents a relatively novel treatment for cancer and other disorders such as cardiovascular disease. It offers significant application to disease control in an isolated environment such as space flight. In studying PDT in the laboratory, low energy lasers such as HeNe lasers are used to activate the photosensitized cellular target. A major problem associated with these studies is that events occurring during actual exposure of the target cells to the system cannot be examined in real time. In this study HeLa cells were photosensitized and photodynamic activation was accomplished using the scanning microbeam from a confocal laser scanning microscope. This form of activation allowed for simultaneous photoactivation and observation and facilitated the recording of events at a microscopic level during photoactivation. Effects of photodynamic activation on the target cells were monitored using the fluorophores rhodamine 123 and ethidium homodimer-1. Potential applications of these forms of analyses to space medicine and cell biology are discussed.

Lysolecithin-lipid interactions in disruption of the canine gastric mucosal barrier.

Lysolecithin has been implicated in the pathogenesis of gastric ulcer and reflux gastritis. By use of canine Heidenhain pouches, we examined effects of lysolecithin on the gastric mucosal barrier both at neutral (PO4 buffer, pH 7) and acidic (0.15 M HCl) pH with respect to interaction of lysolecithin with membrane lipids. At both pH values, 2 mM lysolecithin significantly increased forward diffusion of Na+ and backdiffusion of H+. Both solutions also caused significant efflux of membrane phospholipid and cholesterol. Saturation of neutral and acidic lysolecithin solutions with either lecithin or cholesterol significantly diminished or completely prevented disruption of the barrier to H+ backdiffusion and Na+ forward diffusion. In agreement with other reports, we also demonstrated formation of soluble lysolecithin-lecithin mixed micelles but formation of insoluble lysolecithin-cholesterol complexes. Finally, by use of [3H]polyethylene glycol and [14C]lysolecithin, we demonstrated more rapid mucosal uptake of lysolecithin at acidic than at neutral pH for any given experimental condition. Specifically, mean mucosal uptake of lysolecithin from acidic solutions saturated with lecithin (which did not disrupt the gastric mucosal barrier) was greater than uptake from neutral solutions without added lipid (which did disrupt the barrier). These studies suggest that lysolecithin-lipid interactions play an important role in lysolecithin-induced injury to the gastric mucosa. The most important of these interactions appears to occur in luminal lysolecithin micelles rather than within mucosal membranes.

Effect of high-energy shock wave frequency on viability of malignant cell lines in vitro.

In this study, the effects of high-energy shock wave (HESW) frequency on the viability of three different malignant cell lines were evaluated using a piezo-electric generator. The cell lines studied were AR42-J, VX-2 and HeLa. These cells were targeted both in free suspension and immobilised in gelatine beads. All cell lines in free suspension were acutely sensitive to HESW over a range of frequencies. When cells were immobilised in gelatine, however, the effect on cell viability was not as pronounced. However, at frequencies of 80- and 160-Hz kill rates approaching 80 and 90%, respectively, were observed. Cell proliferation following HESW treatment was not adversely affected. This study demonstrates that single cell suspensions are not appropriate for assessing the in vitro cellular effects of HESW. However, even when cells were immobilised in gelatine, high-frequency HESW produced substantial kill rates. The effects of high-frequency HESW merit further evaluation particularly using in vivo tumour.