Thermogelling Platform for Baicalin Delivery for Versatile Biomedical Applications.

Baicalin (BG) is a natural glycoside with several promising therapeutic and preventive applications. However, its pharmaceutical potential is compromised by its poor water solubility, complex oral absorption kinetics, and low bioavailability. In this work, BG was incorporated in a series of chitosan (Ch)/glycerophosphate (GP)-based thermosensitive hydrogel formulations to improve its water solubility and control its release profile. Molecular interactions between BG and GP were investigated using Fourier transform infrared spectroscopy (FT-IR), and the ability of GP to enhance the water solubility of BG was studied in different release media. Drug-loaded Ch/GP hydrogels were prepared and characterized for their gelation time, swelling ratio, and rheological properties in addition to surface and internal microstructure. Polyethylene glycol (PEG) 6000 and hydroxypropyl methyl cellulose (HPMC) were incorporated in the formulations at different ratios to study their effect on modulating the sol-gel behavior and the in vitro drug release. In vivo pharmacokinetic (PK) studies were carried out using a rabbit model to study the ability of drug-loaded Ch/GP thermosensitive hydrogels to control the absorption rate and improve the bioavailability of BG. Results showed that the solubility of BG was enhanced in the presence of GP, while the incorporation of PEG and/or HPMC had an impact on gelation time, rheological behavior, and rate of drug release in vitro. PK results obtained following buccal application of drug-loaded Ch/GP thermosensitive hydrogels to rabbits showed that the rate of BG absorption was controlled and the in vivo bioavailability was increased by 330% relative to BG aqueous oral suspension. The proposed Ch/GP thermosensitive hydrogel is an easily modifiable delivery platform that is not only capable of improving the solubility and bioavailability of BG following buccal administration but also can be suited for various local and injectable therapeutic applications.

Efficacy and Safety Profiles of Oral Atorvastatin-Loaded Nanoparticles: Effect of Size Modulation on Biodistribution.

Atorvastatin calcium (AC)-loaded nanoparticles (NPs) of mean particle diameter <100 nm and narrow distribution were prepared and characterized. Their in vivo PK as well as PD measures following oral administration in different dosage regimens in hyperlipidemic rats were evaluated. The results revealed that the oral bioavailability of two selected AC-NPs formulations was 235% and 169% relative to Lipitor. However, the treatment regimens were not superior in reducing serum total cholesterol (TC), low-density lipoproteins (LDL), and triglycerides (TG) levels compared to Lipitor. Moreover, the AC-NPs treatments were associated with significant adverse effects observed biochemically and histologically. These results were contradictory with those obtained from a previous study in which similarly formulated AC-NPs of mean particle diameter >200 nm were found to be more safe and effective in reducing TC, LDL, and TG levels when administered to hyperlipiemic rats at reduced dosing frequency compared to daily dose of Lipitor despite their lower oral bioavailability. The discrepant correlation between pharmacokinetics (PK) and pharmacodynamics (PD) results was suggested to pertain to the different biodistribution profiles of AC-NPs depending on their sizes. Hereby, we provide a simple approach of particle size modulation to enhance the efficacy and safety of atorvastatin.

Effect of lyophilized grapefruit juice on P-glycoprotein-mediated drug transport in-vitro and in-vivo.

The administration of grapefruit juice (GFJ) has been postulated to inhibit the activity of P-glycoprotein (P-gp) transport system and thus can enhance the uptake of substrate drugs. However, for various reasons, the results obtained have been always swaying between confirmation and refutation. This study aims at re-evaluating the effect of lyophilized freshly-prepared grapefruit juice (LGFJ) prepared from the whole peeled fruit on P-gp activity using the model drug doxorubicin (DOX) in-vitro and timolol maleate (TM) in-vivo. Human uterine sarcoma MES-SA/DX5v cells, grown under nanomolar concentration of DOX and highly expressing P-gp, were used as model cells for in-vitro studies whereas white New Zealand male rabbits were used for in-vivo studies. Results showed that the accumulation of DOX in MES-SA/DX5v cells was increased by 18.3 ± 2.0% in presence of LGFJ compared to control experiments. Results from in-vivo absorption studies showed that the relative oral bioavailability of TM ingested with LGFJ was significantly higher by 70% and 43% compared to the oral bioavailability of TM ingested with saline and a commercial GFJ, respectively. This study as such confirms the inhibitory effects of LGFJ on P-gp efflux proteins and highlights the superiority of using lyophilized freshly prepared juices over the commercially available juices in research studies. Also, the results call for further studies to assess the possibility of co-administrating LGFJ with anti-cancer agents to modulate multidrug resistance in their cellular environment or incorporating LGFJ in solid dosage forms to improve oral bioavailability of drugs.

Differential cytotoxicity and sonosensitization by sanazole: effect of cell type and acoustic parameters.

PURPOSE: Although sanazole has been used as a hypoxic radiosensitizer, we recently reported on its ability to sensitize U937 cells to hyperthermia and X-irradiation under aerobic conditions, enhancing apoptotic cell death following the combined treatment. The current study was undertaken to evaluate the effect of sanazole as a sonosensitizer under previously studied acoustic conditions of different pulse repetition frequencies, using two cell lines representative of solid tumours and haematopoietic cancers. METHODS: Cells were treated with different doses of sanazole. Flow-cytometric analysis and DNA fragmentation assay were carried out at different times, and morphological features were also inspected. For ultrasound treatment, cells were pre-incubated with a non-cytotoxic dose of sanazole for 30 min before exposure. Evaluation of cell killing and a parallel examination of intracellular oxidative stress levels in both cell lines were performed using flow cytometry. RESULTS: Sanazole alone displayed selective cytotoxic effects towards solid tumour-derived cancer cells, resulting in complete cell death after 24 h of treatment, and enhanced the ultrasound-induced cell killing 6 h post-treatment. The enhancement seemed to be mediated by an additive increase in intracellular oxidative stress levels. CONCLUSION: Sanazole seems to be an efficient cytotoxic agent for the treatment of solid tumours and a promising sonosensitizer under aerobic conditions.

Mild hyperthermia prior to electroporation increases transfection efficiency in HCT 116, HeLa S3 and SGC 7901 cells.

The change in transfection efficiency of electroporation by the combined treatment with mild preheating (40 degrees C for 30 min) was investigated. HCT 116, HeLa S3 and SGC 7901 cells were treated with electroporation in medium containing pBKCMV-Luc plasmid with or without preheating. After 24 h, luciferase activity was increased by 36, 28 and 77%; luciferase mRNA transcription was increased by 45, 50 and 68%; and fluorescein isothiocyanate-dextran accumulation was increased by 9, 35 and 15% in preheated groups, respectively. These results demonstrate that the transfection efficiency was enhanced by mild preheating. The mechanism partially involves increased macromolecular particle accumulation.

Threshold curves obtained under various gaseous conditions for free radical generation by burst ultrasound - Effects of dissolved gas, microbubbles and gas transport from the air.

To understand the underlying concepts required for the determination of thresholds for free radical generation, effects of gas dissolution in and microbubble addition to sonicated solutions were investigated. Four solutions with different gaseous conditions, air-saturated and degassed solutions with and without microbubbles of 20 microm in diameter with shells, were studied in the presence of an air-liquid interface. These test solutions were exposed to 1 MHz ultrasound of 0.06 MPa(p-p) at various pulse durations (PDs) from 0.1 to 5 ms and pulse repetition frequencies from 0.1 to 2 kHz. Generation of free radicals was evaluated using the electron spin resonance (ESR) spin trapping method and starch-iodine method. Thresholds of duty ratio (DR) corresponding to temporal average intensity of ultrasound for free radical generation were significantly greater in degassed solutions than in air-saturated solutions. Microbubbles had no significant effects in air-saturated solutions but caused a slight decrease in the threshold in degassed solutions. In all of these results, the DR of a threshold curve against pulse repetition period (PRP) was not constant but linearly decreased with it, suggesting that a balance between bubble growth and shrinkage during the ON and OFF times of burst ultrasound is the primary parameter for the interpretation of thresholds. The effect of an air-liquid interface of the solution was also examined, and it was revealed that gas transport from the air is a predominant factor determining the amount of free radicals.

Evaluation and comparison of three novel microbubbles: enhancement of ultrasound-induced cell death and free radicals production.

Three novel lipid-shell-type microbubbles (MBs), AS-0100, BG6356A and BG6356B, have been evaluated for their impact on ultrasound (US)-induced cell death and free radicals production. Previously studied and well-characterized US exposure conditions were employed in which human myelomonocytic lymphoma U937 cells were exposed to 1MHz pulsed US beam (0.3W/cm(2), 10% duty factor) for 1min with or without MBs. Three different concentrations of each MB were used. Apoptosis and cell lysis were assessed by examining phosphatidylserine externalization and by counting viable cells, respectively, 6h post-exposure. Free radicals production and scavenging activities were evaluated using electron paramagnetic resonance (EPR)-spin trapping. The results showed that only AS-0100 and BG6356A were able to enhance the US-induced apoptosis, mainly by increasing the secondary necrosis. Apoptosis and cell lysis seemed to depend more on mechanical forces exerted by oscillating MBs while free radicals played a trivial role. BG series MBs exhibited pronounced scavenging activities. Generally, despite the need for further optimization, AS-0100 and BG6356A appear to be promising as adjuncts in cases where US-induced cell death is required.

Influence of changing pulse repetition frequency on chemical and biological effects induced by low-intensity ultrasound in vitro.

This study was undertaken to examine ultrasound (US) mechanisms and their impact on chemical and biological effects in vitro as a function of changing pulse repetition frequency (PRF) from 0.5 to 100Hz using a 1MHz-generator at low-intensities and 50% duty factor (DF). The presence of inertial cavitation was detected by electron paramagnetic resonance (EPR) spin-trapping of hydroxyl radicals resulting from sonolysis of water. Non-cavitational effects were evaluated by studying the extent of sucrose hydrolysis measured by UV spectrophotometry. Biological effects were assessed by measuring the extent of cell killing and apoptosis induction in U937 cells using Trypan blue dye exclusion test and flow cytometry, respectively. The results indicate significant PRF dependence with respect to hydroxyl radical formation, cell killing and apoptosis induction. The lowest free radical formation and cell killing and the highest cell viability were found at 5Hz (100ms pulse duration). On the other hand, no correlation was found between sucrose hydrolysis and PRF. To our knowledge, this is the first report to be devoted to study the impact of low PRFs at low-intensities on US-induced chemical and biological effects and the mechanisms involved. This study has introduced the role of "US streaming" (convection); a forgotten factor in optimization studies, and explored its importance in comparison to standing waves.

Low-intensity ultrasound adjuvant therapy: enhancement of doxorubicin-induced cytotoxicity and the acoustic mechanisms involved.

PURPOSE: In this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally, the acoustic mechanisms involved were investigated. METHODS: The effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects. RESULTS: The combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect. CONCLUSION: This study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic conditions.

Enhancement of sodium butyrate-induced cell death and apoptosis by X-irradiation in the human colorectal cancer cell line HCT 116.

In this study, we aimed at evaluating the possible enhancing effect exerted by the combined use of sodium butyrate (SB) and X-rays on eradicating the human colorectal cancer cell line HCT 116 containing wild-type p53. We assessed the effect of this combination on the molecular pathways leading to cell death. HCT 116 cells were subjected to SB (1 mM) treatment followed by X-irradiation (5 Gy), and the effects on cell death, cell proliferation and cell cycle were examined. We also analyzed the apoptosis-indicating protein expression, mitochondrial membrane potential and intracellular superoxide formation. Treatment with SB alone significantly induced cell cycle arrest and apoptosis, whereas X-irradiation showed no effect on cell death despite its ability to block cell proliferation. Growth arrest and cell death were enhanced in the combined treatment groups. A marked reduction in the growth rate of the combined-treatment group was observed compared to that of the single-treatment groups. The apoptotic mitochondrial pathway was significantly enhanced with the combined use of the two agents. It was observed to be involved in the increased expression levels of p53 and p21, as well as in the release of cytochrome c and the alteration of the balance of anti- and pro-apoptotic Bcl-2 family proteins. Enhanced superoxide formation was also observed. However, the death receptor pathway was found to play no role in this phenomenon. These results suggest that X-irradiation promotes cell killing in synergy with SB treatment. Thus, the combined treatment led to a mutual potentiation of the killing effects of each agent.

Enhancement of sodium butyrate-induced cell death by hyperthermia in HCT 116 human colorectal cancer cells.

AIM: The possible enhancing effect of the combined use of sodium butyrate (SB) and hyperthermia to kill HCT 116 cells was evaluated. MATERIALS AND METHODS: HCT 116 cells were subjected to SB (1 mM) treatment followed by hyperthermia (44 degrees C 60 min) and the effects on cell death, cell proliferation and the cell cycle were examined. Apoptosis-indicating protein expressions and intracellular superoxide formation were also analysed. RESULTS: A marked reduction in the growth rate of the combined-treatment group was observed compared to those of the single-treatment groups. This involved the increased expression of p53 and p21, the alteration of the balance of anti- and proapoptotic Bcl-2 family proteins and enhanced superoxide formation. However, the death receptor pathway played no role. CONCLUSION: Hyperthermia synergistically promoted cell death induced by SB. Thus, the combined treatment led to mutual potentiation of the killing effects of each agent.

Cellular effects of low-intensity pulsed ultrasound and X-irradiation in combination in two human leukaemia cell lines.

Previously, we have shown that a combination between X-irradiation and low-intensity pulsed ultrasound (US) could synergistically suppress cell survival post exposure (Buldakov et al., 2014). In this study, the cellular effects underlying the enhanced cell killing are investigated. U937 and Molt-4 cell lines were exposed to 1.0 MHz US with 50% duty factor at 0.3 W/cm(2) and pulsed at 1, 5 and 10 Hz immediately after exposure to X-rays at 0, 0.5, 2.5 and 5 Gy. The cells were assayed at different time points to depict the major cellular events that culminated in cell death. For instance, membrane damage and cell lysis were estimated immediately following exposure and 24 h later. Intracellular reactive oxygen species (ROS) were also determined flow cytometrically after treatment. Moreover, the extent of DNA damage and cell cycle progression were determined at 6 and 24 h, respectively. Despite the general trend for synergism, there was a disproportionation of mediating factors depending on the cell type and its specific biological makeup. Immediately, US could induce appreciable necrotic cell death through extensive membrane damage in U937 but induced cell lysis in Molt-4 cells. ROS might have contributed to cell killing in Molt-4 but not in U937 cells. Although both of the physical modalities are significantly DNA-damaging alone, no additional damage was observed in combination. Moreover, override in some arrested cell cycle phases was also observed following combination. Collectively, the interaction between X-rays and US seems to depend mainly on the acoustic environment determined by the setup and this might explain the contradictory data among reports.

Effects of therapeutic ultrasound on the nucleus and genomic DNA.

In recent years, data have been accumulating on the ability of ultrasound to affect at a distance inside the cell. Previous conceptions about therapeutic ultrasound were mainly based on compromising membrane permeability and triggering some biochemical reactions. However, it was shown that ultrasound can access deep to the nuclear territory resulting in enhanced macromolecular localization as well as alterations in gene and protein expression. Recently, we have reported on the occurrence of DNA double-strand breaks in different human cell lines exposed to ultrasound in vitro with some insight into the subsequent DNA damage response and repair pathways. The impact of these observed effects again sways between extremes. It could be advantageous if employed in gene therapy, wound and bone fracture-accelerated healing to promote cellular proliferation, or in cancer eradication if the DNA lesions would culminate in cell death. However, it could be a worrying sign if they were penultimate to further cellular adaptations to stresses and thus shaking the safety of ultrasound application in diagnosis and therapy. In this review, an overview of the rationale of therapeutic ultrasound and the salient knowledge on ultrasound-induced effects on the nucleus and genomic DNA will be presented. The implications of the findings will be discussed hopefully to provide guidance to future ultrasound research.

Enhanced gene transfection using calcium phosphate co-precipitates and low-intensity pulsed ultrasound.

The capability to controllably disrupt the cell membrane by ultrasound (US), thus facilitating entry of exogenous species, has now reached a state of some maturity. However, a compelling question asks whether there is a residual role for US in enhancing transfection: that is, once the genetic material has been delivered to the cytosol, can US assist in its transport into the nucleus? The present experiment was designed with a view to addressing this question. As such, our experimental setup discriminates between: (i) the precursor cell membrane permealization step, and (ii) any subsequent intracellular trafficking into the nucleus. In this study, calcium phosphate co-precipitates (CaP) were used to internalize plasmid DNA encoding for luciferase (pDNA-Luc) (>90%) in HeLa cells. After 2h incubation with the CaP-pDNA-Luc, cells were washed and insonated for varying durations. The results showed that US can indeed enhance the intracellular trafficking of previously internalized genes when longer insonation periods are implemented, culminating with an increased probability for successful nuclear localization, as inferred from an enhanced luciferase expression. Moreover, the results suggest that the intracellular role of US might be mediated through a pathway that appears not to be limited to destabilizing the endosomal vesicles. The study thus provides new information regarding the intracellular effects of US, and in effect represents a new modality combining US and CaP carriers for improved efficiency in gene delivery.

Ultrasound-induced new cellular mechanism involved in drug resistance.

The acoustic effects in a biological milieu offer several scenarios for the reversal of multidrug resistance. In this study, we have observed higher sensitivity of doxorubicin-resistant uterine sarcoma MES-SA/DX5 cells to ultrasound exposure compared to its parent counterpart MES-SA cells; however, the results showed that the acoustic irradiation was genotoxic and could promote neotic division in exposed cells that was more pronounced in the resistant variant. The neotic progeny, imaged microscopically 24 hr post sonication, could contribute in modulating the final cell survival when an apoptotic dose of doxorubicin was combined with ultrasound applied either simultaneously or sequentially in dual-treatment protocols. Depending on the time and order of application of ultrasound and doxorubicin in combination treatments, there was either desensitization of the parent cells or sensitization of the resistant cells to doxorubicin action.

The role of Ca(2+) in ultrasound-elicited bioeffects: progress, perspectives and prospects.

Intracellular calcium (Ca(2+)) transients have been observed in association with exposure to therapeutic ultrasound and correlated to both early- and late-onset bioeffects. For example, it has been suggested that early 'ultra-short' Ca(2+) transients recorded during sonoporation can mediate Ca(2+)-dependent exocytosis and endocytosis processes as complementary mechanisms for membrane self-sealing. Moreover, apoptosis induction has been reported to occur through a partial mediation of a Ca(2+)-dependent pathway. In this review, we attempt to assemble the salient facts into a cogent whole, with special attention given to the relationships arising through altered Ca(2+) levels, which underscore its crucial role during ultrasonic interactions with biological systems and its consequent implications in the context of therapeutics.

Modulation control over ultrasound-mediated gene delivery: evaluating the importance of standing waves.

Low modulation frequencies from 0.5 to 100Hz were shown to alter the characteristics of the ultrasound field producing solution agitation (<5Hz; region of "ultrasound streaming" prevalence) or stagnancy (>5Hz; region of standing waves establishment) (Buldakov et al., Ultrason. Sonochem., 2009). In this study, the same conditions were used to depict the changes in exogenous DNA delivery in these regions. The luciferase expression data revealed that lower modulations were more capable of enhancing delivery at the expense of viability. On the contrary, the viability was conserved at higher modulations whereas delivery was found to be null. Cavitational activity and acoustic streaming were the effecters beyond the observed pattern and delivery enhancement was shown to be mediated mainly through sonopermeation. To promote transfection, the addition of calcium ions or an echo contrast agent (Levovist((R))) was proposed. Depending on the mechanism involved in each approach, differential enhancement was observed in both regions and at the interim zone (5Hz). In both cases, enhancement in standing waves field was significant reaching 16.0 and 3.3 folds increase, respectively. Therefore, it is concluded that although the establishment of standing waves is not the only prerequisite for high transfection rates, yet, it is a key element in optimization when other factors such as proximity and cavitation are considered.