A Novel Approach for Therapeutic Delivery to the Rodent Pancreas Via Its Arterial Blood Supply.

OBJECTIVES: Endovascular techniques can now access the arterial blood supply of the pancreas in humans to enable therapeutics to reach the gland in high concentrations while concurrently avoiding issues related to non-targeted delivery. However, there is no way to replicate this in small animals. In a rat model, we therefore developed a novel non-terminal technique to deliver therapeutics to different regions of the pancreas, via its arterial blood supply. METHODS: In female Wistar rats, selective branches of the celiac artery were temporarily ligated, depending on the region of the pancreas being targeted. Trypan blue dye was then administered as a surrogate marker for a therapeutic agent, via the celiac artery, and its staining/distribution throughout the pancreas determined. Postoperatively, animals were monitored daily, and serum was evaluated for markers of pancreatitis, liver, and metabolic function. RESULTS: Using this technique, we could selectively target the head, body/tail, or entire gland of the pancreas, via its arterial blood supply, with minimal nontarget staining. Following the procedure, all animals recovered with no evidence of pancreatitis or liver/metabolic dysfunction. CONCLUSIONS: Our study demonstrates a novel technique that can be used to selectively deliver therapeutics directly to the rat pancreas in a safe manner with full recovery of the animal.

Grooved and nongrooved clear corneal incisions in phacoemulsification: permeability study.

PURPOSE: To detect the inflow of trypan blue through grooved and nongrooved sutureless self-sealing clear corneal incisions at the end of phacoemulsification as compared to a control group. METHODS: A prospective randomized masked trial considered 52 eyes randomized into 3 groups in which phacoemulsification was performed: group A, nongrooved incisions; group B, grooved incisions; and group C, controls. By the end of each surgery, trypan blue was instilled on the ocular surface in groups A and B and rinsed out after 2 minutes. A sample of the anterior chamber content was collected and analyzed by high-performance liquid chromatography to identify and quantify the trypan blue concentration. The presence of trypan blue was expressed as a specific single peak graphic image. The mean areas of these peaks were used to assess the groups using a nonparametric Mann-Whitney test. RESULTS: There was a statistically significant difference between the nongrooved incisions group and the control group (p = 0.0448). No significant difference was observed between group B (grooved incision) and controls (p = 0.1800). CONCLUSIONS: Trypan blue was detected in the anterior chamber when nongrooved clear corneal incision was used. There was no trypan blue detection in the group with grooved clear corneal main incisions.

Comparison of the effect of torsional and microburst longitudinal ultrasound on clear corneal incisions during phacoemulsification.

PURPOSE: To compare incision integrity after clear corneal microcoaxial phacoemulsification using longitudinal and torsional ultrasound (US). SETTING: Iladevi Cataract & IOL Research Centre, Ahmedabad, India. DESIGN: Prospective randomized experimental clinical trial. METHODS: Part 1 comprised an experimental study of rabbit eyes. Group 1 received longitudinal US. Group 2 received torsional US. The right eye of each rabbit served as a control. Samples were processed for histomorphology and collagen I denaturation by immunofluorescence. Part 2 comprised a clinical trial of patients. Group 1 received torsional US. Group 2 received longitudinal US. At the end of surgery, trypan blue 0.0125% was instilled. After 2 minutes, 0.1 mL of aqueous was aspirated and its optical density measured. RESULTS: In part 1, incision histomorphology was comparable in both modalities. Collagen denaturation tests (immunofluorescence, dot blot analysis) showed no irregularity in collagen arrangement in either group. In Group 2, Descemet membrane was detached and endothelial cells were minimal at the roof of the incision. In part 2, trypan blue ingress into the anterior chamber was significantly greater in Group 1 than in Group 2 (mean 3.40 + 0.6 log units versus and 3.77 + 0.82 log units) (P<.007). CONCLUSIONS: Incision histomorphology in the torsional group showed minimal Descemet membrane detachment and minimal endothelial cell loss at the roof of the incision. Minimal ingress of trypan blue into the anterior chamber was observed with torsional US, indicating better wound integrity than with longitudinal US. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Development of an intrabiliary MR imaging-monitored local agent delivery technique: a feasibility study in pigs.

PURPOSE: To investigate the feasibility of using magnetic resonance (MR) imaging to monitor intrabiliary delivery of motexafin gadolinium (MGd) into pig common bile duct (CBD) walls. MATERIALS AND METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. Initially, human cholangiocarcinoma cells were treated with various concentrations of MGd, a compound serving as a T1-weighted MR imaging contrast agent, chemotherapy drug, and cell marker. These cells were then examined by means of confocal microscopy to confirm the intracellular uptake of MGd. In addition, an MGd/trypan blue mixture was locally infused into CBD walls of six cadaveric pigs using a microporous balloon catheter. CBDs of six pigs were infused with saline to serve as controls. Ex vivo T1-weighted MR imaging of these CBDs was performed. For in vivo technical validation, the microporous balloon catheter was placed in the CBD by means of a transcholecytic access to deliver MGd/trypan blue into CBD walls of six living pigs. T1-weighted images were obtained with both a surface coil and an intrabiliary MR imaging guidewire, and contrast-to-noise ratios of CBD walls before and after MGd/trypan blue infusions were compared in the two groups by means of paired t test, with subsequent histologic analysis to confirm the penetration and distribution of the MGd/trypan blue agent into CBD walls. RESULTS: In vitro experiments confirmed uptake of MGd by human cholangiocarcinoma cells. The ex vivo experiments demonstrated the penetration of MGd/trypan blue into the CBD walls. The in vivo experiment confirmed the uptake of MGd/trypan blue, showing an increased contrast-to-noise ratio for the CBD after administration of the mixture, compared with images obtained prior to MGd/trypan blue administration (11.6 ± 4.2 [standard deviation] vs 5.7 ± 2.8; P = .04). Histologic results depicted the blue dye stains and red fluorescence of MGd in CBD walls, confirming the imaging findings. CONCLUSION: It is feasible to use MR imaging to monitor the penetration of locally delivered MGd into pig CBD walls.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.

Subretinal migration of trypan blue during macular hole and epiretinal membrane peel: an observational case series. Is there a safer method?

PURPOSE: To report the inadvertent subretinal migration and effect of trypan blue (TB) during staining of the epiretinal membrane (ERM) for macular pucker, and internal limiting membrane during macular hole (MH) surgery, and to suggest alternative safe methods of injecting TB. METHODS: Three cases in which TB migrated to the subretinal space were followed up on day 1, day 7, day 21, and at 3 months following the initial operation. Two of the cases were operated for MH and one patient had ERM peel. Colour fundus and optical coherence tomography (OCT) were performed on day 1 and on each subsequent visit. RESULTS: In both cases of MH the hole was closed postoperatively. The patient with ERM had the membrane peeled successfully as documented by OCT. Clinically, all patients demonstrated chorioretinal atrophy in the area of TB migration. There was thinning of the retina as noted by OCT. CONCLUSION: It is difficult to prove whether the chorioretinal atrophy was caused by the subretinal TB or by the accidental forceful dye injection, but subretinal TB and contact of TB with the retinal pigment epithelium should be avoided, and precautions should be taken during intravitreal injection. We suggest a more controlled method of dye injection in such cases using the flute needle rather than the syringe technique that is conventionally used.

A microneedle roller for transdermal drug delivery.

Microneedle rollers have been used to treat large areas of skin for cosmetic purposes and to increase skin permeability for drug delivery. In this study, we introduce a polymer microneedle roller fabricated by inclined rotational UV lithography, replicated by micromolding hydrophobic polylactic acid and hydrophilic carboxy-methyl-cellulose. These microneedles created micron-scale holes in human and porcine cadaver skin that permitted entry of acetylsalicylic acid, Trypan blue and nanoparticles measuring 50nm and 200nm in diameter. The amount of acetylsalicylic acid delivered increased with the number of holes made in the skin and was 1-2 orders of magnitude greater than in untreated skin. Lateral diffusion in the skin between holes made by microneedles followed expected diffusional kinetics, with effective diffusivity values that were 23-160 times smaller than in water. Compared to inserting microneedles on a flat patch, the sequential insertion of microneedles row by row on a roller required less insertion force in full-thickness porcine skin. Overall, polymer microneedle rollers, prepared from replicated polymer films, offer a simple way to increase skin permeability for drug delivery.

Pluronic enhances the robustness and reduces the cell attachment of mammalian cells.

The addition of the non-ionic surfactant, Pluronic F-68, to serum-free CHO cultures causes multi-functional effects that enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. Three independent CHO cell lines were subjected to high liquid shear in assay systems that either included or excluded a liquid-gas interface. In the absence of Pluronic, there was a loss in cell viability in either assay system, although there was an intrinsic variability in sensitivity of the cell lines to shear damage. Supplementation with Pluronic prevented loss of cell viability, indicating protection in either a gas sparged or bubble-free environment. However, we found no evidence of long-term protection of cells once Pluronic was removed. Pluronic was capable of repairing trypsin-damaged cells as evidenced by enhanced growth, reduced membrane porosity, and improved robustness under liquid shear. The proportion of adherent cells was reduced to a minimal level by the presence of Pluronic although its effect was rapidly reversible with a high proportion (70%) of adherent cells observed within a few culture passages of its removal. The observed effects of Pluronic on these cultures are compatible with a mechanism in which the polymer forms a protective layer on the cell membrane, which has a significantly lower hydrophobicity.

Dry eye and blink rate simulation with a pig eye model.

PURPOSE: To simulate medium level "dry eye" and investigate the effect of "blink" rates in "dry eye" condition using a novel porcine dry eye model (pDEM). METHODS: In the first experiment, a 40 s "lacrimation/blink" interval (lacrimation occurring in conjunction with blink) was set in the pDEM to simulate a medium level of "dry eye" condition. In the second experiment, "lacrimation" interval was set at 60 s and three different "inter-blink" intervals of 6, 12, and 20 s were set in groups A, B, and C, respectively. The integrity of each cornea was assessed before and after experiments by slit-lamp microscopy with sodium fluorescein solution. The viability of corneal epithelial cells was assessed by the Trypan blue exclusion test after the experiment. RESULTS: The amount of sodium fluorescein staining was significantly (p < 0.05) lower at the end of the experiment, when the "inter-blink" interval was set at 12 s. The medians of the final fluorescein grades of corneas in the pDEM were grade 1.5, 1.0, and 2.0 when the "inter-blink" intervals were set at 6, 12, and 20 s, respectively. There was no significant difference in the number of damaged cells between the central and peripheral corneas with different "inter-blink" intervals. Although in each case the peripheral area had a lower number of non-viable cells than the central area of the cornea, there was no significant change in the number of Trypan blue stained cells in either area with different "inter-blink" intervals. CONCLUSION: Different severity levels of "dry eye" can be simulated using the newly developed pDEM. Increased blink rate may protect the cornea against desiccation-induced damage; however, increased blink rate may also increase shear force between the cornea and conjunctiva and result in mechanical damage because of increased frictional force.

Prediction of convection-enhanced drug delivery to the human brain.

The treatment for many neurodegenerative diseases of the central nervous system (CNS) involves the delivery of large molecular weight drugs to the brain. The blood brain barrier, however, prevents many therapeutic molecules from entering the CNS. Despite much effort in studying drug dispersion with animal models, accurate drug targeting in humans remains a challenge. This article proposes an engineering approach for the systematic design of targeted drug delivery into the human brain. The proposed method predicts achievable volumes of distribution for therapeutic agents based on first principles transport and chemical kinetics models as well as accurate reconstruction of the brain geometry from patient-specific diffusion tensor magnetic resonance imaging. The predictive capabilities of the methodology will be demonstrated for invasive intraparenchymal drug administration. A systematic procedure to determine the optimal infusion and catheter design parameters to maximize penetration depth and volumes of distribution in the target area will be discussed. The computational results are validated with agarose gel phantom experiments. The methodology integrates interdisciplinary expertise from medical imaging and engineering. This approach will allow physicians and scientists to design and optimize drug administration in a systematic fashion.

Assembled microneedle arrays enhance the transport of compounds varying over a large range of molecular weight across human dermatomed skin.

In this study, we demonstrate the feasibility to use microneedle arrays manufactured from commercially available 30G hypodermal needles to enhance the transport of compounds up to a molecular weight of 72 kDa. Piercing of human dermatomed skin with microneedle arrays was studied by Trypan Blue staining on the SC side of the skin and transepidermal water loss measurements (TEWL). Passive transport studies were conducted with Cascade Blue (CB, Mw 538), Dextran-Cascade Blue (DCB, Mw 10 kDa), and FITC coupled Dextran (FITC-Dex, Mw 72 kDa). Microneedle arrays with needle lengths of 900, 700 and 550 micro m are able to pierce dermatomed human skin as evident from (a) the appearance of blue spots on the dermal side of the skin after Trypan Blue treatment and (b) elevated TEWL levels after piercing compared to non-treated human dermatomed skin. Microneedles with a length of 300 micro m did not pierce human skin in vitro. Transport studies performed with model compounds ranging from 538 Da to 72 kDa revealed that pretreatment with microneedle arrays enhanced the transport across dermatomed human skin. However, some degradation was also observed for FITC-Dex and DCB. We conclude that assembled microneedle arrays can be used to deliver compounds through the skin up to a molecular weight of at least 72 kDa.

Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.

OBJECT: Clinical application of the convection-enhanced delivery (CED) technique is currently limited by low infusion speed and reflux of the delivered agent. The authors developed and evaluated a new step-design cannula to overcome present limitations and to introduce a rapid, reflux-free CED method for future clinical trials. METHODS: The CED of 0.4% trypan blue dye was performed in agarose gel to test cannula needles for distribution and reflux. Infusion rates ranging from 0.5 to 50 microl/minute were used. Agarose gel findings were translated into a study in rats and then in cynomolgus monkeys (Macacafascicularis) by using trypan blue and liposomes to confirm the efficacy of the reflux-free step-design cannula in vivo. Results of agarose gel studies showed reflux-free infusion with high flow rates using the step-design cannula. Data from the study in rats confirmed the agarose gel findings and also revealed increasing tissue damage at a flow rate above 5-microl/minute. Robust reflux-free delivery and distribution of liposomes was achieved using the step-design cannula in brains in both rats and nonhuman primates. CONCLUSIONS: The authors developed a new step-design cannula for CED that effectively prevents reflux in vivo and maximizes the distribution of agents delivered in the brain. Data in the present study show reflux-free infusion with a constant volume of distribution in the rat brain over a broad range of flow rates. Reflux-free delivery of liposomes into nonhuman primate brain was also established using the cannula. This step-design cannula may allow reflux-free distribution and shorten the duration of infusion in future clinical applications of CED in humans.

Superiority of the two-layer method before islet isolation confirmed by in vivo viability assessment.

BACKGROUND: Although the outcome of islet transplantation has improved, there remains a major obstacle in isolating viable islets from prolonged preserved pancreas. We previously reported that the two-layer cold storage method (TLM) improved the yield and in vitro function. In this study, we performed in vivo accurate functional analyses of islets from TLM-preserved pancreas and investigated pancreatic duct cell viability, which may critically affect islet isolation. METHODS: Rat islets isolated from fresh pancreas (group 1), after preservation in the University of Wisconsin (UW) solution (group 2) or by the TLM (group 3), were examined by assessing islet yields, stimulation indices, cure rates after transplantation to diabetic nude mice, and trypan blue uptake of pancreatic duct cells. RESULTS: TLM significantly improved the islet yield compared with UW cold storage. The cure rates after transplantation were 100%, 0%, and 80% for groups 1, 2, and 3, respectively. This indicates that islet viability was well maintained even after 24 hr of TLM preservation. The percentages of nonviable duct cells were 4.1%+/-1.9%, 48.3%+/-8.0%, and 26.1%+/-21.4% in groups 1, 2, and 3, respectively, showing that the TLM was superior to UW as seen by this duct cell viability assessment. CONCLUSIONS: The TLM used for pancreas preservation before islet isolation results in excellent islet function in addition to improved islet yield comparable to freshly isolated islets. The underlying mechanism may be duct cell viability maintained during TLM preservation. Therefore the TLM is an excellent preservation technique for isolating sufficient numbers of highly viable islets.

Astrocytes protect neurons from ethanol-induced oxidative stress and apoptotic death.

Ethanol induces oxidative stress in cultured fetal rat cortical neurons and this is followed by apoptotic death, which can be prevented by normalization of cell content of reduced glutathione (GSH). Because astrocytes can play a central role in maintenance of neuron GSH homeostasis, the following experiments utilized cocultures of neonatal rat cortical astrocytes and fetal cortical neurons to determine if astrocytes could protect neurons from ethanol-mediated apoptotic death via this mechanism. In cortical neurons cultured in the absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6-, 12-, and 24-hr exposures) decreased trypan blue exclusion and the MTT viability measures by up to 45% (P < 0.05), increased levels of reactive oxygen species (ROS) by up to 81% (P < 0.05), and decreased GSH within 1 hr of treatment by 49 and 51% for 2.5 and 4 mg/ml, respectively (P < 0.05). This was followed by onset of apoptotic cell death as determined by increased Annexin V binding and DNA fragmentation by 12 hr of ethanol exposure. Coculturing neurons with astrocytes prevented GSH depletion by 2.5 mg/ml ethanol, whereas GSH content was increased over controls in neurons exposed to 4 mg/ml ethanol (by up to 341%; P < 0.05). Ethanol generated increases in neuron ROS and apoptosis; decreases in viability were also prevented by coculture. Astrocytes were largely insensitive to ethanol, using the same measures. Only exposure to 4.0 mg/ml ethanol decreased GSH content in astrocytes, concomitant with a 204% increase in GSH efflux (P < 0.05). These studies illustrate that astrocytes can protect neurons from ethanol-mediated apoptotic death and that this may be related to maintenance of neuron GSH.

[Experimental study on the penetrability of trypan blue to the rat prostate].

OBJECTIVE: To understand the penetrability of trypan blue to the normal prostate as well as to the inflammatory prostate and the prostate with benign hyperplasia in rats. METHODS: Sixty SD male rats were randomized into 4 groups: NP (normal prostate) group (n = 15), BP (bacterial prostatitis) group (n = 15), BPH (benign prostatic hyperplasia) group (n = 15), and BPH-BP (benign prostatic hyperplasia with bacterial prostatitis) group (n = 15). Five rats were taken from each group as non-staining controls (NC, n = 5 x 4) and the other 10 were injected by tail intravenation with 1% trypan blue and then the prostates were isolated from the rats killed by anaesthesia after 2 hours. The color of the prostates and other tissues of the animals were observed and the contents of the trypan blue in the tissues of the prostates were determined separately by colorimetry. RESULTS: Apart from the tissues of brains and spinal cord the surface and the inner tissues of the prostates with NP, BP, BPH and BPH-BP from the rats injected with the dye were also dyed blue similar to that of the muscles, livers, intestines and others. The content of the trypan blue in the tissues of the prostates with NP, BP and BPH-BP was obviously higher than those with NP and BPH. CONCLUSION: The penetrability of trypan blue with properties of ionization and larger molecular weight is high in either the normal prostate or the prostate with BP, BPH and BPH-BP, and much higher in inflammatory prostate than in the normal prostate and the prostate with BPH.

Time course of lens capsule staining using trypan blue and indocyanine green: in vitro study in porcine eyes.

PURPOSE: To determine whether staining of the lens capsule with trypan blue 0.1% and indocyanine green (ICG) 0.5% diminishes with time and whether it differs between the anterior and posterior capsules. SETTING: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan. METHODS: Crystalline lenses removed from porcine eyes were stained for 10 seconds with 0.1 mL of trypan blue 0.1% or indocyanine green 0.5%. They were then placed in distilled water and observed for the persistence of staining over time. In a second experiment, the anterior chamber and internal aspects of the anterior capsule and internal and vitreous aspects of the posterior capsule were gently irrigated with 0.1 mL of trypan blue 0.1% or ICG 0.5%. After 10 seconds, the capsules were irrigated with distilled water and the staining intensities were compared. RESULTS: Staining was not diminished 30 seconds, 5 minutes, or 1 hour after application of either dye. No difference was evident in staining intensity or diminution with time between the anterior and posterior capsules, but the external aspects were stained more than the internal aspects with both dyes. Trypan blue produced more intense staining than ICG. CONCLUSIONS: Since the intensity of capsule staining in the intact lens did not change during a 1-hour immersion in water, capsule dyes may not dissipate fully during cataract surgery. As possible long-term adverse effects have not been ruled out, capsule dyes should be used in a low concentration for a short exposure time.

A histological analysis of lens capsules stained with trypan blue for capsulorrhexis in phacoemulsification cataract surgery.

PURPOSE: Staining of anterior lens capsules with dye to facilitate completion of continuous curvilinear capsulorrhexis is now being used more frequently in phacoemulsification of white and mature cataracts with poor red reflexes. This study examined the histological characteristics of anterior lens capsules stained with trypan blue. The layer(s) of the lens capsule that stained with dye and the extent of accumulation of dye in these layers of the lens capsule were determined. To the best of our knowledge this has not been described before. METHODS: A series of 10 stained lens capsules were analysed histologically. The dye used in this study consisted of a standard sterile, noninflammatory, nonpyrogenic, 2 ml solution containing 0.6 mg/ml of trypan blue. Following capsulorrhexis, samples were sent to the laboratory for histological analysis. Frozen sections (8 microm) were prepared and examined with the light microscope. All 10 capsules were cut by frozen section to preserve trypan blue staining (which would be leached by processing) and then subjected to immunohistochemistry for collagen IV. Immunohistochemical analysis using markers for type IV collagen were done on formalin-fixed specimens for morphological comparison with the frozen sections. A counterstain highlighted the epithelium. RESULTS: Continuous curvilinear capsulorrhexis was successfully and easily completed in all cases without any complications. Frozen section analysis using light microscopy demonstrated accumulation of trypan blue dye in the basement membrane of the lens capsule. Staining was concentrated in the portion of the membrane adjacent to the lens epithelium. The lens epithelium could not be clearly identified on the frozen sections. Consequently, immunohistochemical analysis with markers for type IV collagen was performed. A counterstain highlighted the epithelium. This confirmed that the layer staining with trypan blue was the basement membrane, a consistent feature on all the specimens. CONCLUSION: Trypan blue selectively stains the basement membrane of the anterior lens capsule. There is a concentration of dye in the basement membrane adjacent to the lens epithelial cell layer. The lens cortex does not appear clinically to stain with trypan blue. This enables surgeons to distinguish the lens capsule from the cortex and provides sufficient contrast for successful completion of continuous curvilinear capsulorrhexis during cataract surgery.

Non-invasive opening of BBB by focused ultrasound.

Blood brain barrier (BBB) is a major barrier for delivering therapeutic agents in the brain. In this study we investigated the feasibility of open the BBB by using focused ultrasound. Rabbit brains were exposed to pulsed focused ultrasound while injecting ultrasound contrast agent containg microbubbles intravenously. The BBB opening was measured after the sonications by injecting MRI contrast agent i.v. and evaluating the local enhancement in the brain. Low ultrasound powers and pressure amplitudes were found to cause focal enhancement. Before sacrificing the animals trypan blue was also injected i.v.. After the sacrifice of the animals blue spots were found in the brain in the sonicated locations. This method may have potential for targeted delivery of macromolecules in the brain.

Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro.

BACKGROUND: Previous work has shown that cutaneous microbial species associated with skin conditions of microbial aetiology are susceptible to killing by photodynamic therapy (PDT) using visible light and methylene blue. Antimicrobial PDT (APDT) in vivo would require a therapeutic regimen where bacteria could be killed without damaging adjacent tissue. OBJECTIVES: To study keratinocyte killing in vitro using APDT. METHODS: We used a combination of methylene blue (100 microg mL(-1)) and visible light (42 mW cm(-2)), previously used for microbial killing, to study cytotoxic effects on keratinocytes. Kill rates and subsequent D-values were determined against a human keratinocyte cell line (H103) using trypan blue and neutral red dye viability tests. RESULTS: The kill rates for keratinocytes were exponential over the 90- and 180-min period of the experiment for neutral red and trypan blue, respectively. The corresponding D-values were shown to be 198 and 205 min using trypan blue exclusion and neutral red uptake viability tests, respectively. CONCLUSIONS: The kill rates for keratinocytes were 18-200-fold slower than those previously determined for cutaneous microbial species, suggesting that in vivo, APDT sufficient to reduce microbes by seven log cycles would have little cytotoxic effect on keratinocytes. This approach may offer a safe alternative to conventional antimicrobial treatment.