Effective elimination of cancer stem cells by magnetic hyperthermia.

Cancer stem cells (CSCs) are a subpopulation of cancer cells that have stem cell-like properties and are thought to be responsible for tumor drug resistance and relapse. Therapies that can effectively eliminate CSCs will, therefore, likely inhibit tumor recurrence. The objective of our study was to determine the susceptibility of CSCs to magnetic hyperthermia, a treatment that utilizes superparamagnetic iron oxide nanoparticles placed in an alternating magnetic field to generate localized heat and achieve selective tumor cell kill. SPIO NPs having a magnetite core of 12 nm were used to induce magnetic hyperthermia in A549 and MDA-MB-231 tumor cells. Multiple assays for CSCs, including side population phenotype, aldehyde dehydrogenase expression, mammosphere formation, and in vivo xenotransplantation, indicated that magnetic hyperthermia reduced or, in some cases, eliminated the CSC subpopulation in treated cells. Interestingly, conventional hyperthermia, induced by subjecting cells to elevated temperature (46 °C) in a water bath, was not effective in eliminating CSCs. Our studies show that magnetic hyperthermia has pleiotropic effects, inducing acute necrosis in some cells while stimulating reactive oxygen species generation and slower cell kill in others. These results suggest the potential for lower rates of tumor recurrence after magnetic hyperthermia compared to conventional cancer therapies.

Distribution of aerosols in murine obliterative bronchiolitis lungs by fluorescent imaging.

UNLABELLED: ABSTRACT Background: Obliterative bronchiolitis (OB) is a major obstacle to the success of lung transplantation and is also a serious complication of hematopoietic stem cell transplant. It has few therapeutic options and respiratory delivery of potential therapeutic drugs is hindered by the narrowed and occluded airways. METHODS: OB was induced in mice using an established protocol and lung function was assessed by plethysmograph. Mice were exposed to four different aerosols of aluminum phthalocyanine tetrasulfonic acid (AlPCS) that ranged in concentration and median particle size distribution (0.2-4.0 µm). The fluorescent intensity and number of pixels were measured for the trachea and lobes at two different compressional thicknesses. With analysis of the fluorescent intensity, the concentration and attenuation coefficient were estimated for each lobe and the trachea as well as individual pixels. The latter allowed generation of images reflective of the concentration. RESULTS: Lungs/trachea from OB mice had lower deposition, which correlated with lung function measurements, and apparent greater variability in the intensity compared to controls. The estimated lung volumes measured by plethysmograph were not different between the OB group and controls; however, total inflational lung capacity was reduced in OB mice. CONCLUSIONS: Despite the variability in disease induction, there is a clear link between aerosol deposition and lung function, which was revealed by fluorescent imaging. The modulation of aerosol deposition in lungs with restrictive airway disease underscores the importance of tailoring aerosolization to optimize drug delivery.

Distribution of aerosols in mouse lobes by fluorescent imaging.

Better methods are needed to quantify the distribution of drug among the airways of the lungs of small animals to facilitate the development of agents that can target specific airways. Mice were exposed to aerosols of aluminum phthalocyanine tetrasulfonic acid (AlPCS) that ranged in concentration and size (0.2-2.8 µm). The trachea and lobes were removed and placed between glass slides, and fluorescent images were obtained at two different compression thicknesses. The intensity, normalized by the area, exposure time, and thickness, was then plotted as a function of compression thickness, from which the concentration and attenuation coefficient were estimated for each lobe and then for each pixel of the image. The latter was then used to generate an image reflective of the concentration. The lobe volume, concentration, and tissue attenuation of AlPCS was consistent among the lobes. The deposition fraction increased with decreasing particle size. The network of lines in the concentration image indicated that connective tissue has a lower concentration. The central airways were clearly evident in the images of mice exposed to the very small and large aerosols. This approach provides a rapid, economical means to obtain high resolution images of mouse lungs from which detailed analysis of the distribution of deposited aerosol particles can be obtained.

Deposition of aerosol particles in a model vitreous chamber.

The purpose of this study was to assess quantitatively the aerosol deposition in a model eye chamber to identify the mechanism(s) of deposition and delivery efficiency for application in retinal disease treated with vitrectomy. Dry aerosol particles were produced with mixtures of fluorescein and a variable concentration of cesium chloride, which ranged in aerodynamic size from 0.6 to 1.3 µm. The aerosol was injected through a small inlet tube into Teflon chambers that had a vented, spherical cavity (diameter ¾"). Two filling times of 60 s and 90 s were used. Although significant loss occurred in the syringe, the mass deposited within the chambers increased with aerosol concentration and ranged from 0.5 to nearly 15 µg. Between 60 and 90% of the mass was deposited on the lower surface of the chamber. The mechanism of deposition was consistent with diffusion through a boundary layer during filling followed by sedimentation of the remaining suspended aerosol particles. Based on these results, an aerosol with a median particle size of 1.3 µm was shown to provide a therapeutically effective dose of 5-fluorouracil. The approach is general and can be applied to the aerosol delivery of other drugs to the vitreous chamber.

Physicochemical properties and antifungal activity of amphotericin B incorporated in cholesteryl carbonate esters.

The antifungal activity of amphotericin B (AmB) incorporated in three cholesteryl carbonate esters (CCEs), sodium cholesteryl carbonate, cholesteryl palmityl carbonate, and dicholesteryl carbonate, was examined to assess their potential for use in a dry powder aerosol. Formulations containing dissolved AmB were stable for 6 months. The particle size varied inversely with liquid crystalline content with observed mass median aerodynamic diameters ranging from 4 to 8 µ m. This was consistent with the visual appearance of the liquid crystals as being low density and free flowing at room temperature. When dispersed in water, the presence of the CCE reduced the rate and extent of AmB release, consistent with the estimated liquid crystal/water partition coefficient. Nevertheless, the rate of AmB release was always sufficient to kill the fungus as established with bioactivity studies. AmB formulated with CCE as a dry powder appears to be promising for use in treating lung fungal infections.

In vitro and in vivo lung deposition of coated magnetic aerosol particles.

The magnetic induced deposition of polydispersed aerosols composed of agglomerated superparamagnetic particles was measured with an in vitro model system and in the mouse trachea and deep lung for the purpose of investigating the potential of site specific respiratory drug delivery. Oleic acid coated superparamagnetic particles were prepared and characterized by TEM, induced magnetic moment, and iron content. The particles were dispersed in cyclohexane, aerosolized with an ultrasonic atomizer and dried by sequential reflux and charcoal columns. The fraction of iron deposited on glass tubes increased with particle size and decreasing flow rate. High deposition occurred with a small diameter tube, but the deposition fraction was largely independent of tube size at larger diameters. Results from computational fluid dynamics qualitatively agreed with the experimental results. Enhanced deposition was observed in the mouse lung but not in the trachea consistent with the analysis of the aerodynamic time allowed for deposition and required magnetic deposition time.

Magnetic activated release of umbelliferone from lipid matrices.

Lipid matrices containing dispersed superparamagnetic iron oxide (SPIO) particles were investigated as a magnetic field-responsive drug delivery system. Lipid matrices were prepared by combining myristyl alcohol, fatty acid coated SPIO particles, and umbelliferone (UMB). With placement of the matrices into the release medium, initial UMB release was fast but fell to zero indicating a burst effect. With application of an alternating magnetic field, additional UMB was released. The rate and extent of magnetic field-stimulated release increased with UMB load but not SPIO content. Differences between oleic and myristic acid coated SPIO appeared to be a result of phase separation. UMB release coincided with matrix melting, which can be controlled by the SPIO content and external magnetic field as shown by theoretical analysis. While significant technological issues remain, the foundation for developing magnetic field-stimulated drug delivery systems has been established.

Measurement of the distribution of aerosols among mouse lobes by fluorescent imaging.

Lung samples were prepared to investigate the perturbing effects of light absorption for quantifying the fluorescence signal of aluminum phthalocyanine tetrasulfonic acid (AlPCS). Standard solutions of known concentration and depth were imaged with different exposure times and analyzed. The intensity was found to be a linear function of concentration, depth, exposure time, and area. Mice were exposed to an aerosol of AlPCS with a mass median aerodynamic diameter of 390 nm and geometric standard deviation of 1.8. Images of intact lung lobes and lung homogenates were obtained and then analyzed to allow quantifying the concentration of AlPCS among the lung lobes and trachea. For the distribution of aerosols, the results indicate that the concentration was uniform among the different lobes. Combining the quantitative analysis of the concentration with image analysis of the area/thickness, the mass deposited in each lobe was readily determined. This approach provides a quantitative means to determine the selectivity of drug delivery to mouse lower respiratory tract.

Magnetic deposition of aerosols composed of aggregated superparamagnetic nanoparticles.

PURPOSE: The deposition of magnetic particles was examined for the possibility of further enhancing the selectivity of inhalation drug administration for the treatment of lung cancer. METHODS: Superparamagnetic magnetite nanoparticles were prepared and ultrasonically atomized, dried, and passed through glass tubes in the presence and absence of a wedge-shaped permanent magnet. The change in the outlet aerosol size distribution due to magnetic deposition under various well-defined aerodynamic conditions and a measured magnetic field was determined by an aerodynamic particle sizer. In addition, computational fluid dynamics (CFD) simulations of magnetic aerosol transport and deposition were conducted. RESULTS: The deposition fraction increased nearly linearly with particle diameter and was greater with lower air flow rates. The effect of tube diameter was complicated but well described by CFD simulations, as was the effect of particle size and air flow rate. CONCLUSIONS: The descriptive power of CFD simulations was demonstrated in the in vitro deposition of magnetic aerosol particles. This suggests that CFD simulations can potentially be used in future studies to design systems for selective drug delivery in vivo as a function of magnetic properties, aerosol characteristics, and respiratory physiology.

Inhalation adjuvant therapy for lung cancer.

BACKGROUND: Lung cancer is the leading cause of new cancer cases and also is the number one cause of cancer death in both male and females in the United States and the world. Lung cancer is histologically separated into either small-cell (SCLC) or nonsmall-cell lung cancer (NSCLC). NSCLC is much more common, and can occur in the periphery (adenocarcinomas) or central airways (squamous cell carcinomas). METHODS: The literature pertaining to lung cancer adjuvant therapy and inhalation lung cancer chemoprevention was reviewed. RESULTS: It is argued that inhalation of chemotherapy as an adjuvant in Stage II NSCC and inhalation of chemopreventive agents in Stage I adenocarcinomas are direct paths to improve lung cancer therapy as well as demonstrate the utility of inhalation therapy. CONCLUSIONS: If successful, inhalation delivery may be extended to the treatment of other types of lung cancer.

Concentration dependent aggregation properties of chlorhexidine salts.

PURPOSE: Chlorhexidine (CHX), a chemical antiseptic, is known to bind to dentin and has been shown to be effective in treating bacterial infections caused by microbes. The solubility and aggregation properties of CHX salts were determined to guide the development of a sustained release formulation for long-term disinfection. METHODS: The amount of CHX in solution was determined as a function of counterion concentration (chloride, acetate (Ac) or gluconate (G)) by UV spectrophotometry at 255nm. The weight average molecular weight was determined from the angular dependence of the scattered light. Proton NMR spectroscopy was used to investigate the dependence of the peak intensity and chemical shift on solution concentration and diffusion measurements were performed by Fourier-transform pulsed-field gradient spin-echo (PFG-SE) (1)H NMR. RESULTS: The observed CHX concentration was highly dependent on the type and concentration of salt present in solution with the greatest CHX concentration achieved with gluconate, moderate to low with diacetate, and very low with dichloride solutions. Addition of sodium gluconate enhanced the amount of CHX-Ac(2) in solution; however, only low concentrations of chlorhexidine can be achieved in the presence of chloride ions. For solutions of CHX-G(2), the aggregate number appeared to range from a dimer at 40mM to perhaps a pentamer at 150mM. In contrast, no aggregation of CHX-Cl(2) or CHX-Ac(2) was detected, which was corroborated by diffusion NMR results. The change in chemical shift of protons is consistent with association of the phenyl group of one CHX with the hexamethylene chain of a second CHX. Based on the analysis of NMR peak intensities of CHX, gluconate, and acetate in saturated solutions, it appears that solubilization of the diacetate species occurs within digluconate aggregates, since the solubility product of chlorhexidine diacetate is such that the concentration of CHX will exceed the critical micelle concentration (CMC). However, no solubilization of CHX-Cl(2) occurs because the solubility product falls below the CMC. CONCLUSIONS: The low concentration of CHX that can be achieved in physiological concentrations of chloride in the oral cavity may be problematic for dental and slow release formulations. Achieving a high concentration of CHX appears to require that the monomer be present at a concentration greater than that required to produce self-association.

Solubility properties of chlorhexidine salts.

Chlorhexidine (CHX) is effective in treating oral bacterial infections. The solubility was shown to be highly dependent on the salt present in solution. Gluconate enhances the amount of CHX diacetate in solution possibly through mixed micelles formation, because the solubility product is such that the concentration of CHX will exceed the critical micelle concentration. However, only low concentrations of CHX dichloride can be obtained, which is not appreciably solubilized by gluconate ions. The low concentration of CHX that can be achieved in physiological concentrations of chloride in the oral cavity may be problematic for dental, slow release formulations.

Collisional solute release from thermally activated lipid particles.

Solid lipid particles were evaluated for their potential as a thermo-activated drug delivery system. Submicron-sized diphenylhexatriene (DPH)/myristyl alcohol particles were produced by an atomization/drying process and the release rate of DPH into sodium dodecyl sulfate (SDS) micellar solutions was measured. The results showed that the presence of micelles and thermal activation was necessary and sufficient for the release of DPH. The initial rate of DPH release was linear for most systems. However, nonlinearity was noted at low micelle concentrations where the rate decreased with time due to the loss of sink conditions with the rising concentration of DPH in the micellar solution. Also at high micelle and particle concentrations, the rate increased with time, which may be due to a loss of particle integrity. Analyzing the data from a design study, the release rate was found to be linearly proportional to particle concentration. In contrast, the rate of release increased with micelle concentration in a nonlinear manner and appeared to approach a plateau at high micelle concentrations. The decrease in rate as the micelle concentration increased is suggestive of a saturable process and may involve a collision-based mechanism.

High-frequency ultrasonic atomization for drug delivery to rodent animal models - optimal particle size for lung inhalation of difluoromethyl ornithine.

A high-(8-MHz) and a low-(1.7-MHz) frequency ultrasonic transducer were compared for delivering aerosols to mouse lung. The aerosol concentration (mass of dry particles/volume of air) rose nonlinearly with solution concentration of difluoromethyl ornithine for both transducers. The particle size was linear with the cube root of the solution concentration, and the slope of the low-frequency transducer was 8 times greater than that of the high-frequency transducer. The deposition fraction assessed by the assayed mass in the lung relative to the calculated inhaled mass was found to decline exponentially with particle size. The lower-frequency transducer provided a higher dose despite a lower deposition fraction, but the high-frequency transducer was more efficient and provides a more selective deposition in the lower respiratory tract while operating with significantly less demands on aerosol drying.

Performance of the vibrating membrane aerosol generation device: Aeroneb Micropump Nebulizer.

The output and particle size distribution of several series of aqueous solutions were measured to define quantitatively the practical limits of the solution properties acceptable for aerosol production by the aeroneb micropump nebulizer. Aerosol output measurements were made gravimetrically and the particle size distributions were obtained by laser diffractometry. Solution properties were obtained from the literature by interpolation of the best-fit curve of the property plotted as a function of composition. For nonionic solutes, addition of sodium chloride dramatically increased the output rate and also decreased the droplet size at low solute concentrations. Increasing viscosity also caused a significant decrease in output. Cesium chloride displayed increased output rate with concentration due to the rising density. Based on calculations with the number of apertures and oscillatory frequency, low output rates appeared to be a consequence of apertures failing to produce a droplet with each oscillation. Overall, ionic strength, density, surface tension, and viscosity affected the output rate in a manner that can be now empirically predicted.

Evaluation of the compaction of sulfathiazole polymorphs.

The aim of this study was to relate the tableting performance assessed by an instrumented tableting machine to the mechanical properties measured by nanoindentation. Three different polymorphic forms of sulfathiazole were prepared by recrystallization, and the density and X-ray powder diffraction patterns were measured and compared with theoretical density and simulated powder patterns, respectively. Tablets were prepared using a series of applied pressures, and the results were subjected to energy analysis, three dimensional (3D) modeling, and the traditional Heckel analysis. With these approaches, form I was found to be consistently the most brittle material, but the subtle differences between forms II and III were only revealed by 3D modeling. The rank order of the crushing force was found to be I is congruent to II < III. From nanoindentation, form III was found to be much harder than forms I and II, and III also had a much higher Young's modulus. The energy calculations of the nanoindentation curves showed that form III was distinct from forms I and II, which is consistent with the presence of slip planes that are only present in form III. However, in this system, there was little correspondence between the macroscopic and microscopic measurements, and thus particle-particle interactions may to be of paramount importance.

Measurement of process-dependent material properties of pharmaceutical solids by nanoindentation.

The purpose of this work was to evaluate nanoindentation as a means to characterize the material properties of pharmaceutical solids. X-ray diffraction of potassium chloride and acetaminophen showed that samples prepared by cooling a melt to a crystalline sample as opposed to slow recrystallization had the same crystal structure. With analysis of the force-displacement curves, the KCl quenched samples had a hardness that was 10 times higher than the recrystallized KCl, while acetaminophen quenched samples were 25% harder than the recrystallized samples. The elastic moduli of the quenched samples were also much greater than that observed for the recrystallized samples. Although the elasticity was independent of load, the hardness increased with load for acetaminophen. With each sample, the flow at constant load increased with applied load. Etching patterns obtained by atomic force microscopy showed that the KCl quenched sample had a higher dislocation density than the recrystallized sample, although there was no evident difference in the acetaminophen samples. Overall, the differences in the observed sample properties may be related to the dislocation density. Thus, nanoindentation has been shown to be a sensitive method for determining a processed-induced change in the hardness, creep, and elasticity of KCl and acetaminophen.

Characterization of pharmaceutical solids by scanning probe microscopy.

The force-displacement profiles of four well-characterized materials that represent both soft/hard and plastic/brittle materials have been obtained using a novel nanoindentation technique. Flat surfaces of acetaminophen, potassium chloride, sucrose, and sodium stearate were prepared by melting or recrystallization, and the melting points were measured. Topographic and the corresponding first derivative images were obtained both before and after indentation. The materials were indented using a 10 s loading time, followed by a 2 s hold, and ending with a 10 s unloading time thereby providing a unique force-displacement profile for each material. The loading profile of acetaminophen was discontinuous, whereas the profiles for the other three materials were smooth. The profiles were analyzed and the rank order of hardness was sucrose > acetaminophen > KCl > sodium stearate, which is consistent with the literature. The rank order of the stiffness, which is related to the modulus of elasticity, was sucrose > KCl > acetaminophen > sodium stearate. Given the flexibility and power of this approach, nanoindentation coupled with atomic force microscopy may be a useful means to characterize materials for evaluating tablet-processing conditions, perhaps at a preformulation stage.

Interaction of bile salts with gastrointestinal mucins.

The properties of three mucins were examined to identify the structural features responsible for their functional difterences. Bovine submaxillary mucin (BSM), porcine gastric mucin (PGM), and rat intestinal mucin (RIM) were each characterized, and high carbohydrate contents were found for RIM and PGM. The amino acid compositions were typical of mucin glycoproteins, with over half comprising small, neutral amino acids. Thereafter, each mucin was equilibrated with three different series of concentrations of the bile salts sodium taurocholate, sodium taurodeoxycholate, and sodium taurochenodeoxycholate. Following multiple centrifugations, the supernatant and mucin pellet concentrations of the bile salts were measured. The bile salt pellet concentration was plotted as a function of supernatant concentration, and from the slopes, the excluded volumes were calculated as 25, 29-44, and 28 55 mL/g for BSM, RIM, and PGM, respectively. The intercepts were 8-10, 2-3, and 1-3 mM for BSM, RIM, and PGM, respectively, which represents an estimate of the bound concentration of bile salt. Differences among the bile salts were observed in the excluded volume and amount bound, but no trends were evident. The bile salts may interact as aggregates with the hydrophobic areas and carbohydrate side chains of the mucins, providing favorable sites for association. The binding at low concentrations with exclusion at high concentrations is significant for modulating the absorption of lipid aggregates from the intestine. Finally, the differences among the mucins reflect the unique structure function relationship of these gastrointestinal mucins.

Excluded volume effect of rat intestinal mucin on taurocholate/phosphatidylcholine mixed micelles.

The interaction of bile salt/phospholipid mixed micelles with an intestinal mucin has been investigated to provide the foundation for the transport of ingested fat and poorly water-soluble drugs through the intestinal mucous layer. Egg phosphatidylcholine (PC) was equilibrated with sodium taurocholate (TC) to generate several series of solutions, which had different intermicellar concentrations of TC. Within each series, each solution had the same IMC and thereby micelle sizes, but varied with respect to micelle concentration. These solutions were combined with isolated rat intestinal mucin, equilibrated, and then separated by centrifugation. The supernatant and mucin pellet were assayed for PC and TC, and the diffusion coefficient of PC was measured in the supernatant by PFG-SE NMR spectroscopy. For each series, four linear relationships were found; TC supernatant concentration plotted as a function of PC supernatant concentration; TC pellet concentration plotted as a function of PC pellet concentration; TC pellet concentration plotted as a function of TC supernatant concentration; and PC pellet concentration plotted as a function of PC supernatant concentration. Theoretical analysis of these results indicated that mucin excludes from 25 to 80% of the bile salt/phospholipid mixed micelles with greater exclusion observed with larger micelle size. There is preferential association of the taurocholate with intestinal mucin, when present in the mixed micelle region of the phase diagram. The association coupled with exclusion would allow mucin to modulate the concentration of bile salt at the epithelial surface.