A new catheter for tumor-targeting with radioactive microspheres in representative hepatic artery systems--part II: solid tumor-targeting in a patient-inspired hepatic artery system

In this second part, the methodology for optimal tumor-targeting is further explored, employing a patient-inspired hepatic artery system which differs significantly from the idealized configuration discussed in Part I. Furthermore, the fluid dynamics of a microsphere supply apparatus is also analyzed. The best radial catheter positions and particle-release intervals for tumor targeting were determined for both the idealized and patient-inspired configurations. This was accomplished by numerically analyzing generated particle release maps (PRMs) for ten equally spaced intervals throughout the pulse. As in Part I, the effects of introducing a catheter were also investigated. In addition to the determination of micro-catheter positioning and, hence, optimal microsphere release, a microsphere-supply apparatus (MSA) was analyzed, which transports the particles to the catheter-nozzle, considering different axial particle injection functions, i.e., step, ramp, and S-curve. A refined targeting methodology was developed which demonstrates how the optimal injection region and interval can be determined with the presence of a catheter for any geometric configuration. Additionally, the less abrupt injection functions (i.e., ramp and S-curve) were shown to provide a more compact particle stream, making them better choices for targeting. The results of this study aid in designing the smart micro-catheter (SMC) in conjunction with the MSA, bringing this innovative treatment procedure one step closer to implementation in clinical practice.     

The blood supply to the abdominal organs of the fetal guinea-pig

Guinea-pigs near term of pregnancy were anaesthetized with diazepam and sodium pentobarbitone. A fetus was exposed and the vitelline artery catheterized to measure blood pressure and heart rate or to render a reference sample of blood for the determination of organ blood flow by the microsphere technique. The radioactive microspheres were injected through a catheter in the right atrium. Mean arterial blood pressure was 4.0 kPa and heart rate was 261 beats min-1. The liver, spleen, pancreas and gut receive most of their blood supply from the same trunk as the vitelline artery. The sample from this vessel was also used to calculate blood flow to the adrenal glands, kidneys, urogenital tract, and placenta, assuming even mixing of microspheres and blood in the abdominal aorta. Umbilical blood flow, corrected to a fetal weight of 100 g, averaged 7.5 ml min-1. The adrenal glands, which are known to increase their cortisol secretion near term, had a very high rate of perfusion. If the microspheres were injected in the umbilical vein, almost all were trapped in the liver, confirming the absence of a ductus venosus in the guinea-pig fetus. Most of these microspheres were found in the quadrate lobe of the liver. Hepatic arterial blood flow was also unequally distributed, with more than two-thirds going to the right lobe of the liver. Although the distribution of portal venous blood flow is not known, it is evident that different areas of the liver are presented with blood of greatly varying oxygen saturation.     

Measurement of carotid body blood flow in cats by use of radioactive microspheres

To resolve the controversy regarding carotid body blood flow, we used the radioactive microsphere technique for determination of tissue blood flow. We also measured the blood flow to several other tissues in the cat. Blood flow experiments were performed on 13 cats that were anesthetized, paralyzed, and mechanically ventilated with air. Different numbers of differently labeled 9-, 15-, and 25-micron microspheres were injected via a catheter into the left atrium. It was determined that one injection of 5 x 10(6) 15-micron microspheres was appropriate for the determination of carotid body blood flow. Flows to the carotid bodies and other organs by use of this protocol were as follows (ml.min-1.100 g-1, means +/- SE): carotid bodies, 1,417 +/- 143; adrenal glands, 406 +/- 89; left kidney, 355 +/- 69; right kidney, 375 +/- 74; heart, 201 +/- 39; liver 81 +/- 14; pancreas, 80 +/- 21; superior cervical ganglia, 62 +/- 9; carotid artery wall, 2.4 +/- 1.1. The blood flow to the carotid bodies was the highest for any organ. This measurement provides new evidence that tissue blood flow to the carotid body is very high. This high flow is consistent with the prompt physiological reflex functions of the carotid body.     

Intracellular degradation of microspheres based on cross-linked dextran hydrogels or amphiphilic block copolymers: a comparative raman microscopy study

Micro- and nanospheres composed of biodegradable polymers show promise as versatile devices for the controlled delivery of biopharmaceuticals. Whereas important properties such as drug release profiles, biocompatibility, and (bio)degradability have been determined for many types of biodegradable particles, information about particle degradation inside phagocytic cells is usually lacking. Here, we report the use of confocal Raman microscopy to obtain chemical information about cross-linked dextran hydrogel microspheres and amphiphilic poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) microspheres inside RAW 264.7 macrophage phagosomes. Using quantitative Raman microspectroscopy, we show that the dextran concentration inside phagocytosed dextran microspheres decreases with cell incubation time. In contrast to dextran microspheres, we did not observe PEGT/PBT microsphere degradation after 1 week of internalization by macrophages, confirming previous studies showing that dextran microsphere degradation proceeds faster than PEGT/PBT degradation. Raman microscopy further showed the conversion of macrophages to lipid-laden foam cells upon prolonged incubation with both types of microspheres, suggesting that a cellular inflammatory response is induced by these biomaterials in cell culture. Our results exemplify the power of Raman microscopy to characterize microsphere degradation in cells and offer exciting prospects for this technique as a noninvasive, label-free optical tool in biomaterials histology and tissue engineering.     

Targeted drug aerosol deposition analysis for a four-generation lung airway model with hemispherical tumors

One important research area of broad interest is the development of highly efficient drug delivery systems for desired site deposition and uptake. For example, controlled drug aerosol release and targeting to specific regions of the lung is a novel way to combat lung diseases, diabetes, virus infections, cancers, etc. Determination of feasible air-particle streams is a prerequisite for the development of such delivery devices, say, smart inhalers. The concept of "controlled particle release and targeting" is introduced and results are discussed for a representative model of bronchial lung airways afflicted with hemispherical tumors of different sizes and locations. It is shown that under normal particle inlet conditions a particle mass fraction of only up to 11% may deposit on the surface of a specific tumor with critical radius r/R approximately 1.25, while a controlled particle release achieves deposition fractions of 35 to 92% for a realistic combination of inlet Stokes and Reynolds numbers, depending mainly on tumor size. Furthermore, with the controlled release and targeting approach nearby healthy tissue is hardly impacted by the typically aggressive drug aerosols. Assuming laminar, quasi-steady, three-dimensional air flow and spherical non-interacting micron-particles in sequentially bifurcating rigid airways, the results were obtained using a validated commercial finite-volume code with user-enhanced programs on a high-end engineering workstation. The new concept is generic and hence should be applicable to other regions of the respiratory system as well.     

Measurement of bronchial blood flow with radioactive microspheres in awake sheep

Distribution of bronchial blood flow was measured in unanesthetized sheep by the use of two modifications of the microsphere reference sample technique that correct for peripheral shunting of microspheres: 1) A double microsphere method in which simultaneous left and right atrial injections of 15-microns microspheres tagged with different isotopes allowed measurement of both pulmonary blood flow and shunt-corrected bronchial blood flow, and 2) a pulmonary arterial occlusion method in which left atrial injection and transient occlusion of the left pulmonary artery prevented delivery to the lung of microspheres shunted through the peripheral circulation and allowed systemic blood flow to the left lung to be measured. Both methods can be performed in unanesthetized sheep. The pulmonary arterial occlusion method is less costly and requires fewer calculations. The double microsphere method requires less surgical preparation and allows measurement without perturbation of pulmonary hemodynamics. There was no statistically significant difference between bronchial blood flow measured with the two methods. However, total bronchial blood flow measured during pulmonary arterial occlusion (1.52 +/- 0.98% of cardiac output, n = 9) was slightly higher than that measured with the double microsphere method (1.39 +/- 0.88% of cardiac output, n = 9). In another series of experiments in which sequential measurements of bronchial blood flow were made, there was a significant increase of 15% in left lung bronchial blood flow during the first minute of occlusion of the left pulmonary artery. Thus pulmonary arterial occlusion should be performed 5 s after microsphere injection as originally described by Baile et al. (1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative disposition of adriamycin delivered via magnetic albumin microspheres in presence and absence of magnetic field in rats

The multiple tissue disposition of adriamycin hydrochloride delivered via magnetic albumin microspheres, in absence (control) and presence of magnetic field (experimental), has been investigated in rats. The animal tail was demarcated into three segments: T1, the dosing-site; T2, the target-site; and T3, the post target-site. Following the arterial cannulation at T1, 0.4 mg/kg of microsphere associated drug was administered to the control as well as the experimental animals. In experimental group, the target-site T2 was exposed to a 8000 G magnetic field for 30 min. In both groups the animals were sacrificed in triplicates over a 48 hr period and their various tissues monitored for drug concentrations using HPLC. In presence of magnetic field, the microspheres demonstrated 16 fold increase in the maximum drug concentration, 6 fold increase in drug exposure and 6 fold increase in the drug targeting efficiency for T2. Drug delivery to most non-target tissues, including heart and liver, was substantially reduced. The results quantitatively suggest that the efficacy of magnetic albumin microspheres in the targeted delivery of incorporated therapeutic agent is predominantly due to the magnetic effects, and not alone due to the characteristics of the micro-carrier system.     

Synthesis and characterization of hydroxyapatite-ciprofloxacin delivery systems by precipitation and spray drying technique

This investigation synthesized and characterized hydroxyapatite (HAP) microspheres, agglomerated microspheres, and implants containing ciprofloxacin. This delivery system is to be used as an implantable drug delivery system for the treatment of bone infections. The HAP microspheres were made by chemical precipitation followed by a spray-drying technique. Agglomerated microspheres were prepared by a wet granulation process using a granulator. Implants were prepared by direct compression of the granules on a Carver press. Ciprofloxacin was analyzed by high-performance liquid chromatography. Characterization of the HAP microspheres include particle size, size distribution, physical state of the drug in the microsphere, and microstructure of the drug delivery system before and after in vitro release. The particle size, porosity, and morphology of the microspheres were dependent on viscosity and concentration of the slurry as well as the atomization pressure used during spray drying. Even at the highest drug load (2% wt/wt), the drug was present in a noncrystalline state. The drug release from the agglomerated microspheres was quick and almost complete within 1 hour. However, compressing the same amount of agglomerated microspheres into an implant greatly reduced the rate of ciprofloxacin release. Only 12% (wt/wt) of the drug was released from the implant within 1 hour. The in vitro release of ciprofloxacin from these implants follows a diffusion-controlled mechanism. This method provides a unique way of producing various shapes and drug loads of HAP microspheres that can be easily manufactured on a commercial scale. Published: January 28, 2002.     

Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

In this study the w/o/w extraction-evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The microspheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical microspheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the microsphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.     

Release of indomethacin from pH-sensitive pullulan acetate microsphere

We studied the pH-sensitive indomethacin (IND) delivery system using pullulan. Hydrophobic pullulan acetate was prepared by chemical modification of hydrophilic pullulan and pullulan acetate microsphere was made by a solvent evaporation method. The size of microspheres was below 5 μm, and the drug loading efficiencies of microspheres were approximately 78 and 65% at the initial amount of drug 40 and 80 mg, respectively. The microsphere showed pH-sensitive swelling behavior in PBS buffer. After 15 hrs, the swelling of the microsphere at pH 7.4 was approximately 20 times greater than that at pH1.2. The pH of the medium significantly influenced on thein vitro release rate. The released amount of drug at pH 7.2 was approximately 90 times greater than that at pH 1.2. The shape of microspheres at pH 1.2 were maintained sphere forms, but at pH 7.4 were disintegrated. The pH-sensitive IND release pattern was due both to the pH-sensitive diffusion of IND from the microspheres and to the release of the drug from the surface which underwent disintegration after swelling, due to the chemical composition of the microspheres and the pH of the release media.     

Blood modeling using polystyrene microspheres

The steady flow viscosity at shear rates 0 to 120 sec-1 and dynamic viscoelasticity at frequencies 0.02 to 0.8 Hz were determined for aqueous suspensions of uniform polystyrene microspheres of 1.0 micron diameter. Rheological properties of the microsphere suspensions were Newtonian for particle concentrations up to 32%. By introducing dextran and calcium chloride into the particle suspensions, non-Newtonian behavior was produced similar to that observed for human blood. The cooperative effects of dextran and calcium ions promoted aggregation of particles at a concentration as low as 12%. Thus, a suspension of uniform sized spherical polystyrene particles in aqueous solution of dextran may be made to mimic blood by controlling the surface charge on the polystyrene spheres using addition of calcium ions to the medium.     

Effects of tumors on inhaled pharmacologic drugs

Lung carcinomas are now the most common form of cancer. Clinical data suggest that tumors are found preferentially in upper airways, perhaps specifically at carina within bifurcations. The disease can be treated by aerosolized pharmacologic drugs. To enhance their efficacies site-specific drugs must be deposited selectively. Since inhaled particles are transported by air, flow patterns will naturally affect their trajectories. Therefore, in Part I of a systematic investigation, we focused on tumor-induced effects on airstreams, in Part II (the following article [p. 245]), particle trajectories were determined. To facilitate the targeted delivery of inhaled drugs, we simulated bifurcations with tumors on carinas using a commercial computational fluid dynamics (CFD) software package (FIDAP) with a Cray T90 supercomputer and studied effects of tumor sizes and ventilatory parameters on localized flow patterns. Critical tumor sizes existed; e.g., tumors had dominant effects when r/R > or = 0.8 for bifurcation 3-4 and r/R > or = 0.6 for bifurcation 7-8 (r = tumor radius and R = airway radius). The findings suggest that computer modeling is a means to integrate alterations to airway structures caused by diseases into aerosol therapy protocols.     

Particle diameter influences adhesion under flow

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.     

Spray-dried poly(D,L-lactide) microspheres containing carboplatin for veterinary use: In vitro and in vivo studies

The aim of this study was the development of a veterinary dosage form constituted by injectable biodegradable microspheres designed for the subcutaneous release of carboplatin, a chemotherapeutic drug. Poly(D,L-lactide) (PDLLA) microspheres were prepared by an emulsification/spray-drying method, using the drug-to-polymer weight ratios 1∶9 and 1∶5; blank microspheres (1% w/v) were prepared as a comparison. Microparticles were characterized in terms of morphology, encapsulation efficiency, and in vitro drug release behavior. In vivo tests were conducted on rats by subcutaneous injection of microsphere aqueous suspensions. Levels of carboplatin were evaluated both in the skin and in serum. The microparticles obtained had a spherical shape; particle size ranged from 5 to 7 μm, dependent on drug loading. Microspheres were able to control the in vitro release of the drug: approximately 90% to 100% of the carboplatin was released over 30 days. In vivo results showed that the microspheres were able to release high drug amounts locally, and sustained serum levels of drug were also achieved. Based on these results, carboplatin-loaded PDLLA microspheres may be useful for local delivery of the antineoplastic drug to the tumor, avoiding tumor recurrence in small animals, and may decrease the formation of distant metastases. Published: September 20, 2005     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation.

Vascular infusions of 15-microm-diameter microspheres are used to study pulmonary blood flow distribution. The sites of microsphere lodging and their effects on microvascular perfusion are debated but unknown. Using intravital microscopy of the subpleural surface of rat lungs, we directly observed deposition of fluorescent microspheres. In a pump-perfused lung model, approximately 0.5 million microspheres were infused over 30 s into the pulmonary artery of seven rats. Microsphere lodging was analyzed for the location in the microvasculature and the effect on local flow after lodging. On average, we observed 3.2 microspheres per 160 alveolar facets. The microspheres always entered the arterioles as singlets and lodged at the inlets to capillaries, either in alveolar corner vessels or small arterioles. In all cases, blood flow continued either around the microspheres or into the capillaries via adjacent pathways. We conclude that 15-microm-diameter microspheres, in doses in excess of those used in typical studies, have no significant impact on pulmonary capillary blood flow distribution.     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

Insulin particle formation in supersaturated aqueous solutions of poly(ethylene glycol)

Protein microspheres are of particular utility in the field of drug delivery. A novel, completely aqueous, process of microsphere fabrication has been devised based on controlled phase separation of protein from water-soluble polymers such as polyethylene glycols. The fabrication process results in the formation of spherical microparticles with narrow particle size distributions. Cooling of preheated human insulin-poly(ethylene glycol)-water solutions results in the facile formation of insulin particles. To map out the supersaturation conditions conducive to particle nucleation and growth, we determined the temperature- and concentration-dependent boundaries of an equilibrium liquid-solid phase separation. The kinetics of formation of microspheres were followed by dynamic and continuous-angle static light scattering techniques. The presence of PEG at a pH that was close to the protein's isoelectric point resulted in rapid nucleation and growth. The time elapsed from the moment of creation of a supersaturated solution and the detection of a solid phase in the system (the induction period, t(ind)) ranged from tens to several hundreds of seconds. The dependence of t(ind) on supersaturation could be described within the framework of classical nucleation theory, with the time needed for the formation of a critical nucleus (size <10 nm) being much longer than the time of the onset of particle growth. The growth was limited by cluster diffusion kinetics. The interfacial energies of the insulin particles were determined to be 3.2-3.4 and 2.2 mJ/m(2) at equilibrium temperatures of 25 and 37 degrees C, respectively. The insulin particles formed as a result of the process were monodisperse and uniformly spherical, in clear distinction to previously reported processes of microcrystalline insulin particle formation.     

Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

In this study the w/o/w extraction–evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The micro-spheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical micro-spheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the mi-crosphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.     

A miniaturized nebulization catheter for improved gene delivery to the mouse lung

BACKGROUND: The available methods for administration of gene delivery systems to the lungs of small animals via nebulization have several drawbacks. These include lack of control over the delivered dose and a negative impact on the stability of the formulation. This paper describes a new nebulization catheter device for the administration of plasmid-based gene delivery systems (polyplexes) as aerosols to the mouse lung in vivo. METHODS: The physical stability of naked pDNA and polyplexes formulated with chitosan oligomers and PEI was examined following nebulization with the catheter device. We also examined the in vitro transfection efficiency of the polyplexes recovered after nebulization. Lung distribution and gene expression after administration of the selected gene delivery systems to the mouse lung were also investigated. RESULTS: In contrast to previously described nebulization methods, the structural integrity of the unprotected naked pDNA was maintained following nebulization by the catheter device, which indicates relatively mild nebulization conditions. In addition, the nebulization procedure did not affect the physical stability of the formulated polyplexes. Small volumes of the pDNA aerosol (10-20 microl) were delivered in a highly controlled and reproducible manner. The aerosol droplet size varied with the molecular weight of the polycations. Aerosol delivery via this method resulted in improved lung distribution of pDNA polyplexes and a six-fold increase in the efficiency of gene delivery in vivo over that seen with the commonly used intratracheal instillation method. CONCLUSION: The use of the nebulization catheter device provides a promising alternative for aerosol gene delivery to the mouse lung.