Nanogel DDS enables sustained release of IL-12 for tumor immunotherapy

For a valid cytokine immunotherapy of malignancies, a suitable delivery system that ensures slow-release of cytokines is required, because short half-life in vivo of the molecules ruins therapeutic efficacy while causing severe systemic toxic effects. We previously showed that the cholesterol-bearing pullulan (CHP)-based hydrogel nanoparticles, or nanogel, encapsulates, stabilizes and releases various molecules. Here we applied this nanogel to administration in vivo of interleukin-12 (IL-12). Recombinant murine IL-12 (rmIL-12) was successfully incorporated into CHP nanogel simply by incubated with CHP at room temperature. After subcutaneously injected into mice, the CHP/rmIL-12 complex led to a prolonged elevation in IL-12 concentration in the sera. Repetitive administrations of the CHP/rmIL-12, but not rmIL-12 alone, induced drastic growth retardation of preestablished subcutaneous fibrosarcoma without causing any serious toxic event. The present study proposes a novel therapeutic intervention technology, taking advantage of slow and sustained release of bioactive cytokines from the self-assembling biocompatible nanoparticles.     

Comparison of refolding activities between nanogel artificial chaperone and GroEL systems

The chaperone-like activity of a nanogel of cholesteryl group-bearing pullulan (CHP) was compared with that of GroEL for refolding acid-denatured green fluorescent protein (GFP). The refolding of denatured GFP was carried out by dilution of acid-denatured GFP in the presence of the nanogel or GroEL. GFP fluorescence was increasingly repressed with increases in the concentration of the CHP nanogel or GroEL added to the dilution buffer. The concentrations of 50% inhibition of recovery of GFP fluorescence were 0.03 microM (GroEL) and 0.08 microM (CHP nanogel), respectively. The refolding was resumed by the addition of ATP into the GroEL (0.20 microM) system or by the addition of methyl-beta-cyclodextrin into the nanogel (0.20 microM) system. In the nanogel-GFP system, about 90% of the intensity was recovered within 10 min. The half time (t(1/2)) for refolding in the CHP nanogel system (36 s) is almost equal to that of the natural chaperone GroEL-GroES system.     

Nanogels for oligonucleotide delivery to the brain

Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood-brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine ("nanogel"). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain.     

Interactions between platelet activating factor and eicosanoids during endotoxic shock in anaesthetized pigs

The effects of platelet activating factor (PAF) on eicosanoid release during endotoxic shock was investigated in anaesthetized pigs receiving 5 mug kg(-1) Escherichia coli endotoxin (LPS) into the superior mesenteric artery over a 60 min period, by measuring plasma levels of a variety of mediators. Fifteen of the 31 animals infused with LPS and not treated with BN 52021, a PAF receptor antagonist, died within 30 min after the commencement of LPS infusion (non-survivors), while the other 16 survived the experimental period of 3 h, though in a state of shock (survivors). No alterations were observed in plasma concentrations of eicosanoids in the non-survivors. A significant, though transient, increase in eicosanoid concentrations occurred only in the survivors. Treatment with BN 52021 (4 mg kg-1, i.v.) injected 5 min prior to LPS infusion, failed to exert any effect on the survival rate. However, pretreatment with BN 52021 prevented circulatory collapse in the survivors and reduced the concentration of cyclooxygenase enzyme products, without affecting LTB(4) release. Exogenous administration of PAF (0.01 mug kg(-1)) caused hypotension and increased TXB(2) levels although 6-keto PGF(1alpha) and LTB(4) concentrations were unchanged. The data suggest that prostanoid formation may be secondary to PAF release in circulatory collapse evoked by LPS infusion in survivors, and give further support to the suggestion that PAF prostanoid interaction is important during endotoxic shock. However, their role in early death seems to be negligible, indicating the importance of other mediators.     

Cardiovascular, respiratory and glottic effects of sodium salicylate administered into different parts of the circulation in rabbits.

The authors studied the effect of sodium salicylate administered into different parts of the circulatory system on various cardiovascular, respiratory and glottic parameters in Pentobarbital-anaesthetized rabbits. The results show that apnoea, bradycardia and hypotension, followed by hypertension, can also be caused by the extrathoracic action of salicylate. Cardiovascular responses induced by injecting salicylate into the carotid circulation are qualitatively the same, even after vagotomy, as in injection into the femoral vein. Salicylate injected into the common carotid artery, the internal carotid artery or the femoral vein causes inspiratory apnoea in rabbits, with powerful electrical activity of the diaphragm and an intrapleural pressure shift to marked inspiratory values. Laryngoconstriction occurs simultaneously, despite inspiratory apnoea. The injection of salicylate into the common carotid artery after bilateral vagotomy induces expiratory (not inspiratory) apnoea, indicating that the vagi play an important role in the origination of inspiratory apnoea in rabbits.     

Small doses of intracarotid insulin do not lower plasma glucose in the rat

We sought to confirm the observation that 500 microU of insulin injected into the carotid artery of rats lowers plasma glucose by approximately 20 mg/dl within 2 minutes. In our hands, glucose concentrations fell gradually by approximately 20-25 mg/dl over a 45-60 minute period after insertion of a carotid artery cannula. This occurred whether 500 microU of insulin and/or anti-insulin serum or saline were injected toward the heart. There was no change in glucose concentrations following injection of 500 microU of insulin toward the head 45 minutes after insertion of the cannula. Thus, the hypoglycemic response to small amounts of insulin administered to the head via the carotid artery must be very sensitive to factors that are currently difficult to recognize.     

Optimization of "Photosens" pharmakokinetics by biodegradable nanospheres]

The present study is dedicated to investigation of pharmacokinetics of the colloidal delivery system based on polybutylcyanoacrylate nanoparticles for the II generation photosensitizer Photosense. Free or nanoparticle-bound Photosense was injected intravenously in healthy rats in the dose 15 mg/kg. It was shown that pharmacokinetic curve of the free drug was characterized by peak concentration while plasma concentrations of nanoparticulate Photosense were relatively steady. Elimination of nanoparticulate Photosense was more rapid comparing to the free drug. It is noteworthy that nanoparticles did not enhance liver uptake of the drug. Lung level of nanoparticulate drug was found to be lower and spleen uptake was enhanced. More important is the fact that nanoparticles provided two-fold decrease of Photosense skin concentration which is potentially important for decrease of drug-related skin phototoxicity. The above data provide evidence that optimization of Photosense pharmacokinetic parameters could be achieved by the use of nanoparticles.     

The reaction of Acridine Orange with proteoglycans in the articular cartilage of the rat

Summary Acridine Orange in concentrations from 0.01% to 0.2% was added to the first fixative solution in order to stain vibratome sections and small blocks of the articular cartilage of 2 month old rats. The interterritorial matrix of the radial or deep zone (zone 3) was examined. It contained reaction products with different morphology depending on the specimens used. In vibratome sections filaments were seen arranged in a homogenous pattern and changing in size with the concentration of the dye: diluted solutions produced finer filaments than concentrated ones. In contrast, in tissue blocks the staining pattern was not altered by different concentrations of Acridine Orange. However, with increase of the distance from the surface of the specimens the size of the filaments gradually decreased and formed a finer network. Since after preincubation with chondroitin ABC lyase only minute reaction products remained, an interaction of the dye with the sulphated glycosaminoglycans of the proteoglycans in the articular cartilage is suggested.The experiments show that by using mainly monocationic monomers of Acridine Orange the proteoglycans can be stained in a more expanded state than with polycationic dye polymers.     

Self-assembled cationic nanogels for intracellular protein delivery

An effective intracellular protein delivery system with self-assembled cationic nanogels is reported. Interaction of proteins with self-assembled nanogels of cationic cholesteryl group-bearing pullulans (CHPNH 2) was investigated by dynamic light scattering (DLS), transmission electron micrograph (TEM), fluorescence resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS). The cationic nanogels strongly interacted with bovine serum albumin (BSA) and formed monodispersed nanoparticels (<50 nm). The complex more effectively internalized into HeLa cells than cationic liposomes and a protein transduction domain (PTD) based carrier even in the presence of serum. The higher efficiency of the nanogel carrier is probably due to the formation of colloidally stable nanoparticles with the protein. The enzymatic activity of beta-galactosidase (beta-Gal) was retained after internalization into cells. The nanogel carrier promoted nuclear delivery of a GFP-conjugated nuclear localization signal and Tat as a PTD (Tat-NLS-GFP). A blocking experiment with chemical inhibitors revealed the possible involvement of macropinocytosis in the uptake of the nanogel complex. After cellular uptake, the complex of the nanogel-protein was dissociated and the protein was released inside the cell. Such a self-assembled cationic nanogel system should create opportunities for novel applications of protein delivery.     

Effects of Albumin, Amino Acids and Clofibrate on the Uptake of Tryptophan by the Rat Brain

Abstract: The influences of total tryptophan concentration, albumin binding and amino acid competition on the rate of tryptophan influx into rat brain were compared using a single-pass injection technique with tritiated water as a freely diffusible reference. Omission of 3% bovine albumin from a bolus containing tryptophan in Krebs–Ringer bicarbonate buffer injected into the carotid artery increased non-albumin bound (free) tryptophan concentration threefold but tryptophan uptake by only 35% and 30% into forebrain and hypothalamus, respectively. However, tryptophan uptake from injected rat plasma was more markedly elevated when free tryptophan concentration was raised. Thus, when free tryptophan was doubled, but total tryptophan unchanged, by in vitro addition of clofibrate to a plasma bolus, uptake was increased by 53% and 28% into forebrain and hypothalamus respectively. When clofibrate was injected in vivo so that plasma total tryptophan concentration was decreased by 45% but neither free tryptophan nor competing amino acid concentrations were altered, then uptake from a bolus of the rat's own plasma was unchanged. Addition of competing amino acids at physiological concentrations to tryptophan in Krebs-Ringer buffer significantly reduced tryptophan influx into both brain regions, but did not increase the effect of albumin binding. The results indicate that tryptophan uptake into rat forebrain is substantially influenced by albumin binding and competition from other amino acids, but that hypothalamic uptake is less influenced by these factors.     

Pulmonary arterial transit times

To begin to characterize the pulmonary arterial transport function we rapidly injected a bolus containing a radiopaque dye and a fluorescence dye into the right atrium of anesthetized dogs. The concentrations of the dye indicators were measured in the main pulmonary artery (fluoroscopically) and in a subpleural pulmonary arteriole (by fluorescence microscopy). The resulting concentration vs. time curves were subjected to numerical deconvolution and moment analysis to determine how the bolus was dispersed as it traveled through the arteriole stream tube from the main pulmonary artery to the arteriole. The mean transit time and standard deviation of the transport function from the main pulmonary artery to the arterioles studied averaged 1.94 and 1.23 s, respectively, and the relative dispersion (ratio of standard deviation to mean transit time) was approximately 64%. This relative dispersion is at least as large as those reported for the whole dog lung, indicating that relative to their respective mean transit times the dispersion upstream from the arterioles is comparable to that taking place in capillaries and/or veins. The standard deviations of the transport functions were proportional to their mean transit times. Thus the relative dispersion from the main pulmonary artery to the various arterioles studied was fairly consistent. However, there were variations in mean transit time even between closely adjacent arterioles, suggesting that variations in mean transit times between arteriole stream tubes also contribute to the dispersion in the pulmonary arterial tree.     

Effect of the ligation of hepatic artery on the microcirculation in the cirrhotic liver in the rat.

The significance of the hepatic arterial supply in the intrahepatic microcirculation in normal and carbon tetrachloride-induced cirrhotic livers was studied by dye injection method and by ligation of the hepatic artery. The in vivo distribution of dye injected into the hepatic artery evidenced the presence of arterio-venous shunts in the cirrhotic liver. When the hepatic artery of the cirrhotic liver was ligated, the elevated portal venous pressure dropped significantly, and the fast-flowing population of microvessels and sinusoids in the bimodal frequency distribution plot disappeared. The fast-flowing microvessel and sinusoids appeared to be the "arterial" microvessels and sinusoids, and they were converted into the slow-flowing venous channels after hepatic arterial ligation. The transmission of arterial pressure via the A-V shunts may be of greater significance in the pathophysiology of portal hypertension than previously believed.     

Lung serotonin uptake kinetics from indicator-dilution and constant-infusion methods

The kinetics of the pulmonary endothelial uptake of serotonin (5-HT) were evaluated in isolated dog lung lobes using three methods. In method A serotonin was infused at various constant rates to provide a range of capillary concentrations that included Km. The arterial and venous concentrations measured by high-performance liquid chromatography were then used to determine the effect of concentration on the rate of 5-HT uptake. In method B trace doses of 5-[3H]HT and a reference indicator (indocyanine green dye) were injected during each constant infusion of unlabeled 5-HT to provide a measure of unidirectional 5-HT uptake at each background concentration. In method C boluses containing different amounts of unlabeled 5-HT, along with the 5-[3H]HT and the dye, were injected such that each bolus resulted in a range of concentrations and provided a measure of the unidirectional uptake at each concentration. Each method provided the data needed to calculate the maximum uptake rate (Vmax) and the concentration at Vmax/2 (Km), assuming that the uptake kinetics can be represented by the Michaelis-Menten equation. However, the mathematical model underlying each method involved different assumptions about the returning flux of the 5-HT which entered the endothelial cell and the heterogeneity of vascular transit times. The results obtained, considered in light of the different assumptions involved, indicate that all three methods can provide reasonable estimates of the mass transfer kinetic constants if the constant infusions of 5-HT are of short duration and/or the boluses are adequately dispersed prior to reaching the capillary bed.     

Stabilization of alpha-chymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel

Stabilization of alpha-chymotrypsin (CT) by covalent immobilization on the amine-functionalized magnetic nanogel was studied. The amino groups containing superparamagnetic nanogel was obtained by Hoffman degradation of the polyacrylamide (PAM)-coated Fe(3)O(4) nanoparticles prepared by facile photochemical in situ polymerization. CT was then covalently bound to the magnetic nanogel with reactive amino groups by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide as coupling reagent. The binding capacity was determined to be 61mg enzyme/g nanogel by BCA protein assay. Specific activity of the immobilized CT was measured to be 0.93U/(mgmin), 59.3% as that of free CT. The obtained immobilized enzyme had better resistance to temperature and pH inactivation in comparison to free enzyme and thus widened the ranges of reaction pH and temperature. The immobilized enzyme exhibited good thermostability, storage stability and reusability. Kinetic parameters were determined for both the immobilized and free enzyme. The value of K(m) of the immobilized enzyme was larger than did the free form, whereas the V(max) was smaller for the immobilized enzyme.     

Dye Transfer Between Cells of the Lens

Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide dynamic range-cooled CCD camera, H₂O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer between deep fiber cells was also observed. Dye transfer between ≈10% of epithelial cells and their underlying fiber cells was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial junction. Received: 14 September 1995/Revised: 13 November 1995     

Gap junctional communication in the post-implantation mouse embryo.

We studied the extent of cell-to-cell communication via junctional channels in in vitro-implanted mouse blastocysts by monitoring ionic coupling and the spread of two injected low molecular weight dyes, fluorescein and Lucifer yellow. In the early attached embryos, both trophoblasts and cells of the inner cell mass (ICM) were ionically coupled to one another. Dye injections in either trophoblasts or ICM cells resulted in spread to the entire embryo. As older and more developed embryos were examined, the spread of injected dye was progressively more limited. In the most developed embryos examined, dye injected into a cell in the ICM region resulted in spread throughout the ICM but not into the surrounding trophoblast cells, while dye injected into a trophoblast cell did not spread to any other cell in the embryo. Simultaneous monitoring of ionic coupling and dye injections in embryos of intermediate stages in this transition revealed that the trophoblast and ICM cells were ionically coupled, even across the apparent boundary where no dye was observed to pass. In the latest stage embryos examined in which no injected dye was observed to move out of the ICM, ionic coupling was still observed between the cells of the ICM and the trophoblasts. Furthermore, in the more developed embryos, dye injected into the ICM region frequently was not transferred to all the cells of the ICM, thus suggesting a further compartmentalization of due spread within the ICM. Our observations that ionic coupling is more extensive than the detectable spread of injected dyes may perhaps reflect a reduced number of junctional channels. With fewer channels less dye would pass between cells, so that, together with continuous quenching, the transfer of injected dye would not be detectable. This partial segregation of cell-to-cell communication as indicated by the limited dye spread may parallel specific differentiation processes, in particular that of giant trophoblast, embryonic ectoderm and extraembryonic endoderm differentiation.     

Interstitial albumin-bound dye concentrations in mice with a protein-rich effusion

Levels of intravenously injected Evans blue dye in eluates of the lung and kidney, an index of interstitial fluid albumin concentration, together with water content of these tissues and levels of serum albumin were measured in Ha-icr mice with a tumor cell-induced protein-rich peritoneal effusion. By the fourth day after the intraperitoneal injection of tumor cells, when mean serum albumin levels had fallen to 76% of control values, mean albumin bound dye concentrations in lung and kidney had decreased to 63 and 58%, respectively, of control values. By the tenth day when serum albumin levels had decreased further to 67% of control values, albumin-bound dye concentrations in the lung and kidney had decreased to 58 and 43%, respectively, of control values. During this 10-day period the water content of the lung remained unchanged whereas that of the kidney had decreased by 7%. These observations suggest that the reduction in serum albumin which results from an abnormal distribution of this protein into a nonvascular compartment is accompanied, as in other models of hypoalbuminemia, by a more than proportionate reduction in interstitial albumin concentration in the lung and kidney.     

Design and synthesis of lipase nanogel with interpenetrating polymer networks for enhanced catalysis: Molecular simulation and experimental validation

A temperature-responsive lipase nanogel (denoted as CRL-IPN nanogel), in which lipase is encapsulated into an interpenetrating polymer matrix formed by polyacrylamide and poly(N-isopropylacrylamide) (PNIPAAm) has been designed and synthesized for an enhanced stability and activity in both aqueous and non-polar organic solvents. A three-step method, including acryloylation, polymerization with acrylamide and sequential polymerization with N-isopropylacrylamide, was established to fabricate enzyme nanogel with temperature-sensitive interpenetrating polymer network. It has been shown by an all-atom molecular dynamics simulation that above mentioned polymer matrix forms a more hydrophobic environment, as compared to that obtained with sole polyacrylamide, because of the penetration of N-isopropylacrylamide into the polymer acrylamide network via hydrogen bonding, which is further confirmed by the fluorescence spectrum. This favours the uptake of hydrophobic substrates and thus the overall rate of enzymatic catalysis. The enhanced stability and catalytic performance of this novel lipase nanogel in aqueous and non-polar organic solvent were demonstrated by using hydrolysis reaction of p-NPP in aqueous and esterification reaction of ibuprofen in isooctane. In aqueous solution, the residual activity of CRL-IPN nanogel maintains its 70% activity at 60 °C after 4 h, compared with that free lipase only has 30% at the same condition. In addition, the CRL-IPN nanogel can be reused for 10 cycles with no loss of its activity. In isooctane, CRL-IPN nanogel gave a 33% yield of esterification of ibuprofen, in comparison to 22% using free lipase and less than 5% using lipase encapsulated in a polyacrylamide matrix. The enhanced stability and activity make this CRL-IPN nanogel promising for enzymatic catalysis in non-polar solvents.     

Dye and electrical coupling of endothelial cells in situ

Electron microscopic studies show that endothelial cells of pig coronary arteries are linked by gap junctions. We investigated the dye and electrical coupling of these junctions in a strip of pig coronary artery in vitro. The membrane potential of two neighbouring (about 0.2 mm) endothelial cells were simultaneously recorded with two microelectrodes. The fluorescent dye lucifer yellow was microiontophoretically injected through one of the microelectrodes. The endothelial cells in situ were dye and electrically coupled. The dye coupling extended parallel to the longitudinal axis of the arteries. We conclude that an electrical message like the bradykinin and substance P hyperpolarizations of the endothelial cells can be conveyed electrotonically by the endothelium along the longitudinal axis of arteries.     

Observation of a Flowing Duct in the Abdominal Wall by Using Nanoparticles

The primo vascular system (PVS) is being established as a circulatory system that corresponds to acupuncture meridians. There have been two critical questions in making the PVS accepted as a novel liquid flowing system. The first one was directly to show the flow of liquid in PVS and the second one was to explain why it was not observed in the conventional histological study of animal tissues. Flow in the PVS in the abdominal cavity was previously verified by injecting Alcian blue into a primo node. However, the tracing of the dye to other subsystems of the PVS has not been done. In the current work we injected fluorescent nanoparticles (FNPs) into a primo node and traced them along a primo vessel which was inside a fat tissue in the abdominal wall. Linea alba is a white middle line in the abdominal skin of a mammal and a band of fat tissue is located in parallel to the linea alba in the parietal side of the abdominal wall of a rat. In this fat band a primo vessel runs parallel to the prominent blood vessels in the fat band and is located just inside the parietal peritoneum. About the second question on the reason why the PVS was not in conventional histological study the current work provided the answer. Histological analysis with hematoxyline and eosine, Masson’s trichrome, and Toluidine blue could not discriminate the primo vessel even when we knew the location of the PVS by the trace of the FNPs. This clearly explains why the PVS is hard to observe in conventional histology: it is not a matter of resolution but the contrast. The PVS has very similar structure to the connective tissues that surround the PVS. In the current work we propose a method to find the PVS: Observation of mast cell distribution with toluidine blue staining and the PN has a high density of mast cells, while the lymph node has low density.