Tumor repolarization by an advanced liposomal drug delivery system provides a potent new approach for chemo-immunotherapy.

Immunosuppressive cells in the tumor microenvironment allow cancer cells to escape immune recognition and support cancer progression and dissemination. To improve therapeutic efficacy, we designed a liposomal oxaliplatin formulation (PCL8-U75) that elicits cytotoxic effects toward both cancer and immunosuppressive cells via protease-mediated, intratumoral liposome activation. The PCL8-U75 liposomes displayed superior therapeutic efficacy across all syngeneic cancer models in comparison to free-drug and liposomal controls. The PCL8-U75 depleted myeloid-derived suppressor cells and tumor-associated macrophages in the tumor microenvironment. The combination of improved cancer cell cytotoxicity and depletion of immunosuppressive populations of immune cells is attractive for combination with immune-activating therapy. Combining the PCL8-U75 liposomes with a TLR7 agonist induced immunological rejection of established tumors. This combination therapy increased intratumoral numbers of cancer antigen-specific cytotoxic T cells and Foxp3- T helper cells. These results are encouraging toward advancing liposomal drug delivery systems with anticancer and immune-modulating properties into clinical cancer therapy.

Liposome-encapsulated chemotherapy: Current evidence for its use in companion animals.

Cytotoxic drugs encapsulated into liposomes were originally designed to increase the anticancer response, while minimizing off-target adverse effects. The first liposomal chemotherapeutic drug was approved for use in humans more than 20 years ago, and the first publication regarding its use in a canine cancer patient was published shortly thereafter. Regardless, no general application for liposomal cytotoxic drugs has been established in veterinary oncology till now. Due to the popularity of canines as experimental models for pharmacokinetic analyses and toxicity studies, multiple publications exist describing various liposomal drugs in healthy dogs. Also, some evidence for its use in veterinary cancer patients exists, especially in canine lymphoma, canine splenic hemangiosarcoma and feline soft tissue sarcoma, however, the results have not been overwhelming. Reasons for this may be related to inherent issues with the enhanced permeability and retention effect, the tumour phenomenon which liposomal drugs exploit. This effect seems very heterogeneously distributed in the tumour. Also, it is potentially not as ubiquitously occurring as once thought, and it may prove important to select patients for liposomal therapy on an individual, non-histology-oriented, basis. Concurrently, new developments with active-release modified liposomes in experimental models and humans will likely be relevant for veterinary patients as well, and holds the potential to improve the therapeutic response. It, however, does not resolve the other challenges that liposomal chemotherapy faces, and more work still needs to be done to determine which veterinary patients may benefit the most from liposomal chemotherapy.

An assessment of the importance of exposure routes to the uptake and internal localisation of fluorescent nanoparticles in zebrafish (Danio rerio), using light sheet microscopy.

A major challenge in nanoecotoxicology is finding suitable methods to determine the uptake and localisation of nanoparticles on a whole-organism level. Some uptake methods have been associated with artefacts induced by sample preparation, including staining for electron microscopy. This study used light sheet microscopy (LSM) to define the uptake and localisation of fluorescently labelled nanoparticles in living organisms with minimal sample preparation. Zebrafish (Danio rerio) were exposed to fluorescent gold nanoparticles (Au NPs) and fluorescent polystyrene NPs via aqueous or dietary exposure. The in vivo uptake and localisation of NPs were investigated using LSM at different time points (1, 3 and 7 days). A time-dependent increase in fluorescence was observed in the gut after dietary exposure to both Au NPs and polystyrene NPs. No fluorescence was observed within gut epithelia regardless of the NP exposure route indicating no or limited uptake via intestinal villi. Fish exposed to polystyrene NPs through the aqueous phase emitted fluorescence signals from the gills and intestine. Fluorescence was also detected in the head region of the fish after aqueous exposure to polystyrene NPs. This was not observed for Au NPs. Aqueous exposure to Au NPs resulted in increased relative swimming distance, while no effect was observed for other exposures. This study supports that the route of exposure is essential for the uptake and subsequent localisation of nanoparticles in zebrafish. Furthermore, it demonstrates that the localisation of NPs in whole living organisms can be visualised in real-time, using LSM.

Recent advances in compartmentalized synthetic architectures as drug carriers, cell mimics and artificial organelles.

Compartmentalization is a key feature of biological cells which conduct their metabolic activity in individual steps isolated in distinct, separated compartments. The creation of architectures containing multiple compartments with a structure that resembles that of a biological cell has generated significant research attention and these assemblies are proposed as candidate materials for a range of biomedical applications. In this Review article, the recent successes of multicompartment architectures as carriers for the delivery of therapeutic cargo or the creation of micro- and nanoreactors that mimic metabolic activities, thus acting as artificial cells or organelles, are discussed. The developed technologies to assemble such complex architectures are outlined, the multicompartment carriers' properties which contribute to their performance in diverse applications are discussed, and their successful applications are highlighted. Finally, future directions and developments in the field are suggested.

Injectable silver nanosensors: in vivo dosimetry for external beam radiotherapy using positron emission tomography.

Development of safe and efficient radiotherapy routines requires quantification of the delivered absorbed dose to the cancer tissue in individual patients. In vivo dosimetry can provide accurate information about the absorbed dose delivered during treatment. In the current study, a novel silver-nanosensor formulation based on poly(vinylpyrrolidinone)-coated silver nanoparticles formulated in a gelation matrix composed of sucrose acetate isobutyrate has been developed for use as an in vivo dosimeter for external beam radiotherapy. In situ photonuclear reactions trigger the formation of radioactive (106)Ag, which enables post treatment verification of the delivered dose using positron emission tomography imaging. The silver-nanosensor was investigated in a tissue equivalent thorax phantom using clinical settings and workflow for both standard fractionated radiotherapy (2 Gy) and stereotactic radiotherapy (10- and 22 Gy) in a high-energy beam setting (18 MV). The developed silver-nanosensor provided high radiopacity on the planning CT-scans sufficient for patient positioning in image-guided radiotherapy and provided dosimetric information about the absorbed dose with a 10% and 8% standard deviation for the stereotactic regimens, 10 and 22 Gy, respectively.

A hydrogel based nanosensor with an unprecedented broad sensitivity range for pH measurements in cellular compartments.

Optical pH nanosensors have been applied for monitoring intracellular pH in real-time for about two decades. However, the pH sensitivity range of most nanosensors is too narrow, and measurements that are on the borderline of this range may not be correct. Furthermore, ratiometric measurements of acidic intracellular pH (pH < 4) in living cells are still challenging due to the lack of suitable nanosensors. In this paper we successfully developed a multiple sensor, a fluorophore based nanosensor, with an unprecedented broad measurement range from pH 1.4 to 7.0. In this nanosensor, three pH-sensitive fluorophores (difluoro-Oregon Green, Oregon Green 488, and fluorescein) and one pH-insensitive fluorophore (Alexa 568) were covalently incorporated into a nanoparticle hydrogel matrix. With this broad range quadruple-labelled nanosensor all physiological relevant pH levels in living cells can be measured without being too close to the limits of its pH-range. The nanosensor exhibits no susceptibility to interference by other intracellular ions at physiological concentrations. Due to its positive surface charge it is spontaneously internalized by HeLa cells and localizes to the lysosomes where the mean pH was measured at 4.6. This quadruple-labelled nanosensor performs accurate measurements of fluctuations of lysosomal pH in both directions, which was shown by treatment with the V-ATPase inhibitor bafilomycin A1 or its substrate ATP in HeLa cells. These measurements indicate that this novel quadruple-labelled nanosensor is a promising new tool for measuring the pH of acidic compartments in living cells.

Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay.

We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 µM doxorubicin concentration, whereas this was not observed at 5 or 100 µM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 µM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.

Propargylamine-isothiocyanate reaction: efficient conjugation chemistry in aqueous media.

A coupling reaction between secondary propargyl amines and isothiocyanates in aqueous media is described. The reaction is high-yielding and affords cyclized products within 2-24 h. A functionalized ether lipid was synthesized in 8 steps, formulated as liposomes with POPC and conjugated to FITC under mild conditions using this method.

Particulate systems for targeting of macrophages: basic and therapeutic concepts.

Particulate systems in the form of liposomes, polymeric micelles, polymeric nano- and microparticles, and many others offer a rational approach for selective delivery of therapeutic agents to the macrophage from different physiological portals of entry. Particulate targeting of macrophages and intracellular drug release processes can be optimized through modifications of the drug carrier physicochemical properties, which include hydrodynamic size, shape, composition and surface characteristics. Through such modifications together with understanding of macrophage cell biology, targeting may be aimed at a particular subset of macrophages. Advances in basic and therapeutic concepts of particulate targeting of macrophages and related nanotechnology approaches for immune cell modifications are discussed.

Factors controlling nanoparticle pharmacokinetics: an integrated analysis and perspective.

Intravenously injected nanoparticulate drug carriers provide a wide range of unique opportunities for site-specific targeting of therapeutic agents to many areas within the vasculature and beyond. Pharmacokinetics and biodistribution of these carriers are controlled by a complex array of interrelated core and interfacial physicochemical and biological factors. Pertinent to realizing therapeutic goals, definitive maps that establish the interdependency of nanoparticle size, shape, and surface characteristics in relation to interfacial forces, biodistribution, controlled drug release, excretion, and adverse effects must be outlined. These concepts are critically evaluated and an integrated perspective is provided on the basis of the recent application of nanoscience approaches to nanocarrier design and engineering. The future of this exciting field is bright; some regulatory-approved products are already on the market and many are in late-phase clinical trials. With concomitant advances in extensive computational knowledge of the genomics and epigenomics of interindividual variations in drug responses, the boundaries toward development of personalized nanomedicines can be pushed further.

Thermodynamic profiling of peptide membrane interactions by isothermal titration calorimetry: a search for pores and micelles.

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T(zero)). This allows for an exclusive study of the pore-formation process. The use of ITC to find T(zero) allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.

Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: the challenges ahead.

Since their introduction, poly(ethylene glycol)-phospholipid (PEG-PL) conjugates have found many applications in design and engineering of nanosized delivery systems for controlled delivery of pharmaceuticals especially to non-macrophage targets. However, there are reports of idiosyncratic reactions to certain PEG-PL engineered nanomedicines in both experimental animals and man. These reactions are classified as pseudoallergy and may be associated with cardiopulmonary disturbance and other related symptoms of anaphylaxis. Recent studies suggest that complement activation may be a contributing, but not a rate limiting factor, in eliciting hypersensitivity reactions to such nanomedicines in sensitive individuals. This is rather surprising since PEGylated structures are generally assumed to suppress protein adsorption and blood opsonization events including complement. Here, we examine the molecular basis of complement activation by PEG-PL engineered nanomedicines and carbon nanotubes and discuss the challenges ahead.

Complement-mediated tumour growth: implications for cancer nanotechnology and nanomedicines.

The recent unexpected observation that complement activation helps tumour growth and progression has an important bearing on the future development of cancer nanomedicines for site-specific tumour targeting as these entities are capable of triggering complement. These issues are discussed and suggestions are provided for future design and development of safer and effective cancer nanomedicines.

Effects of hyperoxia and beta-adrenergic stimulation on pulmonary surfactant in neonatal rabbits.

To study the effects of hyperoxia and beta-adrenergic stimulation on pulmonary surfactant in the neonatal lung, we measured disaturated phosphatidylcholine (DSPC) and [14C]choline incorporation into DSPC, obtained from alveolar lavage and lung tissue. We used an isolated salt-perfused rabbit lung preparation from neonatal rabbits exposed to room air or greater than 95% oxygen for 3 days. There were four experimental groups: room air, basal condition; room air, beta-adrenergic stimulation; hyperoxia, basal conditions; and hyperoxia, beta-adrenergic stimulation. Hyperoxia caused a significant decrease in lavage and intracellular [14C]DSPC specific activity, and a decrease in intracellular DSPC suggesting depressed surfactant synthesis. Beta-stimulation in room air caused a decrease in lavage DSPC, an increase in DSPC, and [14C]DSPC fraction released, consistent with increased uptake for reutilization. With hyperoxia and beta-stimulation, there is an increase in total DSPC in the lavage; lavage [14C]DSPC specific activity is similar to that of the basal hyperoxia group (i.e., depressed compared with the room air state); intracellular [14C]DSPC specific activity does not differ from basal, hyperoxia, or beta-stimulated, room air groups, all being depressed compared with basal, room air conditions. Intracellular DSPC in the beta-stimulated group is less affected by hyperoxia than the basal groups. It appears that prolonged exposure to hyperoxia is manifested primarily by a decrease in [14C]DSPC specific activity suggesting alterations in surfactant synthesis, though DSPC in the lavage is not altered. Beta-adrenergic stimulation may enhance release of newly synthesized surfactant into the alveoli, and possibly enhances uptake for reutilization. The enhancement of surfactant release seems to be preserved after prolonged hyperoxia.

Effect of hyperoxia on 5-hydroxytryptamine uptake by the neonatal rabbit lung.

Neonatal rabbits were exposed to either normoxia (21% oxygen) or hyperoxia (. 95% oxygen) for 2-4 days, and isolated ventilated perfused lung preparations from the various animals were studied. 5-Hydroxytryptamine (5HT) uptake, perfusion pressure, alveolar lavage protein and lung tissue vitamin E concentrations were measured. There was no difference in mortality between the two groups at any time point. There was no difference in perfusion pressures at any time point. There were no differences between normoxic and hyperoxic animals in alveolar lavage protein or 5 HT uptake at 2 and 3 days. At 4 days, 5HT uptake (fractional) was lower in the hyperoxia group than in controls (0.65 +/- 0.033 v. 0.75 +/- 0.013 (mean +/- SE); p less than or equal to 0.05) and alveolar lavage protein was higher compared to normoxia (1111 +/- 415 micrograms/ml v. 481 +/- 78 micrograms/ml; p less than or equal to 0.05). Lung vitamin E concentrations were higher at 3 days in rabbits exposed to hyperoxia compared to normoxia (16.5 +/- 1.8 micrograms/gm v. 12.3 +/- 0.6 micrograms/gm; p less than or equal to 0.05). In air exposed animals there was a decrease in lung vitamin E concentration after 2 days, whereas hyperoxia exposed animals had no significant decrease in lung vitamin E concentrations from 2-4 days exposure. These studies establish that the decrease in 5HT uptake, albeit delayed compared to that described previously in adult animals, is a reasonable measure of pulmonary oxygen toxicity in newborn rabbits.

Conjunctival fungal flora in horses, cattle, dogs, and cats.

Conjunctival swab specimens were obtained from both eyes of 43 horses, 25 cows, 50 dogs, and 25 cats without keratitis or other ophthalmologic problems. Fungi were isolated from 95% of the horses, 100% of the cows, 22% of the dogs, and 40% of the cats. Aspergillus spp were isolated from 56% of the horses, 12% of the cows, 8% of the cats, and none of the dogs. Penicillium spp and Cladosporium spp were isolated ubiquitously. Collectively, 28 species from 209 isolants were identified.

Biometry and clinical characteristics of congenital cataracts and microphthalmia in the Miniature Schnauzer.

Forty-two Miniature Schnauzer pups and adults with congenital cataracts and microphthalmia were evaluated by serial ophthalmic examinations, slit lamp biomicroscopic photography, and A-scan ultrasonography. The cataracts were evident when the eyelids opened at 2 weeks, affecting predominantly the lens nucleus and posterior cortex. Lenticonus was evident in 19% of the cataractous lenses. Progression of the cataracts was variable and related to involvement of the equatorial and posterior cortices. Lens-induced uveitis developed in some adult dogs with advanced hypermature cataracts. The globe and lens were smaller than normal in the cataractous eyes, as ascertained by A-scan ultrasonography. Age-matched comparisons of clear lens carrier Miniature Schnauzers and normal Beagles with the cataractous Miniature Schnauzers indicated affected globes and cataractous lenses were reduced 10% to 20% in their anteroposterior lengths. The microphthalmia appeared related to the congenital microphakic cataract.

Inheritance of congenital cataracts and microphthalmia in the Miniature Schnauzer.

Congenital cataracts and microphthalmia in the Miniature Schnauzer were inherited as an autosomal recessive trait. Eighteen matings of affected X affected Miniature Schnauzers resulted in 87 offspring with congenital cataracts and microphthalmia (49 males/38 females). Two matings of congenital cataractous and microphthalmic Miniature Schnauzers (2 females) X a normal Miniature Schnauzer (1 male) yielded 11 clinically normal Miniature Schnauzers (7 males/4 females). Eighteen matings of congenital cataractous and microphthalmic Miniature Schnauzers (6 males) X carrier Miniature Schnauzers (9 females) produced 81 offspring; 39 exhibited congenital cataracts and microphthalmia (20 males/19 females) and 42 had clinically normal eyes (17 males/25 females).