Conrad Coester

Optimized dispersion of nanoparticles for biological in vitro and in vivo studies.

BackgroundThe aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies.ResultsTiO2 (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 × 105 kJ/m3 was sufficient to disaggregate TiO2 (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO2 (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO2 (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO2 (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO2 (rutile), ZnO, Ag, SiOx, SWNT, MWNT, and diesel SRM2975 particulate matter.ConclusionThe optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates.

Targeting CpG Oligonucleotides to the Lymph Node by Nanoparticles Elicits Efficient Antitumoral Immunity

Viral nucleic acids are recognized by specific pattern-recognition receptors of the Toll-like and RIG-I-like receptor families. Synthetic DNA and RNA oligonucleotides can activate the immune system through these receptors and potentiate Ab and CD8 cytotoxic responses to Ags. Systemic application of immunostimulatory oligonucleotides however also results in a generalized, non-Ag-specific stimulation of the immune system. In this study, we have dissociated the induction of an Ag-specific response from the systemic immune activation generally associated with immunostimulatory oligonucleotides. Delivery of CpG oligodeoxynucleotides that bind TLR9 by cationized gelatin-based nanoparticles potentiates the in vivo generation of an Ag-specific cytotoxic T cell and Ab response. Furthermore, immunization with CpG-loaded nanoparticles induces a protective antitumoral response in a murine model of melanoma. The systemic release of proinflammatory cytokines and widespread immunostimulation associated with free CpG is however completely abolished. In addition, we show that gelatin nanoparticle formulation prevents the destruction of lymphoid follicles mediated by CpG. Nanoparticle-delivered CpG, in contrast to free CpG, are selectively targeted to APCs in the lymph nodes where they mediate local immune stimulation. We describe a novel strategy to target immunostimulatory oligonucleotides to the initiation site of the immune response while at the same time protecting from an indiscriminate and generalized activation of the immune system.

Delivery by Cationic Gelatin Nanoparticles Strongly Increases the Immunostimulatory Effects of CpG Oligonucleotides

PurposeCationized gelatin nanoparticles (GNPs) were used as carrier to improve delivery of immunostimulatory CpG oligonucleotides (CpG ODN) both in vitro and in vivo.MethodsUptake of CpG ODN-loaded cationized gelatin nanoparticles (CpG-GNPs) into murine myeloid dendritic cells (DCs) and their respective immunostimulatory activity was monitored. In vivo, induction of cytokine secretion by CpG-GNPs was measured. For experiments on primary human cells, prototypes of the three CpG ODN classes were adsorbed onto GNPs. Uptake and induction of proinflammatory cytokines were assessed in human plasmacytoid DCs and B cells, the only existing human target cells for CpG ODN.ResultsIn the murine system, gelatin nanoparticle formulations enhanced the uptake and immunostimulatory activity of CpG ODN both in vitro and in vivo. Furthermore, delivery by cationized gelatin nanoparticles of CpG ODN of the classes B and C to primary human plasmacytoid DCs increased production of IFN-α, a key cytokine in the driving of both the innate and adaptive immune responses.ConclusionGNPs can be used as a biodegradable and well tolerated carrier to deliver CpG ODN to their target cells and strongly increase activation of the immune system. This concept may be applied as novel adjuvant for antiviral and antitumoral vaccines.

Matrix-loaded biodegradable gelatin nanoparticles as new approach to improve drug loading and delivery

The long-term objective of this study is to develop a nanoparticulate formulation based on gelatin or its admixtures with other polyelectrolytes, under very gentle nanoprecipitation conditions, for the delivery of fragile macromolecules such as proteins and peptide drugs. However, the objective of the present study was to achieve drug loading into the matrices of gelatin-based nanoparticles through incubation of the drug-gelatin solution prior to formation and cross-linking of the nanoparticles in situ. Two molecular weight types (4 kDa and 20 kDa) of fluorescein isothiocyanate dextran (FITC-D) were used as surrogate macromolecules to study the loading and in vitro release behavior of gelatin nanoparticles. Unloaded and FITC-D-loaded gelatin nanoparticles were prepared by the one-step desolvation technique using ethanol-water mixture as the non-solvent. The preparation method was optimized with respect to the amount of cross-linking agent and cross-linking time. The nanoparticles formed were further characterized for mean size, size distribution and zeta potential using a Zetasizer nano while the morphology of the particles was evaluated by scanning electron microscopy (SEM). For cell uptake studies, FITC-D-labeled nanoparticles were incubated with Caco-2 cell monolayers and then evaluated using fluorescence microscopy. Results obtained showed the formation of very smooth and spherical particles with a unimodal distribution. Zeta potential measurements revealed that both the unloaded and FITC-D-loaded nanoparticles had a surface charge of -23.0 mV at pH 7.0. The loading capacity of the nanoparticles was found to be approximately 93.0 microg FITC-D (20 kDa) and 86 microg FITC-D (4 kDa) per milligram gelatin nanoparticles. Up to 16.5% of the 20 kDa FITC-D was loaded on the surface of the nanoparticles while 76.8% was entrapped into the matrices of the particles. For the 4 kDa FITC-D, 10.8% was bound to the surface of the particles while 75.6% was entrapped into the core of the nanoparticles. The release profile of FITC-D from the nanoparticles over a 168-h period showed a low release in phosphate-buffered saline (PBS), pH 7.4 while more than 80% was released after 3h for both types of FITC-D in PBS containing trypsin. Release of the 4 kDa FITC-D from the nanoparticles was generally more rapid than that of the 20 kDa indicating that its entrapment into gelatin nanoparticles was based on weaker interactions when compared to that of the higher molecular weight FITC-D. Bio-imaging using fluorescence microscopy demonstrated uptake and internalization of the nanoparticles, notably into the nucleus and the cytoplasm, by Caco-2 cells.

New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: in-vivo characterization.

Doxorubicin(DOX) is a potent chemotherapy drug that is often limited by severe adverse effects such as cardiac toxicity and myelosupression. Drug targeting with non invasive techniques would be desirable, aiming at increased local drug concentration and reduced systemic side effects. Ultrasound(US) targeted destruction of drug loaded microbubbles(MBs) has evolved as a promising strategy for non invasive local gene and drug delivery. A recently developed novel DOX-loaded microbubble (DOX-MB) formulation was previously tested in-vitro, with optimal DOX loading capacity, ideal physical characteristics and preserved antiproliferative efficacy. The aim of this study was to evaluate applicability and efficacy of DOX-loaded MBs in a pancreas carcinoma model of the rat. First, immediate toxicity was tested in rats ruling out in-vivo MB agglomeration/capillary adhesion with subsequent embolisation/occlusion of the pulmonary vasculature. In a second set of experiments, tumors derived from pancreas carcinomas were implanted in both flanks of Lewis rats. After establishing the tumors, DOX-MBs were administered intravenously while one of the two tumors was exposed to US (1.3 MHz; mechanical index 1.6). DOX tissue concentration was measured in tumors and control organs after the experiment. Finally, efficacy of US targeted destruction of DOX-MBs in tumors was studied, looking at tumor growth after two therapeutic applications. All rats survived the DOX-MB administration without any sign of embolisation/occlusion of the pulmonary vasculature. US targeted destruction of DOX-MBs leads to a 12-fold higher tissue concentration of DOX and a significantly lower tumor growth in the target tumor compared to the contralateral control tumor. In conclusion, novel DOX-loaded MBs can be safely administered to rats, leading to a relevant increase in local drug concentration and reduction in tumor growth.

New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: Part I--Formulation development and in-vitro characterization.

Despite high antitumor efficacy and a broad application spectrum, clinical treatment with anthracycline chemotherapeutics is often limited by severe adverse effects such as cardiotoxicity and myelosupression. In recent years, tumor drug targeting has evolved as a promising strategy to increase local drug concentration and reduce systemic side effects. One recent approach for targeting solid tumors is the application of microbubbles, loaded with chemotherapeutic drugs. These advanced drug carriers can be safely administered to the patient by intravenous infusion, and will circulate through the entire vasculature. Their drug load can be locally released by ultrasound targeted microbubble destruction. In addition, tumors can be precisely localized by diagnostic ultrasound since microbubbles act as contrast agents. In the present work a novel microbubble carrier for doxorubicin has been developed and characterized in-vitro. In contrast to many recent tumor-targeting MB designs the newly developed doxorubicin-loaded microbubbles possess a soft but stable phospholipid monolayer shell. Importantly, the active drug is embedded in the microbubble shell and is complexed to the phospholipids by both electrostatic and hydrophobic interactions. Despite their drug load, these novel microbubbles retained all important physical characteristics for ultrasound targeted microbubble destruction, comparable with the commercially available ultrasound contrast agents. In cell culture studies doxorubicin-loaded microbubbles in combination with ultrasound demonstrated an about 3 fold increase of the anti-proliferative activity compared to free doxorubicin and doxorubicin-loaded liposomes. For the first time in the literature the intracellular partition of free doxorubicin and phospholipid-complexed doxorubicin were compared. In conclusion, new doxorubicin-loaded microbubbles with ideal physical characteristics were developed. In-vitro studies show enhanced cytotoxic activity compared to free doxorubicin and doxorubicin-loaded liposomes.

Formulation development of freeze-dried oligonucleotide-loaded gelatin nanoparticles

The freeze-drying properties of gelatin nanoparticles were investigated with the goal of providing practicable nanoparticle formulations for in vitro applications or clinical studies. Various excipients and rehydration protocols were assessed, and gelatin nanoparticles loaded with oligonucleotides were successfully freeze-dried and rehydrated. An NF-kappaB decoy oligonucleotide-loaded gelatin nanoparticle formulation was developed and applied in a drug targeting approach in an animal model. The high concentrations of nanoparticles achieved after rehydration with reduced volumes proved to be critical for the in vivo effect. Finally, short term storage stability under accelerated conditions was assessed for dried gelatin nanoparticles formulated in sucrose, trehalose, mannitol, or a mannitol/sucrose mixture. Size, size distribution, and residual moisture content were investigated. Sucrose- and trehalose-containing formulations exhibited the greatest stability, but mannitol-containing formulations also showed notable stabilization despite their crystalline nature.

A novel technique for selective NF-κB inhibition in Kupffer cells: contrary effects in fulminant hepatitis and ischaemia–reperfusion

Background and aims: The transcription factor nuclear factor kappa B (NF-κB) has risen as a promising target for anti-inflammatory therapeutics. In the liver, however, NF-κB inhibition mediates both damaging and protective effects. The outcome is deemed to depend on the liver cell type addressed. Recent gene knock-out studies focused on the role of NF-κB in hepatocytes, whereas the role of NF-κB in Kupffer cells has not yet been investigated in vivo. Here we present a novel approach, which may be suitable for clinical application, to selectively target NF-κB in Kupffer cells and analyse the effects in experimental models of liver injury. Methods: NF-κB inhibiting decoy oligodeoxynucleotides were loaded upon gelatin nanoparticles (D-NPs) and their in vivo distribution was determined by confocal microscopy. Liver damage, NF-κB activity, cytokine levels and apoptotic protein expression were evaluated after lipopolysaccharide (LPS), d-galactosamine (GalN)/LPS, or concanavalin A (ConA) challenge and partial warm ischaemia and subsequent reperfusion, respectively. Results: D-NPs were selectively taken up by Kupffer cells and inhibited NF-κB activation. Inhibition of NF-κB in Kupffer cells improved survival and reduced liver injury after GalN/LPS as well as after ConA challenge. While anti-apoptotic protein expression in liver tissue was not reduced, pro-apoptotic players such as cJun N-terminal kinase (JNK) were inhibited. In contrast, selective inhibition of NF-κB augmented reperfusion injury. Conclusions: NF-κB inhibiting decoy oligodeoxynucleotide-loaded gelatin nanoparticles is a novel tool to selectively inhibit NF-κB activation in Kupffer cells in vivo. Thus, liver injury can be reduced in experimental fulminant hepatitis, but increased at ischaemia–reperfusion.

Delivery of immunostimulatory RNA oligonucleotides by gelatin nanoparticles triggers an efficient antitumoral response.

RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleotides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA oligonucleotides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-α. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8+ T-cell and antibody responses. Indeed, immunization with RNA-loaded nanoparticles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic.

A Nebulized Gelatin Nanoparticle-Based CpG Formulation is Effective in Immunotherapy of Allergic Horses

ABSTRACTPurposeIn the recent years, nanotechnology has boosted the development of potential drug delivery systems and material engineering on nanoscale basis in order to increase drug specificity and reduce side effects. A potential delivery system for immunostimulating agents such as Cytosine-Phosphate-Guanine-Oligodeoxynucleotides (CpG-ODN) needs to be developed to maximize the efficacy of immunotherapy against hypersensitivity. In this study, an aerosol formulation of biodegradable, biocompatible and nontoxic gelatin nanoparticle-bound CpG-ODN 2216 was used to treat equine recurrent airway obstruction in a clinical study.MethodsBronchoalveolar lavage fluid was obtained from healthy and allergic horses to quantify Th1/Th2 cytokine levels before and after inhalation regimen. Full clinical examinations were performed to evaluate the therapeutic potential of this nebulized gelatin nanoparticle-based CpG formulation.ResultsMost remarkable was that regulatory anti-inflammatory and anti-allergic cytokine IL-10 expression was significantly triggered by five consecutive inhalations. Thorough assessment of clinical parameters following nanoparticle treatment indicated a partial remission of the allergic condition.ConclusionThus this study, for the first time, showed effectiveness of colloidal nanocarrier-mediated immunotherapy in food-producing animals with potential future applicability to other species including humans.