Monolithic precolumns as efficient tools for guiding the design of nanoparticulate drug-delivery formulations.

The development of nanomedicines for improved diagnosis and treatment of diseases is pushing current analytical methods to their limits. More efficient, quantitative high-throughput screening methods are needed to guide the optimization of promising nanoparticulate drug delivery formulations. In response to this need, we present herein a novel approach using monolithic separation media. The unique porosity of our capillary monolithic precolumns allows the direct injection and online removal of protamine-oligonucleotide nanoparticles ("proticles") without column clogging, thus avoiding the need for time-consuming off-line sample workup. Furthermore, ring-opening metathesis polymerization (ROMP)-derived monoliths show equivalent preconcentration efficiency for the target drug vasoactive intestinal peptide (VIP) as conventional particle-packed precolumns. The performance of the ROMP-derived monolithic precolumns was constant over at least 100 injections of crude proticle-containing and 300 injections of highly acidic samples. Applying a validated LC-MS/MS capillary monolithic column switching method, we demonstrate the rapid determination of both drug load and in vitro drug release kinetics of proticles within the critical first 2 h and investigate the stability of VIP-loaded proticles in aqueous storage medium intended for inhalation therapy.

Apolipoprotein A-I coating of protamine-oligonucleotide nanoparticles increases particle uptake and transcytosis in an in vitro model of the blood-brain barrier.

Drug delivery to the brain is severely restricted by formation of tight junctions between adjacent brain capillary endothelial cells (BCEC). In the present study we have evaluated the effects of protamine-oligonucleotide nanoparticles (proticles) on the functional properties of primary porcine BCEC and characterized uptake and transcytosis of proticles by these cells. Proticles had no adverse effects on BCEC properties relevant to blood-brain barrier (BBB) function. Transcytosis of (125)I-labeled proticles across polarized BCEC cultures occurred in a time- and concentration-dependent manner. As apolipoproteins were suggested to enhance cellular proticle uptake, proticle coating was performed with apoA-I, the major apolipoprotein component of high density lipoproteins. Adsorption of apoA-I on the surface of proticles resulted in significantly improved uptake and transcytosis properties as compared to uncoated proticles. ApoA-I coating enhanced proticle delivery to astrocytes in an in vitro model of the BBB almost twofold. Blocking of scavenger receptor class B, type I (the prime receptor for high density lipoprotein/apoA-I that is expressed on BCEC) reduced transcytosis of apoA-I-coated proticles to levels observed for uncoated proticles. Our data indicate that apoA-I-coating of proticles could be a feasible targeting technology to improve delivery across the BBB.

Adiponectin-coated nanoparticles for enhanced imaging of atherosclerotic plaques.

BACKGROUND: Atherosclerosis is a leading cause of mortality in the Western world, and plaque diagnosis is still a challenge in cardiovascular medicine. The main focus of this study was to make atherosclerotic plaques visible using targeted nanoparticles for improved imaging. Today various biomarkers are known to be involved in the pathophysiologic scenario of atherosclerotic plaques. One promising new candidate is the globular domain of the adipocytokine adiponectin (gAd), which was used as a targeting sequence in this study. METHODS: gAd was coupled to two different types of nanoparticles, namely protamine-oligonucleotide nanoparticles, known as proticles, and sterically stabilized liposomes. Both gAd-targeted nanoparticles were investigated for their potency to characterize critical scenarios within early and advanced atherosclerotic plaque lesions using an atherosclerotic mouse model. Aortic tissue from wild type and apolipoprotein E-deficient mice, both fed a high-fat diet, were stained with either fluorescent-labeled gAd or gAd-coupled nanoparticles. Ex vivo imaging was performed using confocal laser scanning microscopy. RESULTS: gAd-targeted sterically stabilized liposomes generated a strong signal by accumulating at the surface of atherosclerotic plaques, while gAd-targeted proticles became internalized and showed more spotted plaque staining. CONCLUSION: Our results offer a promising perspective for enhanced in vivo imaging using gAd-targeted nanoparticles. By means of nanoparticles, a higher payload of signal emitting molecules could be transported to atherosclerotic plaques. Additionally, the opportunity is opened up to visualize different regions in the plaque scenario, depending on the nature of the nanoparticle used.

VPAC receptor mediated tumor cell targeting by protamine based nanoparticles.

The receptors for vasoactive intestinal peptide (VIP), VPAC1-, VPAC2-, and PAC1-receptor are overexpressed by various tumor cells. VIP can target these receptors and transport conjugates into the cell. However, the use of VIP for tumor cell targeting is hampered by the peptides short half-lives due to enzymatic degradation. Because protamine-based nanoparticles (proticles) protect the peptide and serve as peptide depot, we explored the potential of proticles as carrier for VIP-conjugated molecules. The VIP-loaded proticles were stable as shown by Fluorescence Correlation Spectroscopy. With Confocal Laser Scanning Microscopy, we observed VIP-loaded proticles to specifically target the tumor cells. The cell binding triggered the substance release and conjugate internalization of VIP-Cy3 in vitro and ex vivo by human tumors. We observed VIP releasing proticle depots distributed in rat tissue and human tumors. Our findings warrant further studies to explore the proticles potential to enable peptide-mediated targeting for in vivo and clinical applications.

Depot formulation of vasoactive intestinal peptide by protamine-based biodegradable nanoparticles.

Drug delivery of protein and peptide-based drugs, which represent a growing and important therapeutic class, is hampered by these drugs' very short half-lives. High susceptibility towards enzymatic degradation necessitates frequent drug administration followed by poor adherence to therapy. Among these drugs is vasoactive intestinal peptide (VIP), a potent systemic and pulmonary vasodilator, which is a promising drug for the treatment of idiopathic pulmonary arterial hypertension (IPAH). Encapsulation of VIP into the nanoparticle matrix of biodegradable protamine-oligonucleotide nanoparticles (proticles) protects the peptide against rapid enzymatic degradation. Additionally, the nanoparticle matrix will be able to sustain drug release. Proticles consist of 18mer non-sense oligonucleotides and protamine, a polycationic arginine-rich peptide. VIP encapsulation occurs during self-assembly of the components. Within the present study, we evaluate nanoparticle size (hydrodynamic diameter) and zeta potential of VIP-loaded proticles as well as encapsulation efficiency and VIP release. Further, the pharmacological VIP response of "encapsulated VIP" is investigated using an ex vivo lung arterial model system. We found satisfying encapsulation efficiency (up to 80%), VIP release (77-87%), and an appropriate nanoparticle size (177-251 nm). Investigations on rat pulmonary arteries showed a modified VIP response of proticle-associated VIP. We noted differences in the profile of artery relaxation where VIP proticles lead to a 20-30% lower relaxation maximum than aqueous VIP solutions followed by prolonged vasodilatation. Our data indicate that proticles could be a feasible drug delivery system for a pulmonary VIP depot formulation.