Synthesis of polymer-lipid nanoparticles for image-guided delivery of dual modality therapy.

For advanced treatment of diseases such as cancer, multicomponent, multifunctional nanoparticles hold great promise. In the current study we report the synthesis of a complex nanoparticle (NP) system with dual drug loading as well as diagnostic properties. To that aim we present a methodology where chemically modified poly(lactic-co-glycolic) acid (PLGA) polymer is formulated into a polymer-lipid NP that contains a cytotoxic drug doxorubicin (DOX) in the polymeric core and an anti-angiogenic drug sorafenib (SRF) in the lipidic corona. The NP core also contains gold nanocrystals (AuNCs) for imaging purposes and cyclodextrin molecules to maximize the DOX encapsulation in the NP core. In addition, a near-infrared (NIR) Cy7 dye was incorporated in the coating. To fabricate the NP we used a microfluidics-based technique that offers unique NP synthesis conditions, which allowed for encapsulation and fine-tuning of optimal ratios of all the NP components. NP phantoms could be visualized with computed tomography (CT) and near-infrared (NIR) fluorescence imaging. We observed timed release of the encapsulated drugs, with fast release of the corona drug SRF and delayed release of a core drug DOX. In tumor bearing mice intravenously administered NPs were found to accumulate at the tumor site by fluorescence imaging.

Attaching the phage display-selected GLA peptide to liposomes: factors influencing target binding.

In our previous study, phage display selections were performed by in situ perfusion of a random peptide library through a mouse brain. This yielded two peptides (GLA and GYR) that showed significant binding to human brain endothelial cells (hCMEC/D3) when displayed on phage particles, but not to human umbilical vein endothelial cells (HUVECs). In the present study, these peptides were produced synthetically and coupled to liposomes to investigate the capacity of the peptides to act as ligands for targeting to hCMEC/D3 cells. Flow cytometry studies showed that these peptides when coupled to liposomes showed weak binding to the target brain endothelial cells. We hypothesized that the weak endothelial cell binding of the selected peptides when coupled to liposomes as compared to the binding of the peptides displayed on phage particles may be ascribed to: change of vehicle shape, change of peptide density, or change of peptide conformation. Peptide density on the liposomes influenced binding of the liposomes to the cells, however, this effect was minor. To study the influence of the peptide conformation, the GLA peptide was recombinantly produced fused to the N1-N2 domains of the phage p3 minor coat protein (p3-GLA) to mimic its conformation when displayed on phage particles. Binding of liposomes modified with either the GLA peptide or the p3-GLA protein to hCMEC/D3 cells was studied, and the p3-GLA-liposomes showed a higher binding to the cells compared to the GLA-liposomes. The experiments demonstrate that bringing the GLA peptide into the original phage protein environment restores and improves the peptide binding capacity and suggest that the GLA peptide, with some modifications, may be used as a brain-targeting ligand in the future.

Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain.

In many different studies nanocarriers modified with targeting ligands have been used to target to the brain. Many ligands have been successful, but it is difficult to compare results from different studies to determine which targeting ligand is the best. Therefore, we selected five targeting ligands (transferrin, RI7217, COG133, angiopep-2, and CRM197) and compared their ability to target liposomes to the brain in vitro and in vivo. In vitro, only CRM197-modified liposomes were able to bind to murine endothelial cells (bEnd.3). Both CRM197 and RI7217-modified liposomes associated with human endothelial cells (hCMEC/D3). In vivo, uptake of targeted liposomes was tested at 12h after iv injection. For some of the ligands, additional time points of 1 and 6h were tested. Only the RI7217 was able to significantly enhance brain uptake in vivo at all time points. Uptake in the brain capillaries was up to 10 times higher compared to untargeted liposomes, and uptake in the brain parenchyma was up to 4.3 times higher. Additionally, these results show that many targeting ligands that have been described for brain targeting, do not target to the brain in vivo when coupled to a liposomal delivery vehicle.

Preparation and characterization of liposomal formulations of neurotensin-degrading enzyme inhibitors.

Neurotensin-degrading enzyme (NTDE) inhibitors hold great potential for treating psychotic disorders. However, brain uptake of such compounds in vivo is generally low due to the presence of the blood-brain barrier. In this study, liposomal formulations of two NTDE inhibitors, named compound 1 (C1) and compound 2 (C2) were prepared. Association of these compounds with the liposomal bilayer, subsequent liposomal stability, and compound release in the presence of albumin was studied. Entrapment of the compounds in the liposomal bilayer showed the solubilizing properties of the liposomes. Size and polydispersity index of the compound-entrapped liposomes did not change over 1 month, showing colloidal stability of the liposomal drug formulations. The amount of compounds associated with the liposomes decreased within one day. After this, the association remained stable at 4°C. For C1, association remained stable at 37°C in HEPES buffered saline, and the compound was gradually released in the presence of bovine serum albumin. For C2, the release was rapid in both HBS and BSA at 37°C. In conclusion, the formulation of NTDE inhibitors C1 and C2 in liposomes has been demonstrated and holds promise to deliver NTDE inhibitors in vivo.