No abstract

PEGylated liposomes are attractive pharmaceutical nanocarriers; however, literature reports of ligand-targeted nanoparticles have not consistently shown successful results. Here, we employed a multifaceted synthetic strategy to prepare peptide-targeted liposomal nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities to evaluate the role of liposomal PEG coating, peptide EG-linker length, and peptide valency on cellular uptake in a systematic manner. We analyzed these parameters in two distinct disease models where the liposomes were functionalized with either HER2- or VLA-4-antagonistic peptides to target HER2-overexpressing breast cancer cells or VLA-4-overexpressing myeloma cells, respectively. When targeting peptides were tethered to nanoparticles with an EG45 (∼PEG2000) linker in a manner similar to a more traditional formulation, their cellular uptake was not enhanced compared to non-targeted versions regardless of the liposomal PEG coating used. Conversely, reduction of the liposomal PEG to PEG350 and the peptide linker to EG12 dramatically enhanced cellular uptake by ∼9 fold and ∼100 fold in the breast cancer and multiple myeloma cells, respectively. Uptake efficiency reached a maximum and a plateau with ∼2% peptide density in both disease models. Taken together, these results demonstrate the significance of using the right design elements such as the appropriate peptide EG-linker length in coordination with the appropriate liposomal PEG coating and optimal ligand density in efficient cellular uptake of liposomal nanoparticles.

The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of ''gel shifting'' has perplexed biochemists and biologists for decades. We identify a mechanism for ''gel shifting'' that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues~80-101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80-101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80-101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).

Ligand-targeted nanoparticles are emerging drug delivery vehicles for cancer therapy. Here, we demonstrate that the cellular uptake of peptide-targeted liposomes and micelles can be significantly enhanced by increasing the hydrophilicity of the targeting peptide sequence while simultaneously optimizing the EG peptide-linker length. Two distinct disease models were analyzed, as the nanoparticles were functionalized with either VLA-4 or HER2 antagonistic peptides to target multiple myeloma or breast cancer cells, respectively. Our results demonstrated that including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake of targeted nanoparticles up to ∼80-fold using an EG6 peptide-linker in liposomes and ∼27-fold using an EG18 peptide-linker in micelles for the VLA-4/multiple myeloma system. Similar trends were also observed in the HER2/breast cancer system with the EG18 peptide-linker resulting in optimal uptake for both types of nanoparticles. Cellular uptake efficiency of these formulations was also confirmed under fluidic conditions mimicking physiological systems. Taken together, these results demonstrated the significance of using the right design elements to improve the cellular uptake of nanoparticles.

In the continuing search for effective cancer treatments, we report the rational engineering of a multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-adhesion functionalities. Very late antigen-4 (VLA-4) mediated adhesion of multiple myeloma (MM) cells to bone marrow stroma confers MM cells with cell-adhesion-mediated drug resistance (CAM-DR). In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin (Dox) conjugates, simultaneously, to selectively target MM cells and to overcome CAM-DR. Dox was conjugated to the nanoparticles through an acid-sensitive hydrazone bond. VLA-4-antagonist peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities. The nanoparticles were efficiently internalized by MM cells and induced cytotoxicity. Mechanistic studies revealed that nanoparticles induced DNA double-strand breaks and apoptosis in MM cells. Importantly, multifunctional nanoparticles overcame CAM-DR, and were more efficacious than Dox when MM cells were cultured on fibronectin-coated plates. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors with ∼10 fold more drug accumulation and demonstrated dramatic tumor growth inhibition with a reduced overall systemic toxicity. Altogether, we demonstrate the disease driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM.