Optimizing design parameters of a peptide targeted liposomal nanoparticle in an in vivo multiple myeloma disease model after initial evaluation in vitro.

Despite ligand-targeted liposomes long garnering interest as drug delivery vehicles for cancer therapeutics, inconsistency in successful outcomes have hindered their translation into the clinic. This is in part due to discrepancies between in vitro design evaluations and final in vivo outcomes. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles of high purity, reproducibility, and with precisely controlled quantity of functionalities, we systematically evaluated the individual roles that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on cancer cell uptake and tumor targeting both in vitro and in vivo, and how the results correlated and contrasted. These parameters were analyzed using a VLA-4-targeted liposome system in a multiple myeloma mouse xenograft model to evaluate in vivo biodistribution and tumor cell uptake. The results showed that using in vitro models to optimize targeted-nanoparticles for maximum cellular uptake was helpful in narrowing down the particle characteristics. However, in vitro optimization fell short of achieving enhanced results in animal models, rather had negative consequences for in vivo targeting. This outcome is not surprising considering that the receptor being targeted is also present on healthy lymphocytes and increasing targeting peptide valency on particle surfaces results in an increase in non-selective, off-target binding to healthy cells. Hence, further optimization using in vivo models was absolutely necessary, through which we were able to increase the uptake of peptide-targeted liposomes by cancerous cells overexpressing VLA-4 to 15-fold over that of non-targeted liposomes in vivo. The results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints including both in vitro and in vivo in determining the therapeutic potential of peptide-targeted liposomes.

Dual Carfilzomib and Doxorubicin-Loaded Liposomal Nanoparticles for Synergistic Efficacy in Multiple Myeloma.

Here, we report the synthesis and evaluation of dual drug-loaded nanoparticles as an effective means to deliver carfilzomib and doxorubicin to multiple myeloma tumor cells at their optimal synergistic ratio. First, various molar ratios of carfilzomib to doxorubicin were screened against multiple myeloma cell lines to determine the molar ratio that elicited the greatest synergy using the Chou-Talalay method. The therapeutic agents were then incorporated into liposomes at the optimal synergistic ratio of 1:1 to yield dual drug-loaded nanoparticles with a narrow size range of 115 nm and high reproducibility. Our results demonstrated that the dual drug-loaded liposomes exhibited synergy in vitro and were more efficacious in inhibiting tumor growth in vivo than a combination of free drugs, while at the same time reducing systemic toxicity. Taken together, this study presents the synthesis and preclinical evaluation of dual drug-loaded liposomes containing carfilzomib and doxorubicin for enhanced therapeutic efficacy to improve patient outcome in multiple myeloma. Mol Cancer Ther; 15(7); 1452-9. ©2016 AACR.

Liposomal carfilzomib nanoparticles effectively target multiple myeloma cells and demonstrate enhanced efficacy in vivo.

Carfilzomib, a recently FDA-approved proteasome inhibitor, has remarkable anti-myeloma (MM) activity. However, its effectiveness is limited by associated severe side-effects, short circulation half-life, and limited solubility. Here, we report the engineering of liposomal carfilzomib nanoparticles to overcome these problems and enhance the therapeutic efficacy of carfilzomib by increasing tumoral drug accumulation while decreasing systemic toxicity. In our design, carfilzomib was loaded into the bilayer of liposomes to yield stable and reproducible liposomal nanoparticles. Liposomal carfilzomib nanoparticles were efficiently taken up by MM cells, demonstrated proteasome inhibition, induced apoptosis, and exhibited enhanced cytotoxicity against MM cells. In vivo, liposomal carfilzomib demonstrated significant tumor growth inhibition and dramatically reduced overall systemic toxicity compared to free carfilzomib. Finally, liposomal carfilzomib demonstrated enhanced synergy in combination with doxorubicin. Taken together, this study establishes the successful synthesis of liposomal carfilzomib nanoparticles that demonstrates improved therapeutic index and the potential to improve patient outcome in MM.

Liposomal bortezomib nanoparticles via boronic ester prodrug formulation for improved therapeutic efficacy in vivo.

In this study, we describe the development of liposomal bortezomib nanoparticles, which was accomplished by synthesizing bortezomib prodrugs with reversible boronic ester bonds and then incorporating the resulting prodrugs into the nanoparticles via surface conjugation. Initially, several prodrug candidates were screened based upon boronic ester stability using isobutylboronic acid as a model boronic acid compound. The two most stable candidates were then selected to create surface conjugated bortezomib prodrugs on the liposomes. Our strategy yielded stable liposomal bortezomib nanoparticles with a narrow size range of 100 nm and with high reproducibility. These liposomal bortezomib nanoparticles demonstrated significant proteasome inhibition and cytotoxicity against multiple myeloma cell lines in vitro and remarkable tumor growth inhibition with reduced systemic toxicity compared to free bortezomib in vivo. Taken together, this study demonstrates the incorporation of bortezomib into liposomal nanoparticles via reversible boronic ester bond formation to enhance the therapeutic index for improved patient outcome.

Ligand-targeted liposome design: challenges and fundamental considerations.

Nanomedicine, particularly liposomal drug delivery, has expanded considerably over the past few decades, and several liposomal drugs are already providing improved clinical outcomes. Liposomes have now progressed beyond simple, inert drug carriers and can be designed to be highly responsive in vivo, with active targeting, increased stealth, and controlled drug-release properties. Ligand-targeted liposomes (LTLs) have the potential to revolutionize the treatment of cancer. However, these highly engineered liposomes generate new problems, such as accelerated clearance from circulation, compromised targeting owing to non-specific serum protein binding, and hindered tumor penetration. This article highlights recent challenges facing LTL strategies and describes the advanced design elements used to circumvent them.

Enhanced cellular uptake of peptide-targeted nanoparticles through increased peptide hydrophilicity and optimized ethylene glycol peptide-linker length.

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.

A systematic analysis of peptide linker length and liposomal polyethylene glycol coating on cellular uptake of peptide-targeted liposomes.

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.