Controlled loading of albumin-drug conjugates ex vivo for enhanced drug delivery and antitumor efficacy.

To harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Here, model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drug would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native secondary structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that better recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to better maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy.

Electrostatic driven transport enhances penetration of positively charged peptide surfaces through tumor extracellular matrix.

Drug carriers achieve poor and heterogeneous distribution within solid tumors due to limited transport through the tumor extracellular matrix (ECM). The tumor ECM forms a net negatively charged network that interacts with and hinders the transport of molecules in part due to electrostatic interactions. Traditionally, the surfaces of drug delivery systems are passivated to minimize these interactions, but the mechanism of how charge interactions impact transport and penetration within the tumor microenvironment (TME) is not well understood. Here, we used T7 bacteriophage as a model biological nanoparticle to display peptides of different charges on its surface and elucidate how charge-based binding drives transport, uptake, and retention within tumor tissue. In contrast to current studies with neutrally charged surfaces, we discovered that a positively charged peptide displayed on T7 enhanced its penetration through a tumor-like ECM when compared to neutrally and negatively charged peptides. The positively charged peptide displayed on T7 facilitated weak and reversible binding with the TME to achieve Donnan partitioning and deep penetration into ex vivo tumor tissue. Additionally, the positively charged peptide-presenting T7 has a high number of intra-tissue binding sites in the TME (~4 µM) that enables almost 100% retention in the tumor tissue for up to 24 h. These results, coupled with transport studies of systematically mutated T7, show that electrostatic interactions can be responsible for uptake and retention of the positively charged peptide-presenting T7 within the net negatively charged TME. STATEMENT OF SIGNIFICANCE: The TME selectively hinders the transport of drugs and drug delivery systems due to their size, shape, and intermolecular interactions. Typically, the focus in drug delivery has been to develop delivery systems smaller than the pore size of the tumor ECM and/or develop inert surface coatings that have negligible interactions with the tumor ECM for diffusive transport. While there is an association of the surface charge of carriers with their transport through the tumor ECM, the mechanism of charge-driven transport is poorly understood. In this work, we elucidate the mechanism and find that interestingly, particles with a weakly positive surface charge interact with the net negatively charged tumor ECM to significantly improve their uptake, penetration, and retention in tumor tissue.

Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment.

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts as a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) affect their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and quantify their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and spatial ordering impact diffusive transport. The P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited ∼40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to functionalized control particles. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.