Multifunctional albumin nanoparticles as combination drug carriers for intra-tumoral chemotherapy

Abstract

Current cancer therapies are challenged by weakly soluble drugs and by drug combinations that exhibit non-uniform biodistribution and poor bioavailability. In this study, we have presented a new platform of advanced healthcare materials based on albumin nanoparticles (ANPs) engineered as tumor penetrating, delivery vehicles of combinatorially applied factors to solid tumors. These materials were designed to overcome three sequential key barriers: tissue level transport across solid tumor matrix; uptake kinetics into individual cancer cells; therapeutic resistance to single chemotherapeutic drugs. The ANPs were designed to penetrate deeper into solid tumor matrices using collagenase decoration and evaluated using a three-dimensional multicellular melanoma tumor spheroid model. Collagenase modified ANPs exhibited 1-2 orders of magnitude greater tumor penetration than unmodified ANPs into the spheroid mass after 96 hours, and showed preferential uptake into individual cancer cells for smaller sized ANPs (<100 nm). For enhanced efficacy, collagenase coated ANPs were modified with two therapeutic agents, curcumin and riluzole, with complementary mechanisms of action for combined cell cycle arrest and apoptosis in melanoma. The collagenase coated, drug loaded nanoparticles induced significantly more cell death within 3-D tumor models than the unmodified, dual drug loaded ANP particles and the kinetics of cytotoxicity was further influenced by the ANP size. Thus, multifunctional nanoparticles can be imbued with complementary size and protease activity features that allow them to penetrate solid tumors and deliver combinatorial therapeutic payload with enhanced cancer cytotoxicity but minimal collateral damage to healthy primary cells.

Keywords: albumin nanoparticles; collagenase; drug delivery; tumor penetration.

Figure 1. Schematic summarizing the overall design of albumin nanoparticles as advanced healthcare materials for enhanced “intratumoral” chemotherapy

(A) Albumin nanoparticles (ANPs) fabricated by controlled coacervation can be engineered to overcome multiple barriers to chemotherapy. The synthesis process was modified to display an extracellular matrix protease (cANP). Drugs can be adsorbed onto the surface after fabrication to further enhance chemotherapeutic efficacy. (B) On a multi-cellular tumor level, collagenase coating of ANPs allows for rapid penetration through the dense extracellular matrix to allow for increased intratumoral drug availability. (C) ANP size can be modulated to engineer size-dependent uptake by individual cancer cells, thus triggering controlled intracellular drug release. Smaller ANPs are hypothesized to exhibit enhanced cellular uptake over the larger ANPs. (D) A novel dual-drug regimen of ANP-based presentation and release of complementary melanoma drugs, curcumin and riluzole, was implemented. These drugs would act on distinct but convergent pathways that promote apoptosis and inhibit cellular proliferation, thus enhancing cancer cell chemotherapy intratumorally.

Figure 2. Multimodular Design of Albumin Nanoparticles as Monodisperse Substrates for Size Modulation, Intracellular Uptake, Protease Modification, and Dual Drug Binding

Scanning electron microscopy (SEM) images of (A) small and (B) large ANPs reveal monodisperse, spherical nanoparticles. Collagenase modification altered the morphology of the (C) small and (D) large ANPs. This change in morphology was more evident after both drugs were loaded on the (E) small and (F) large collagenase ANPs. (G) ANPs were synthesized and characterized at two different sizes by modifying the concentration of NaCl used during synthesis. Both sizes exhibit narrow size distributions and negative zeta potentials in PBS. Hydrodynamic diameter, polydispersity and loading efficiency of ANP formulations with and without collagenase incorporation were characterized. (H) Large Collagenase ANPs carried 2 fold greater levels of collagenase compared to smaller C-ANPs. One unit of collagenolytic activity was defined as the cleavage of 1 mg of collagen substrate per minute. (I) Cellular uptake of collagenase conjugated and unmodified ANPs in C8161+ cell line was analyzed by flow cytometry for up to 24 h. The decrease in size and collagenase conjugation accelerates the uptake of nanoparticles (I). ± Values: standard deviation (n=3); Error bars represent standard deviation (SD) for n=3. *** p<0.005 (t test).

Figure 3. Protease Modified ANPs Exhibit Increased Transport Into Melanoma Tumor Spheroid Model

Confocal microscopy images of multicellular spheroids stained with Hoechst (blue) treated with various nanoparticles (green) for 96 hours (A–D): (A) larger ANPs, (B) small ANPs, (C) large collagenase conjugated ANPs, and (D) small collagenase conjugated ANPs. Images indicate increased penetration of both collagenase modified ANPs compared to ANPs. (E) Large C-ANPs penetrate further into the spheroids than the small ones, while the smaller C-ANPs displayed deeper penetration by 96 hours. Error bars represent standard deviation for n=5. ** p<0.02; *** p<0.005 (t test).

Figure 4. Dual Drug Combination of Curcumin and Riluzole Elicits Increased Melanoma Cytotoxicity

In vitro cytotoxicity of riluzole, curcumin, and dual drugs on (A) C8161+ melanoma cells and (B) AR7119 melanocytes. (C) Apoptotic effects of 40 μM curcumin, 10 μM riluzole, and dual drugs (10 μM riluzole + 40 μM curcumin) on C8161+ melanoma cells after 48 hours of incubation were obtained by flow cytometry. The dual drug treatment resulted in more apoptotic and dividing (G2) cells than either single drug therapy. Error bars represent standard deviation for n=3.

Figure 5. Multimodular ANPs exhibit enhanced tumor cytotoxicity in a size, protease-modification, and dual drug-display dependent manner

A dual drug (DD) combination of 10 μM riluzole and 40 μM curcumin bound ANPs nanoformulations were tested within 2-D monolayer cultures and showed enhanced cytotoxicity of (A) C8161+ human melanoma cells compared to (B) AR7119 healthy human melanocytes. The cANPs showed no inherent cytotoxicity in either cell line. (C) The multimodular ANPs showed unique effects in the 3-D multicellular tumor spheroid model as a function of time and ANP size, collagenase modification, and dual drug treatment. ANP-based dual drug delivery showed enhanced efficacy over free drugs. The collagenase modification enhanced efficacy for both large and smaller ANPs but particularly for smaller ANPs. The smaller cANPs showed the most pronounced time dependent therapeutic effect. Overall, the ANPs can be designed as versatile chemotherapy tools to modulate matrix transport rates as well as intracellular uptake rates and effect short-term and longer-term tumor killing. Error bars represent standard deviation for n=3. * p<0.05; ** p<0.02 (t test).