Multistage Nanovectors Enhance the Delivery of Free and Encapsulated Drugs

Abstract

Nanoparticles have considerable potential for cancer imaging and therapy due to their small size and prolonged circulation. However, biological barriers can impede the delivery of a sufficient dose of a drug to the target site, thereby also resulting in the accumulation of toxic compounds within healthy tissues, and systemic toxicity. Multistage nanovectors (MSV) preferentially accumulate on inflamed endothelium, and can thus serve as carriers for drugs and nanoparticles. Herein, we describe the loading of free (i.e., melittin) and nano-encapsulated (i.e., doxorubicin-loaded micelles) drugs into MSV, and report the impact of surface charge and pore size on drug loading. For both drug formulations, negatively charged MSV (i.e., oxidized) with larger pores were shown to retain higher concentrations of payloads compared to positively charged (i.e., APTES-modified) MSV with small pores. Treatment of human umbilical vein endothelial cells (HUVEC) with melittin-loaded MSV (MEL@MSV) resulted in an 80% reduction in cell viability after 3 days. Furthermore, MEL@MSV conjugated with antivascular endothelial growth factor receptor 2 (VEGFR2) antibodies displayed preferential targeting and delivery of MEL to activated HUVEC expressing VEGFR2. Treatment of HUVEC and MCF7 cells with doxorubicin-loaded micelles (DOXNP@MSV) resulted in a 23% and 47% reduction in cell viability, respectively. Taken together, these results demonstrate increased loading of a payload in oxidized, large pore MSV, and effective delivery of free and nano-encapsulated drugs to endothelial and cancer cells.

Fig. (1). Free melittin loading into MSV

A. Loading of MEL into oxidized and (B) APTES SP and LP MSV comparing the drug content (μg of MEL/μg of MSV). (** = p < 0.01, *** = p < 0.001) C. Fluorescent images of MEL loaded into oxidized and (D) APTES LP MSV, where white outline encircles the MSV (scale bar: 1 μm).

Fig. (2). Cellular proliferation of HUVEC upon treatment with MEL@MSV

A. MTT assay to evaluate the cell viability of HUVEC treated with oxidized SP and (B) LP MEL@MSV at LOW, MED, and HIGH concentrations corresponding to HUVEC to MSV ratios of 1:5, 1:10, and 1:20, respectively. C. HUVEC treated with APTES SP and (D) LP MEL@MSV at low, medium, and high. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Fig. (3). Targeted delivery of MEL@MSV using a-VEGFR2 on activated HUVEC

A. Fluorescence of MSV measured by means of flow cytometry comparing oxidized, APTES, anti-IgG, and FITC anti-VEGFR2. B. Fluorescent microscopy images of anti-IgG MSV (upper, green) and anti-VEGFR2 MSV (bottom, green) loaded with MEL (red). C. Flow cytometry analysis of simultaneous MEL loading (red, left axis) and conserved antibody labeling (green, right axis) for both anti-IgG and anti-VEGFR2 MSV. D. Fluorescent microscopy images of control (top) and activated (bottom) HUVEC following treatment for 19 hours with untargeted (anti-IgG) and targeted (anti-VEGFR2) MSV. MSV are in green, microtubules (f-actin) in red, and white arrows indicate location of MSV. (scale bar, 25 μm). E. Docking (orange arrows) and internalization (white arrows) of targeted MSV on activated HUVEC. MSV are in green, microtubules (f-actin) in red. (scale bar, 25 μm). F. Cellular proliferation of activated HUVEC following treatment with untargeted and targeted MEL@MSV after three days comparing LOW (1:5), MED (1:10), and HIGH (1:20) ratios of MSV. (** = p < 0.01, *** = p < 0.001).

Fig. (4). DOXNP loading in MSV

A. Dynamic light scattering size analysis of DOXNP showing a mean diameter of 13 nm with sizes ranging from 10 to 20 nm. B. Transmission electron micrograph of DOXNP micelles showing nanoparticles of uniform size and shape. (scale bar, 100 nm). C. Quantitative analysis of cationic DOXNP loading into SP and (D) XLP comparing oxidized and APTES MSV. E-G. Fluorescent images of DOXNP@MSV showing (E) bright field, (F) DOX, and (G) merged channels. (scale bar, 1 μm) H. Release of DOXNP from MSV at 1 and 2 hours for SP and XLP.

Fig. (5). Assessment of DOXNP@MSV toxicity on endothelial and cancer cells

A. Cell viability was measured using an MTT assay on HUVEC and (B) MCF-7 cancer cells after treatment with LOW and HIGH concentrations of DOX, DOXNP, and DOXNP@MSV and monitored for three days. C. Analysis of HUVEC and (D) MCF7 comparing day 2 and day 3 treatments normalized to day 1 treatments of the same group. (** = p < 0.01, *** = p < 0.001).

Fig. (6). Internalization of MSV on HUVEC and MCF7

Fluorescent images depicting nucleus (blue), MSV (green), microtubules (red), and merge of MSV internalized in HUVEC (A) and (B) MCF-7 (B) after 4 hours. (scale bar, 25 μm).