Targeted albumin-based nanoparticles for delivery of amphipathic drugs

Abstract

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)β(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)β(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.

Figure 1

The preparation of RPA-Dox.

Figure 2

The reaction progress of RPA-Dox. The intermediates and final products were analyzed by FPLC-QELS. Dotted line: molar mass. Solid line: Rayleigh ratio.

Figure 3

TEM image of RPA-Dox (170,000x). The sample is counterstained by uranyl acetate. The average particle size is around 30 nm.

Figure 4

Stability of RPA-Dox. RPA-Dox sample were analyzed by FPLC-QELS in 3 days (A) and 5 weeks (B) postsynthesis.

Figure 5

Doxorubicin release kinetics. (A) RPA-DOX and MPA-DOX were incubated with PBS only or PBS with 10% serum at 37 °C. (B) RPA-DOX and MPA-DOX were incubated at in PBS with thiols from 10 mM glutathione and 100 uM cysteine or PBS only at 37 °C.

Figure 6

Cellular uptake of doxorubicin formulation. In (A), M21 cells were incubated 4 hours in Opti-MEM medium containing medium only, 1 micromolar doxorubicin, RPA-DOX and MPA-DOX with 1 micromolar doxorubicin, and RPA-DOX with 1 micromolar doxorubicin with pre-incubation of 100 micromolar of cyclic(RGDfV), FL2 channel of flow cytometry was set to collect doxorubicin fluorescence. Figure (B) shows the dynamic cellular uptake of doxorubicin formulation. (mean +/− SD, from about 5000 cells).

Figure 7

Confocal microscopy of doxorubicin uptake. Cells were incubated for 4 hours with 2 micromolar doxorubicin in free form (A), RPA-DOX (B) or MPA-DOX (C), or further cultured in drug-free medium for 12 hours, see (D), (E) and (F), respectively. Arrows point to the doxorubicin subcellular distributions.

Figure 8

Cytotoxicity of RPA-DOX, MPA-DOX and free doxorubicin on M21+ cells. The cytotoxicity of MPA and RPA equivalent to RPA-DOX and MPA-DOX containing 0.5 uM to 50 uM of doxorubicin was evaluated in a parallel assay, see inset.