Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection - PubMed (original) (raw)

Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

J Andrew MacKay et al. Nat Mater. 2009 Dec.

Abstract

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

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Figures

Figure 1. Structure of CP-Dox conjugate

a) High molecular weight ELPs were synthesized by genetically encoded biosynthesis and conjugated to Dox at Cys residues via a heterobifunctional linker. b) The hydrophobic-drug block triggers self-assembly of chimeric polypeptide (CP) nanoparticles with a drug ( formula image ) rich core surrounded by a hydrophilic polypeptide corona.

Figure 2. Characterization of CP-Dox nanoparticles

a) Freeze fracture transmission electron microscopy image of CP-Dox nanoparticles (bar = 200 nm). b) Distribution of hydrodynamic radii for CP and CP-Dox nanoparticles at 25 μM in PBS at 37 °C by dynamic light scattering (DLS). c) CP-Dox nanoparticles have a stable hydrodynamic radius over time in PBS at pH 7.4, PBS with 0.1 mM BSA, and in buffer at pH 5.0; however, the distribution of hydrodynamic radii (error bars) increases over time at low pH. (Mean ± SD). d) The kinetics of particle diameter broadening are in agreement with the pH dependent release of Dox from CP-Dox nanoparticles as determined using size exclusion chromatography at pH 7.4 and 5.0. The fit line (solid) and 95% confidence interval lines (dash) are provided.

Figure. 3. Internalization of CP-Dox and delivery of drug to the nucleus

Confocal laser scanning microscopy images through C26 cells (blue) that show cellular uptake for free Dox, free CP-Oregon Green, and CP-Dox at 5 mins, 30 mins, and 24 hrs. a-c) Free Dox (red) rapidly localizes to the nucleus at all time points. d-f) CP-Oregon Green (green) internalizes into the cytoplasm with intense punctate staining after 30 mins. g-i) CP-Dox (red) nanoparticles produce intense nuclear staining with drug after 30 mins. (bar = 20 μm).

Figure 4. Plasma pharmacokinetics and tissue biodistribution

a) Plasma Dox concentrations as a function of time post-injection. A two-compartment model was fit to the plasma Dox concentration, which yielded a terminal half-life of 9.3 ± 2.1 hrs (CI 95%) for CP-Dox. The concentration of free Dox has been confirmed at two time points experimentally; furthermore, the observed concentrations correlate with the prediction of a pharmacokinetic model of free Dox in mice (Supplementary Table 3). (Mean ± 95% CI; n=5-8). b-c) The Dox concentration in b) tumor tissue and c) heart tissue at 2 and 24 hours post-administration was determined. *indicates p<0.0005 (ANOVA, Tukey HSD). (Mean ± SD; n=4 to 6).

Figure 5. Anti-tumor activity of CP-Dox nanoparticles

a-b)Tumors cells (C26) were implanted subcutaneously on day zero. Mice were treated on day 8 (↑) at MTD with PBS (n=10), free Dox (5 mg kg-1 BW; n=10), and CP-Dox (20 mg Dox Equiv kg-1 BW; n=9). a) Tumor volume up to day 30 (Mean ± SD; n=6 to 10). *indicates p= 0.03, 0.00002 for CP-Dox vs. Dox and PBS (day 15) respectively (Mann Whitney). b) Cumulative survival of mice **indicates p=0.0001, 0.00004 for CP-Dox vs. Dox and PBS respectively (Kaplan Meier). c) Cell viability for CP-Dox (n=4; IC50 =1.8 μM) and free Dox (n=14; IC50 =0.41 μM) in C26 cells. (Mean ± 95% CI).

Figure 6. Genomic profiles of CP-Dox and free Dox treated tumor tissues

a) Volcano plot of gene expression contrasting CP-Dox treated mice vs. Dox treated mice. Expression level is denoted by a square colored to reflect the P-value. The horizontal green line indicates the cutoff of P-value (P<0.00012), which corrects for multiple comparisons. The vertical green lines indicate the cutoff for significant fold change (>1.5 fold). In CP-Dox treated mice, 12 genes were significantly down-regulated, and 2 genes were up-regulated in contrast to Dox treated mice. b) A hierarchical clustering analysis was performed based on the expression pattern. The genes were linked together according to their expression patterns (dendrogram on left). Individual mice were also clustered (dendrogram on top). For all mice, the clustering results match their treatment: CP-Dox (red bar); Dox (blue bar); and PBS (green bar). The gene expression intensities were standardized between -2.0 (blue) and 2.0 (red). LOC*7155 is an abbreviation of an untitled gene LOC100047155.

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Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection - PubMed (original) (raw)