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. 2018 Dec 12;18(12):7784-7793.
doi: 10.1021/acs.nanolett.8b03558. Epub 2018 Nov 21.

Genetically Encoding Albumin Binding into Chemotherapeutic-loaded Polypeptide Nanoparticles Enhances Their Antitumor Efficacy

Parisa Yousefpour, Jonathan R McDaniel, Varun Prasad, Lucie Ahn, Xinghai Li, Rishi Subrahmanyan, Isaac Weitzhandler, Steven Suter  1 , Ashutosh Chilkoti
Affiliations

Genetically Encoding Albumin Binding into Chemotherapeutic-loaded Polypeptide Nanoparticles Enhances Their Antitumor Efficacy

Parisa Yousefpour et al. Nano Lett. .

Abstract

We report the development of drug-encapsulating nanoparticles that bind endogenous albumin upon intravenous injection and evaluate their in vivo performance in a murine as well as canine animal model. The gene encoding a protein-G derived albumin binding domain (ABD) was fused to that of a chimeric polypeptide (CP), and the ABD-CP fusion was recombinantly synthesized by bacterial expression of the gene. Doxorubicin (DOX) was conjugated to the C-terminus of the ABD-CP fusion, and conjugation of multiple copies of the drug to one end of the ABD-CP triggered its self-assembly into ∼100 nm diameter spherical micelles. ABD-decorated micelles exhibited submicromolar binding affinity for albumin and also preserved their spherical morphology in the presence of albumin. In a murine model, albumin-binding micelles exhibited dose-independent pharmacokinetics, whereas naked micelles exhibited dose-dependent pharmacokinetics. In addition, in a canine model, albumin binding micelles resulted in a 3-fold increase in plasma half-life and 6-fold increase in plasma exposure as defined by the area under the curve (AUC) of the drug, compared with naked micelles. Furthermore, in a murine colon carcinoma model, albumin-binding nanoparticles demonstrated lower uptake by the reticuloendothelial system (RES) system organs, the liver and spleen, that are the main target organs of toxicity for nanoparticulate delivery systems and higher uptake by the tumor than naked micelles. The increased uptake by s.c. C26 colon carcinoma tumors in mice translated to a wider therapeutic window of doses ranging from 20 to 60 mg equivalent of DOX per kg body weight (mg DOX equiv·kg-1 BW) for albumin-binding ABD-CP-DOX micelles, as compared to naked micelles that were only effective at their maximum tolerated dose of 40 mg DOX equiv·kg-1 BW.

Keywords: Micelles; cancer; canine model; drug delivery; elastin like polypeptide; endogenous albumin.

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Conflict of interest statement

Conflicts of Interest

A.C. serves as a scientific advisor and board member for PhaseBio Pharmaceuticals, Inc., which has licensed the ELP technology for drug delivery from Duke University. The other authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Design of the albumin binding nanoparticle drug carrier. (A) Structure of ABD-CP unimers and ABD-CP-DOX micelles. ABD was genetically fused to the N-terminus of a hydrophilic CP and was recombinantly synthesized in E. coli. DOX was conjugated via a pH-sensitive linker (EMCH) to the thiol groups of the cysteine rich segment at the C-terminus of the CP. The amphiphilic ABD-CP-DOX conjugates self-assembled into micelles with DOX molecules sequestered in the core, and ABDs in the corona that can bind endogenous albumin. (B-D) Characterization of ABD-CP unimers: (B) SDS-PAGE analysis of purified ABDN-CP, ABDH-CP and the CP (control). Successful purification of ABDN-CP and ABDH-CP by inverse transition cycling is confirmed by SDS-PAGE. The primary band corresponds to the molecular weight of CP (63.6 kDa), ABDN-CP (68.5 kDa), and ABDH-CP (68.6 kDa) and a second faint band at higher molecular weight indicates the presence of disulfide linked polypeptide dimers. (C) Native-PAGE analysis of the interaction of human serum albumin (HSA) and mouse serum albumin (MSA) with (ABDN/H-) CP. For the samples in lanes 5–7 and lanes 9–11, polypeptides were mixed at a molar ratio of ~1:1 with MSA and HSA, respectively. (D) Pharmacokinetics of CP, ABDN-CP and ABDH-CP. Polypeptides were labeled with Alexa488 and were administered via tail vein to BALB/c mice. Error bars represent standard error of the mean (n = 5 for ABDN-CP, 6 for CP and MSA, and 7 for ABDH-CP) and plasma concentrations were measured at intervals over 72 h. The data were fitted with a two-compartment model, from which the pharmacokinetic parameters were estimated, as shown in Table 1.
Figure 2.
Figure 2.
In vitro characterization of ABD-CP-DOX micelles: (A) Cryo-TEM micrographs of CP–DOX and ABDN-CP-DOX micelles (inset: magnified view); (I) ABDN-CP–DOX micelles (II) CP-DOX micelles. (B) In vitro drug release profile from CP-DOX and ABDN-CP-DOX micelles. The micelles released the loaded DOX under acidic conditions at pH 5.0 corresponding to the pH of late endosomes, but remained stable at pH 7.4 corresponding to vascular and extracellular space. Error bars represent standard error of the mean (n=3). (C) Native-PAGE analysis of interaction of human serum albumin (HSA) and mouse serum albumin (MSA) with ABDN-CP-DOX and ABDH-CP-DOX. For lanes 5–7 and lanes 9–11, polypeptide carriers were mixed at a molar ratio of 1:1 with MSA and HSA, respectively. (D) Cytotoxicity of ABDN-CP-DOX vs. CP-DOX and free DOX in C26 cells after 72 h incubation. The IC50 was 0.09 μM for free DOX, 0.30 μM for CP-DOX, and 0.31 μM for ABDN-CP-DOX. Error bars represent standard error of the mean (n=3). (E) Calorimetric titration of ABDN-CP-DOX and ABDH-CP-DOX micelles with MSA. The experiments were performed in phosphate buffered saline (pH 7.4) at 37 °C. The solid red line indicates the best-fit binding of the binding isotherm. MSA at 500 μM was titrated into the sample cell containing 50 μM of CP-DOX (I), ABDN-CP-DOX (II), and ABDH-CP-DOX (III). The integrated heat data were fit to a single site binding model and the binding stoichiometry (N) and dissociation constant (KD) were calculated, as shown in Table 3.
Figure 3.
Figure 3.
Pharmacokinetics of ABDN-CP-DOX micelles in dogs. Free DOX, CP-DOX micelles and ABDN-CP-DOX micelles were administered via the cephalic vein at the clinical dose of DOX in dogs reduced by 10% i.e. 27 mg DOX Equiv.m−2 body surface area (BSA) for dogs weighing greater than 10 kg and 0.9 mg DOX Equiv.kg−1 of body weight (BW) for dogs weighing less than 10 kg, and plasma DOX concentration was measured at intervals over 72 h. Error bars represent standard error of the mean (n=2 for ABDN-CP-DOX, and 3 for CP-DOX and DOX). The data were fit to a two-compartment model from which pharmacokinetic parameters were estimated as shown in Table 5.
Figure 4.
Figure 4.
In vivo characterization of ABD-CP-DOX micelles: (A) Pharmacokinetics of ABDN-CP-DOX micelles vs CP-DOX micelles. CP-DOX and ABDN-CP-DOX micelles were administered via tail vein to BALB/c mice at 10 and 20 mg DOX Equiv.kg−1 BW, and plasma DOX concentration was measured at intervals over 72 h. Error bars represent standard error of the mean (n=5–6). The data was fit to a two-compartment model from which pharmacokinetic parameters were estimated as shown in Table 5. (B) Biodistribution of ABDN-CP-DOX micelles, CP-DOX micelles and free DOX at 24 h post administration in the (I) tumor, (II) liver, and (III) spleen. C26 tumor cells were implanted subcutaneously and allowed to grow to approximately 75–100 mm3. Mice were treated with free DOX at 10 mg.kg−1 BW, and CP–DOX, and ABDN-CP-DOX both at 10 and 20 mg DOX Equiv. kg−1 BW. The DOX concentration was measured in tumor and normal tissues at 24 h post-administration. Error bars represent standard error of the mean (n=6–8). (C and D) Anti-tumor activity of (ABDN-)CP–DOX micelles. C26 tumor cells were implanted subcutaneously and allowed to grow to approximately 75–100 mm3. Mice were treated on day 0 with free DOX (10 mg.kg−1 BW), CP–DOX (20 mg DOX Equiv.kg−1 BW), and ABDN-CP-DOX (20 mg DOX Equiv.kg−1 BW). (C) Tumor volume up to day 60 (mean ± SEM.; n=7 for DOX, 8 for ABDN-CP-DOX, and 9 for CP-DOX). (D) Cumulative survival of mice up to day 60 (n=7 for DOX, 8 for ABDN-CP-DOX, and 9 for CP-DOX). * for P < 0.05, ** for P < 0.01, and *** for P < 0.001.
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References

    1. Choi HS; Liu W; Misra P; Tanaka E; Zimmer JP; Ipe BI; Bawendi MG; Frangioni JV Nat. Biotechnol. 2007, 25 (10), 1165–1170. - PMC - PubMed
    1. Cho K; Wang X; Nie S; Chen Z. (Georgia); Shin DM. Clin. Cancer Res. 2008, 14 (5), 1310 LP–1316. - PubMed
    1. Maeda H; Wu J; Sawa T; Matsumura Y; Hori KJ Control. Release 2000, 65 (1), 271–284. - PubMed
    1. Nie S Nanomedicine (Lond). 2010, 5 (4), 523–528. - PMC - PubMed
    1. Albanese A; Tang PS; Chan WCW Annu. Rev. Biomed. Eng. 2012, 14, 1–16. - PubMed

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