Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

Abstract

Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.

Figure 1

Characterization of the split luciferase reporter. (A) A crystallographic model of FlucN-FRB and FKBP12-FlucC domains in the reporter system. In the absence of rapamycin, there is no luciferase activity. Upon treatment with rapamycin, the dimerization of FRB and FKBP12 will lead to the activation of the luciferase reporter. The Fluc, FRB and FKBP12 images were adapted from RCSB PDB (www.pdb.org) using ID# 1LUC, 1AUE and 2PPN. (B) Bioluminescence imaging analyzed the linearity of recovered luciferase activity with cell numbers after addition of free rapamycin (20 nM) for 6 h. (C) Linear regression analysis between luminescence intensity and cell numbers.

Figure 2

Synthesis and characterization of HSA Rapa nanoparticles. (A) Scheme of the HSA Rapa nanoparticles, which was synthesized by two phases emulsion method using high pressure homogenizer. (B) A representative picture of HSA Rapa nanoparticles obtained by transmission electron microscopy (bar = 200 nm). (C) Mean particle size of HSA Rapa nanoparticles incubated with PBS for 1 day or FBS for 6 days. (D) The polydispersity analysis of the HSA Rapa nanoparticles incubated with PBS or FBS for 6 days.

Figure 3

Concentration dependent and specificity of HSA Rapa induced luciferase recovery. (A) Bioluminescence imaging and (B) quantification of the recovered luciferase activity in cells on addition of different concentration of HSA Rapa (0.195 nM to 50 nM). The error bars represent the mean ± SD for triplicate experiments. (C) The cells pretreating with 0.78 nM HSA Rapa subsequently received different concentration of ascomycin and bioluminescence imaging were performed. (D) The quantification of bioluminescence imaging data. The error bars represent the mean ± SD for triplicate experiments.

Figure 4

Time-dependent cellular uptake of Cy5 labeled HSA Rapa nanoparticles. (A) The Cy5 labeled HSA Rapa nanoparticles were incubated with HN12#2 cells. 4 h later, the cells were investigated with confocal microscopy. The lower right picture is a magnification. (B) The nanoparticles were incubated with cells for different time periods (0.5, 1, 2, 4, 6, 24, 48, 72 h), then the cells were harvested and subjected to flow cytometer analysis. (C) The HN12#2 stable cells received the HSA Rapa for different time periods (0.5, 1, 2, 4, 6, 24, 48, 72 h). At the end of time point, bioluminescence imaging was used to investigate the luciferase activity. The graph is the qualification of the bioluminescence imaging results. The error bars represent the mean ± SD for triplicate experiments. (D) The cells were treated with HSA Rapa for different time periods (1, 6, 24 h), then western blot was performed with primary antibodies for P-AKT, AKT and β-actin.

Figure 5

In vivo imaging and pharmacokinetics analysis of HSA Rapa. Cy5 labeled HSA Rapa (10 mg/kg) was intravenously injected into the HN12#2 tumor bearing mice (n=6). At different time points (0.5, 1, 2, 4, 6, 24, 48, 72 h) after injection, (A) fluorescence imaging was performed following by (B) bioluminescence imaging. (C) The qualification of the fluorescence imaging results. (D) The qualification of the bioluminescence imaging results. (E) After imaging, the tumors and major organs as well as plasma at 6 h or 24 h time point were harvested and subjected to LC-MS analysis. (F) After intravenous injection of HSA Rapa into nude mice with a dosage of 10 mg/kg (n=6), the concentration of rapamycin in the blood over time was measured by LC-MS. Blood was drawn from the tail vein of mice at different time point, and the content of rapamycin was measured at each time point.

Figure 6

In vivo evaluation of the antitumor activity of HSA Rapa. HN12 tumor bearing mice were treated i.v. with HSA Rapa (10 mg/kg; n=10) or an equivalent volume of saline (n=10) for five times every other day. (A) An example of tumor regression in HSA Rapa or saline treated animals is showed. (B) A representative of three lesions dissected from HN12 xenograft 21 days after treatment with HSA Rapa or saline control. (C) tumor size from HN12 xenograft in both HSA Rapa and saline treated groups was measured every 2 days as indicated, and tumor volume was calculated as described in Materials and Methods. (D) The body weight of all animals was measured every 2 days for up to 3 weeks as indicated. (E) TUNEL assay in tumor tissues from xenograft model after 5 days of intravenous injection of HSA-Rapa or saline. DAPI was used to stain nuclei of tumor cells. Green: apoptotic cells; blue: DAPI.