1,3-Bis(2-chloroethyl)-1-nitrosourea-loaded bovine serum albumin nanoparticles with dual magnetic resonance-fluorescence imaging for tracking of chemotherapeutic agents

Abstract

To date, knowing how to identify the location of chemotherapeutic agents in the human body after injection is still a challenge. Therefore, it is urgent to develop a drug delivery system with molecular imaging tracking ability to accurately understand the distribution, location, and concentration of a drug in living organisms. In this study, we developed bovine serum albumin (BSA)-based nanoparticles (NPs) with dual magnetic resonance (MR) and fluorescence imaging modalities (fluorescein isothiocyanate [FITC]-BSA-Gd/1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU] NPs) to deliver BCNU for inhibition of brain tumor cells (MBR 261-2). These BSA-based NPs are water dispersible, stable, and biocompatible as confirmed by XTT cell viability assay. In vitro phantoms and in vivo MR and fluorescence imaging experiments show that the developed FITC-BSA-Gd/BCNU NPs enable dual MR and fluorescence imaging for monitoring cellular uptake and distribution in tumors. The T1 relaxivity (R1) of FITC-BSA-Gd/BCNU NPs was 3.25 mM(-1) s(-1), which was similar to that of the commercial T1 contrast agent (R1 =3.36 mM(-1) s(-1)). The results indicate that this multifunctional drug delivery system has potential bioimaging tracking of chemotherapeutic agents ability in vitro and in vivo for cancer therapy.

Keywords: BSA nanoparticles; MR imaging; cancer therapy; drug tracking; fluorescence imaging.

Figure 1

BCNU-loading BSA NPs preparation and the morphology of NPs. Notes: (A) The procedure for preparation of BCNU-loaded BSA NPs with dual MR-fluorescence imaging. (B) SEM images of FITC-BSA NPs (top) and FITC-BSA-Gd/BCNU NPs (bottom). Abbreviations: BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BSA, bovine serum albumin; SEM, scanning electron microscope; FITC, fluorescein isothiocyanate; NPs, nanoparticles; DTPA-Gd, diethylenetriaminepentaacetic acid gadolinium(III) dihydrogen salt hydrate.

Figure 2

Characterizations of BCNU-encapsulated BSA NPs. Notes: (A) FTIR spectra of BSA NPs, BSA/BCNU NPs, and BCNU. (B) XPS surface survey spectra of BSA NPs and BSA/BCNU NPs. Abbreviations: FTIR, Fourier transform infrared spectroscopy; BSA, bovine serum albumin; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; NPs, nanoparticles; XPS, X-ray photoelectron spectroscopy.

Figure 3

Drug loading and release studies. Notes: (A) The encapsulation amount, encapsulation efficiency (EE), and loading efficiency (LE) of FITC-BSA-Gd/BCNU NPs. Values are the mean ± SD (n=6). (B) In vitro release profiles of FITC-BSA/BCNU and FITC-BSA-Gd/BCNU in PBS (pH 7.4) at 37°C. Abbreviations: FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; NPs, nanoparticles; PBS, phosphate-buffered saline; h, hours.

Figure 4

The characterizations of contrast ability for FITC-BSA-Gd/BCNU NPs. Notes: (A) Fluorescence phantom imaging of FITC-BSA-Gd/BCNU (a: 400 μg/mL; b: 200 μg/mL; c: 100 μg/mL; and d: 50 μg/mL). (B) Concentration dependence of FITC-BSA-Gd/BCNU fluorescence intensity. (C) MR T1 phantom imaging of commercial DTPA-Gd (a: 3.13 mM; b: 1.56 mM; c: 0.78 mM; d: 0.39 mM; e: 0.2 mM; and f: 0.1 mM DTPA-Gd). (D) MR T1 phantom imaging of FITC-BSA-Gd/BCNU (a: 3.13 mM; b: 1.56 mM; c: 0.78 mM; d: 0.39 mM; e: 0.2 mM; and f: 0.1 mM DTPA-Gd). (E) MR T1 relaxivity of DTPA-Gd (mM⁻¹ s⁻¹) is indicated by the slope. (F) MR T1 relaxivity of FITC-BSA-Gd/BCNU (mM⁻¹ s⁻¹) is indicated by the slope. Abbreviations: FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; NPs, nanoparticles; MR, magnetic resonance; DTPA-Gd, diethylenetriaminepentaacetic acid gadolinium(III) dihydrogen salt hydrate.

Figure 5

Typical MR contrast-enhanced T1 images of (B) before and (A) after FITC-BSA-Gd/BCNU NPs injection in normal animals. Abbreviations: MR, magnetic resonance; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; NPs, nanoparticles.

Figure 6

In vitro cell uptake and anti-proliferation studies. Notes: (A) Fluorescence image of MBR 261-2 cells after treatment with FITC-BSA-Gd NPs for 8 hours (green: FITC and blue: nuclei stained using Hoechst). (B) Cytotoxicity of MBR 261-2 cells after incubation with BSA NPs or BSA-Gd NPs for 36 hours. (C) DNA interstrand crosslinking in MBR 261-2 cells after treatment with different concentrations of BSA-Gd/BCNU NPs for 12 hours or 36 hours. (D) Viability of MBR 261-2 cells after treatment with different concentrations of BSA-Gd/BCNU NPs for 12 hours or 36 hours. Values are the mean ± SD (n=6). Abbreviations: FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; NPs, nanoparticles; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; h, hours.

Figure 7

In vivo anti-proliferation study. Notes: (A) Quantitative analysis of the effects of various treatments on tumor size. Values are mean ± SD (n=5). (B) Survival plots of animal experiments. FITC-BSA-Gd/BCNU NPs provided significant tumor inhibition and increase in animal survival relative to the other groups. Values are represented as mean ± SD (n=5). Animals were euthanized when the implanted tumor volume reached 3 cm³. Abbreviations: FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; NPs, nanoparticles; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea.