Development and evaluation of a new modular nanotransporter for drug delivery into nuclei of pathological cells expressing folate receptors

Abstract

Purpose: Modular nanotransporters (MNTs) are artificial multifunctional systems designed to facilitate receptor-specific transport from the cell surface into the cell nucleus through inclusion of polypeptide domains for accomplishing receptor binding and internalization, as well as sequential endosomal escape and nuclear translocation. The objective of this study was to develop a new MNT targeted at folate receptors (FRs) for precise delivery of therapeutic cargo to the nuclei of FR-positive cells and to evaluate its potential, particularly for delivery of therapeutic agents (eg, the Auger electron emitter ¹¹¹In) into the nuclei of target cancer cells.

Methods: A FR-targeted MNT was developed by site-specific derivatization of ligand-free MNT with maleimide-polyethylene glycol-folic acid. The ability of FR-targeted MNT to accumulate in target FR-expressing cells was evaluated using flow cytometry, and intracellular localization of this MNT was assessed using confocal laser scanning microscopy of cells. The cytotoxicity of the ¹¹¹In-labeled FR-targeted MNT was evaluated on HeLa and U87MG cancer cell lines expressing FR. In vivo micro-single-photon emission computed tomography/CT imaging and antitumor efficacy studies were performed with intratumoral injection of ¹¹¹In-labeled FR-targeted MNT in HeLa xenograft-bearing mice.

Results: The resulting FR-targeted MNT accumulated in FR-positive HeLa cancer cell lines specifically and demonstrated the ability to reach its target destination - the cell nuclei. ¹¹¹In-labeled FR-targeted MNT demonstrated efficient and specific FR-positive cancer cell eradication. A HeLa xenograft in vivo model revealed prolonged retention of ¹¹¹In delivered by FR-targeted MNT and significant tumor growth delay (up to 80% growth inhibition).

Conclusion: The FR-targeted MNT met expectations of its ability to deliver active cargo into the nuclei of target FR-positive cells efficiently and specifically. As a result of this finding the new FR-targeted MNT approach warrants broad evaluation.

Keywords: cancer; folic acid; indium-111; nuclear delivery; radionuclide therapy.

Figure 1

Schematic presentation of the targeting delivery approach using targeted intranuclear delivery of cytotoxic drug (here, Auger electron emitter ¹¹¹In) by folate receptor-targeted MNT. Notes: (A) Auger electron range and the efficiency of Auger electron emitters located inside and outside the cell. (B) MNT module functions. Abbreviations: DTox, diphtheria toxin translocation domain; FA, folic acid; MNTs, modular nanotransporters; NLS, nuclear localization sequence; PEG, polyethylene glycol.

Figure 1

Schematic presentation of the targeting delivery approach using targeted intranuclear delivery of cytotoxic drug (here, Auger electron emitter ¹¹¹In) by folate receptor-targeted MNT. Notes: (A) Auger electron range and the efficiency of Auger electron emitters located inside and outside the cell. (B) MNT module functions. Abbreviations: DTox, diphtheria toxin translocation domain; FA, folic acid; MNTs, modular nanotransporters; NLS, nuclear localization sequence; PEG, polyethylene glycol.

Figure 2

Development of FR-targeted MNT (MNT-PEG-FA). Notes: (A) Schematic presentation of MNT derivatization with FA moiety with a SDS-PAGE gel under nonreducing conditions of MNT and the resulting FR-targeted MNT-PEG-FA. (B) Schematic presentation of MNT-PEG-FA labeling with Auger electron emitter ¹¹¹In with autoradiography of SDS-PAGE gels of ¹¹¹In-MNT-PEG-FA. Labels on the left indicate the bands from ColorBurst™ Electrophoresis Markers. Abbreviations: DTox, diphtheria toxin translocation domain; FA, folic acid; HMP, hemoglobin-like protein of E. coli; MNTs, modular nanotransporters; NLS, nuclear localization sequence; PEG, polyethylene glycol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3

Intracellular accumulation of MNT-PEG-FA labeled with Alexa Fluor 647 and measured by flow cytometry. Notes: (A) Flow cytometry histograms of HeLa (FR-positive) or A549 (FR-negative) cells incubated with either 50 nM MNT-PEG-FA-Alexa647 alone or in the presence of 1 mM FA or with control non-targeted MNT-PEG-Alexa647, lacking FA alone or in the presence 1 mM FA for 48 h. (B, C) Mean fluorescence intensities for HeLa or control A549 cells incubated with either 50 nM MNT-PEG-FA-Alexa647 alone or in the presence of 1 mM FA or with control non-targeted MNT-PEG-Alexa647, lacking FA alone or in the presence 1 mM FA for 24 h (B) or 48 h (C). Untreated cells served as autofluorescence controls. Error bars represent standard errors of mean (n=3–6). Abbreviations: FA, folic acid; MNTs, modular nanotransporters; PEG, polyethylene glycol.

Figure 4

Intracellular distribution of MNT-PEG-FA labeled with Alexa Fluor 647 and visualized by confocal laser scanning microscopy. Notes: Confocal laser scanning microscopy images (slices made across the nuclei) of HeLa (FR-positive) or A549 (FR-negative) cells incubated with either 50 nM MNT-PEG-FA-Alexa647 alone (A, A′, G, G′) or in the presence 1 mM FA (B, B′, H, H′) or with control non-targeted MNT-PEG-Alexa647, lacking FA alone or in the presence 1mM FA for 48 h. Untreated cells served autofluorescence controls; Alexa Fluor 647 fluorescence corresponding to MNT-PEG-FA or MNT-PEG is in red pseudo-color (A–C, D–F, G–I), and overlay of Alexa Fluor 647 fluorescence and DAPI staining for nuclei is presented in blue pseudo-color (A′–C′, D′–F′, G′–I′). Bar =10 µm. Plan-Apochromat ×63/1.4 Oil DIC lens, zoom 2×. Abbreviations: FA, folic acid; MNTs, modular nanotransporters; PEG, polyethylene glycol.

Figure 5

Cytotoxic efficiency of ¹¹¹In delivered by FR-targeted MNTs. Notes: (A) Cytotoxicity of ¹¹¹In-MNT-FA on HeLa cells compared to ¹¹¹In-MNT-PEG-FA. (B, C) Cytotoxicity of ¹¹¹In-MNT-PEG-FA compared to unlabeled MNT-PEG-FA on HeLa (B) and U87MG (C) cells; (D, E) Cytotoxicity of ¹¹¹In-MNT-PEG-FA compared to control ¹¹¹In on HeLa (D) and U87MG (E) cells. (F) Cytotoxicity of ¹¹¹In-MNT-PEG-FA and control ¹¹¹In in the presence or absence of 500 µM free FA on HeLa cells. The solid lines represent fits of the data to a mono-exponential model. Error bars represent standard errors of mean (n=3–6). Abbreviations: FA, folic acid; MNTs, modular nanotransporters; PEG, polyethylene glycol.

Figure 6

Serial SPECT/CT imaging of nude mice bearing HeLa human cervical carcinoma xenografts after intratumoral injection of ¹¹¹In-MNT-PEG-FA. Notes: (A) SPECT/CT (color/gray) images of a representative animal at indicated times after injection of ¹¹¹In-MNT-PEG-FA. (B) Decay-corrected retention of ¹¹¹In activity in tumor following ¹¹¹In-MNT-PEG-FA injection. The solid line represents fit of the data to a monoexponential model. The ring in the abdominal region presented on the CT scans of the mice is the breathing sensor. (C) Tumor retention and normal tissue redistribution of decay-corrected ¹¹¹In activity with time. Abbreviations: FA, folic acid; MNTs, modular nanotransporters; PEG, polyethylene glycol; SPECT, single-photon emission computed tomography.

Figure 7

In vivo therapy of HeLa human cervical carcinoma-bearing mice with ¹¹¹In-MNT-PEG-FA. Notes: (A) Tumor growth kinetics of HeLa-bearing mice following a single intratumoral injection of ¹¹¹In-MNT-PEG-FA (15 MBq/7.1 µg and 7.5 MBq/3.5 µg), control ¹¹¹In (15 MBq), or unlabeled MNT-PEG-FA (7.1 µg). (B) Survival curves for the selected treatment groups. Abbreviations: FA, folic acid; MNTs, modular nanotransporters; PEG, polyethylene glycol.