Synthesis of doxorubicin-loaded peptosomes hybridized with gold nanorod for targeted drug delivery and CT imaging of metastatic breast cancer

Abstract

Background: Cancer nanomedicines based on synthetic polypeptides have attracted much attention due to their superior biocompatibility and biodegradability, stimuli responsive capability through secondary conformation change, adjustable functionalities for various cargos such as peptides, proteins, nucleic acids and small therapeutic molecules. Recently, a few nanoformulations based on polypeptides comprising NK105, NC6004, NK911, CT2103, have entered phase I-III clinical trials for advanced solid tumors therapy. In the current study, we prepared polypeptide-based vesicles called peptosome via self-assembly of amphiphilic polypeptide-based PEG-PBLG diblock copolymer.

Results: In this regard, poly(γ-benzyl L-glutamate (PBLG) was synthesized via ring opening polymerization (ROP) of γ-benzyl L-glutamate-N-carboxyanhydride (BLG-NCA) using N-hexylamine as initiator. Then amine-terminated PBLG was covalently conjugated to heterofuctional maleimide PEG-carboxylic acid or methyl-PEG-carboxylic acid. The PEG-PBLG peptosomes were prepared through double emulsion method for the co-delivery of doxorubicin.HCl and gold nanorods as hydrophilic and hydrophobic agents in interior compartment and membrane of peptosomes, respectively (Pep@MUA.GNR-DOX) that DOX encapsulation efficiency and loading capacity were determined 42 ± 3.6 and 1.68 ± 3.6. Then, theranostic peptosomes were decorated with thiol-functionalized EpCAM aptamer throught thiol-maleimide reaction producing Apt-Pep@MUA.GNR-DOX for targeted delivery. The non-targeted and targeted peptosomes showed 165.5 ± 1.1 and 185 ± 4.7 nm diameters, respectively while providing sustained, controlled release of DOX. Furthermore, non-targeted and targeted peptosomes showed considerable serum stability. In vitro study on MCF-7 and 4T1 cells showed significantly higher cytotoxicity for Apt-Pep@MUA.GNR-DOX in comparison with Pep@MUA.GNR-DOX while both system did not show any difference in cytotoxicity against CHO cell line. Furthermore, Apt-Pep@MUA.GNR-DOX illustrated higher cellular uptake toward EpCAM-overexpressing 4T1 cells compared to Pep@MUA.GNR-DOX. In preclinical stage, therapeutic and diagnostic capability of the prepared Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX were investigated implementing subcutaneous 4T1 tumor model in BALB/c mice. The obtained data indicated highest therapeutic index for Apt-Pep@MUA.GNR-DOX compared to Pep@MUA.GNR-DOX and free DOX. Moreover, the prepared system showed capability of CT imaging of tumor tissue in 4T1 tumorized mice through tumor accumulation even 24 h post-administration.

Conclusion: In this regard, the synthesized theranostic peptosomes offer innovative hybrid multipurpose platform for fighting against breast cancer.

Keywords: Breast cancer; Doxorubicin; Gold nanorod; Peptosome; Theranostics.

Fig. 1

A ¹HNMR spectrum of PBLG B GPC chromatogram of PBLG

Fig. 2

A ¹HNMR spectrum of Mal-PEG-PBLG diblock copolymer. B ¹³CNMR spectrum of PBLG (red), Mal-PEG-COOH (blue), and Mal-PEG-PBLG block copolymer (green)

Fig. 3

FTIR spectra of PBLG a, Mal-PEG-COOH b, and Mal-PEG-PBLG block copolymer c

Fig. 4

A Differential Scanning Calorimeter (DSC) of PEG (red), PBLG (blue), and PEG-PBLG (black), B TGA of PEG (green), PBLG (red), PEG-PBLG (blue)

Fig. 5

A UV spectra of GNR and MUA-GNR, B TEM image of MUA-GNR

Fig. 6

FTIR spectra of A CTAB (red) and CTAB-GNR (blue). FTIR spectra of B MUA (blue) and MUA-GNR (red)

Fig. 7

Release patterns of DOX from Pep@MUA.GNR-DOX in various release media including PBS (pH = 7.4), PBS supplemented with 30% v/v FBS, and citrate buffer (pH = 5.4). (Error bars show the standard error of mean for two different experiments in the same conditions).

Fig. 8

Serum stability of the Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX in PBS supplemented with 10% FBS after 0, 1 and 2 days incubation in a shaker incubator at 37 °C in terms of nanoparticle size (A) and polydispersity index

Fig. 9

FE-SEM images of Pep@MUA.GNR-DOX (A) and Apt-Pep@MUA.GNR-DOX (B)

Fig. 10

AFM analysis of Pep@MUA.GNR-DOX (A); Apt-Pep@MUA.GNR-DOX (B), Height profile of Pep@MUA.GNR-DOX (C) and Apt-Pep@MUA.GNR-DOX (D)

Fig. 11

Flow cytometry analysis of CHO, and 4T1 cell lines for DOX cellular internalization evaluation after 2 h of exposure with either free DOX, Apt-Pep@MUA.GNR-DOX or Pep@MUA.GNR-DOX

Fig. 12

MTT assay of 4T1 (A), MCF-7 (B) and CHO (C) cell lines after 48 h exposure to free DOX, Apt-Pep@MUA.GNR-DOX, Pep@MUA.GNR-DOX at 37 °C

Fig. 13

In vivo therapeutic efficacy study of Pep@MUA.GNR, Apt-Pep@MUA.GNR-DOX, Pep@MUA.GNR-DOX and free DOX with identical DOX (5 mg/kg) and MUA.GNR (1 mg/kg) concentration and PBS during 30 days after single dose i.v administration in 4T1 tumorized BALB/c mice. Tumor volume (A); Survival percentage (B) and Body weight (g) (C)

Fig. 14

Ex vivo imaging of tumor tissues and mice organs. 6 h (A) and 24 h (B) after i.v injection of free DOX, Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX with equal DOX (5 mg/kg) and MUA.GNR (1 mg/kg) concentration. The Quantitative ROI analysis of DOX in tumor tissues and mice organs after 6 h (C) and 24 h (D) i.v. administration of either Apt-Pep@MUA.GNR-DOX, Pep@MUA.GNR-DOX or free DOX

Fig. 15

Clinical CT scan imaging of 4T1 tumor-bearing mice 6 and 24 h post-injection of either Pep@MUA.GNR-DOX or Apt-Pep@MUA.GNR-DOX

Fig. 16

The in vivo CT scan coronal view of the tumor site 6 and 24 h post-injection of either Pep@MUA.GNR-DOX or Apt-Pep@MUA.GNR-DOX

Fig. 17

Hematoxylin and eosin staining of tumor tissue and mice organs in 4T1 tumoric mice, 20 days i.v. injection of either PBS, free DOX, Pep@MUA.GNR, Pep@MUA.GNR-DOX or Apt-Pep@MUA.GNR-DOX with equal concentration of DOX (5 mg/kg) and MUA.GNR (1 mg/kg)