Dual-Target Peptide-Modified Erythrocyte Membrane-Enveloped PLGA Nanoparticles for the Treatment of Glioma

Abstract

Penetration of the blood-brain barrier (BBB) and the blood-brain tumor barrier (BBTB) remains a significant challenge for the delivery of drugs in the treatment of glioma. Therefore, the development of targeted preparations with the ability to penetrate the BBB and BBTB, and target gliomas, is an important approach if we are to improve the efficacy of glioma treatment. In the current study, an active targeting preparation based on PLGA nanoparticles coated with erythrocyte membranes (RBCNPs) and dual-modified with ^DWSW and NGR peptide ligands (^DWSW/NGR-RBCNPs). Euphorbia factor L1 (EFL1) extracted from euphorbiae semen was used as the model drug. The final nanoparticles were characterized by in vivo and in vitro tests. In vitro results showed that EFL1-loaded ^DWSW/NGR-RBCNPs were taken up by cells and had the ability to penetrate the BBB and BBTB and produce cytotoxic effects. Furthermore, in vivo studies in mice showed that when injected intravenously, these specialized NPs could enter the brain, target tumor tissue, and significantly extend life span. The results showed that dual-targeting EFL1-loaded ^DWSW/NGR-RBCNPs have significant potential as a nanotherapeutic tool for the treatment of brain glioma.

Keywords: DWSW; NGR; biomimetic nanoparticles; blood–brain barrier; blood–brain tumor barrier; dual-targeting; euphorbia factor L1; glioma.

Figure 1

Graphical abstract of this study. Schematic illustration of dual-target peptides modified using erythrocyte membrane-enveloped PLGA nanoparticles for the treatment of glioma. The nanoparticles were designed to penetrate the BBB and BBTB and then to aggregate at tumor sites. ^DWSW peptide was used to penetrate BBB and NGR was used to target tumor. ^DWSW/NGR-RBCNPs were observed to selectively accumulate in tumor tissue and exert a therapeutic effect.

Figure 2

Preparation of ^DWSW/NGR-RBCNPs. First, the drug was loaded into PLGA, coated with erythrocyte membrane, and finally target-modified (A). The targeting ligand was then synthesized by conjugating DSPE-PEG₂₀₀₀-Mal to the cysteine residue on both ^DWSW and NGR (B). The Michael addition reaction was used in chemical synthesis.

Figure 3

Characterization of nanoparticles. Transmission electron microscopy (TEM) photos of ^DWSW/NGR-RBCNPs. (A) Nanoparticles showing the core-shell structure. (B) Erythrocyte membrane proteins in different nanoparticles, showing the protein had not been lost. Similar bands of protein appear in the same location. (C) Stability of EFL1-loaded ^DWSW/NGR-RBCNPs in the presence of FBS; stability fluctuated within a range of 2%. No serious aggregation or sedimentation of the nanoparticles during the measurement period.

Figure 4

Targeting ability test with different cells. Cellular uptake of different DiI-labelled RBCNPs by bEND.3 cells (A, B). ^DWSW exhibited the ability to cross the BBB and the free peptide was able to compete for the target. NGR exhibited the ability to cross the BBTB and the free peptide was able to compete for the receptor in HUVECs cells (C, D). ^DWSW- and NGR-modified nanoparticles showed good tumor-targeting in C6 cells (E, F). DiI-positive cells were counted by FCM and intracellular fluorescence was captured by CLSM. Nanoparticles show different targeting capabilities. Scale bars, 10 μm. * indicates P < 0.05.

Figure 5

In vivo targeting ability test. In vivo real-time imaging of different DiR-encapsulated nanoparticles in the brain showing the biodistribution of nanocarriers in animals (A), brain tissue (B) and distribution of different DiR-encapsulated nanoparticles in different organs (C). The enhancement of targeted modification leads to increased brain fluorescence. As the liver plays a predominant role in the elimination of these nanoparticles, it was no surprise that the liver showed the highest levels of accumulation when compared to other organs. These data show that these nanoparticles can target drugs to the brain tissue in an effective manner.

Figure 6

Targeted distribution of nanoparticles in brain tissue. Distribution of DiI-encapsulated nanoparticles in the brain of mice bearing intracranial C6 gliomas, as determined by confocal laser microscopy. The white line shows the margin of the intracranial glioma while the arrows indicate glioma cells. The green color represents DiI-encapsulated nanoparticles, while nuclei are shown in blue (DAPI). 784 DWSW/NGR-RBCNPs showed the strongest targeting ability. Scale bar, 20 μm.

Figure 7

Antitumor effects of nanoparticles in vivo. Kaplan-Meier survival curves of nude mice (A), brain MRI images (B), and histological changes in gliomas (C) following treatment with different nanoparticles. The strongest therapeutic effect was achieved by the dual-targeted modified nanoparticles.