Transferrin-Conjugated PLGA Nanoparticles for Co-Delivery of Temozolomide and Bortezomib to Glioblastoma Cells

Abstract

Glioblastoma (GBM) represents almost half of primary brain tumors, and its standard treatment with the alkylating agent temozolomide (TMZ) is not curative. Treatment failure is partially related to intrinsic resistance mechanisms mediated by the O6-methylguanine-DNA methyltransferase (MGMT) protein, frequently overexpressed in GBM patients. Clinical trials have shown that the anticancer agent bortezomib (BTZ) can increase TMZ's therapeutic efficacy in GBM patients by downregulating MGMT expression. However, the clinical application of this therapeutic strategy has been stalled due to the high toxicity of the combined therapy. The co-delivery of TMZ and BTZ through nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) is proposed in this work, aiming to explore their synergistic effect while decreasing the drug's toxicity. The developed NPs were optimized by central composite design (CCD), then further conjugated with transferrin (Tf) to enhance their GBM targeting ability by targeting the blood-brain barrier (BBB) and the cancer cells. The obtained NPs exhibited suitable GBM cell delivery features (sizes lower than 200 nm, low polydispersity, and negative surface charge) and a controlled and sustained release for 20 days. The uptake and antiproliferative effect of the developed NPs were evaluated in in vitro human GBM models. The obtained results disclosed that the NPs are rapidly taken up by the GBM cells, promoting synergistic drug effects in inhibiting tumor cell survival and proliferation. This cytotoxicity was associated with significant cellular morphological changes. Additionally, the biocompatibility of unloaded NPs was evaluated in healthy brain cells, demonstrating the safety of the nanocarrier. These findings prove that co-delivery of BTZ and TMZ in Tf-conjugated PLGA NPs is a promising approach to treat GBM, overcoming the limitations of current therapeutic strategies, such as drug resistance and increased side effects.

Figure 1

Synthesis of PLGA NPs containing TMZ and BTZ and their conjugation with Tf by the EDC click reaction.

Figure 2

FTIR absorbance spectrum of Tf-TMZ+BTZ PLGA NPs, TMZ+BTZ PLGA NPs, and Tf stock solution recorded from 350 to 4000 cm^–1. The peaks of PLGA are identified as green, and the peaks from Tf are identified as red.

Figure 3

TEM images and corresponding size histograms with Gaussian distribution fitting of (A) Tf-modified and (B) nonmodified BTZ+TMZ PLGA NPs. Scale bar: 500 nm.

Figure 4

TMZ and BTZ release from BTZ+TMZ coloaded Tf-conjugated and nonconjugated PLGA NPs in different in vitro physiological conditions. (A) Simulated blood-circulation conditions (PBS, pH 7.4, 0.01 M at 37 °C). (B) Simulated GBM tumor acidic microenvironment (PBS, pH 6.4, 0.01 M at 37 °C). The results are shown as mean value ± SD.

Figure 5

Fluorescence quantification of C6-labeled NPs uptake by human cells quantified by fluorescence. (A) U251, T98G, and NHA were incubated for 2 h with 20 μM C6-PLGA NPs cells after pretreatment with excess Tf. Evaluation of the effect of incubation period and Tf-modification of the C6-NPs uptake in (B) U251, (C) T98G, and (D) NHA cells. The cells were treated with 20 μM Tf-modified and nonmodified C6-PLGA NPs for two different incubation periods (30 and 120 min). Control refers to the autofluorescence of the nontreated cells.

Figure 6

(A) U251 and (B) T98G cell survival inhibition curve after 72 h of co-treatment with TMZ and BTZ, free or loaded in nonconjugated and Tf-conjugated PLGA NPs. Data represent the mean values ± SD (n = 3).

Figure 7

Morphological analysis of U251 and T98G cells after 72 h of treatment with combination therapy of TMZ+BTZ in the free form or entrapped in Tf-conjugated and nonconjugated PLGA NPs at IC₅₀ drug dose. Control (CTR) cells were left untreated. Scale bar, 200 μm.