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

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.


PLANIFICATION OF DESIGN OF EXPERIMENT
In this work, a design of experiment (DoE) was implemented for the optimization of the NPs preparation protocol faster and more accurately by simultaneously evaluating the effect of various experimental factors while requiring a lower set of experiments.
A 2 4 full factorial design was initially implemented using the Design Expert software (11.1.2.0 version, Stat-Ease Inc., Minneapolis, USA). The chosen experimental variables were the quantity of PLGA, the PVA percentage (w/v), the sonication cycle number, and the organic solvent/water ratio, and each experimental variable was varied in a high (+1) and low level (-1), as shown in Table S1. The independent variables and experimental levels were selected based on preliminary experiments. Table S1. Planification of the Experimental Design.
Note: mPLGA -PLGA mass; PVA -percentage of PVA; sonication cycles -number of cycles of 10 seconds each; O/W volume ratio -ratio the organic solvent and the aqueous PVA solution.
Then the factorial design was augmented by applying a Central Composite Design (CCD). For that, 2 extra levels (star points, -α and +α) were added to the model, with an α = 1.54671 (table 1). The design expert software automatically determined the α value after choosing a factorial orthogonal quadratic design augmentation.
Then, the effect of the experimental factors on each of the evaluated responses was determined by the following polynomial regression [1]: Where Y is the predicted response; β0 is the intercept term; Xi,,j are the independent variable levels; and βj,i are the coefficients for the levels of the variable.

Parameters Component Units
Applied   Table S2. In Figure S1 are represented the 2Dcontour and 3D-response surface plots that provided a visual representation of the influence of the different experimental variables on the NP's diameter. As shown in Figure S1 (and Table S6), the PLGA mass (X1) and the O/W ratio (X4) showed to influence the NPs' size positively. Decreasing the amount of PLGA leads to forming smaller NPs, since a reduced polymer concentration leads to a decreased viscosity of the organic phase. This facilitates organic solvent diffusion into the aqueous S-6 phase, creating smaller oil droplets during emulsification [2]. The O/W ratio also increased the organic phase viscosity, positively affecting the NPs' size. On the other hand, the %PVA (X2) and the sonication cycle number (X3) negatively affected the NPs' size. Increasing the amount of PVA leads to smaller NPs' since PVA promotes the steric stabilization of the emulsion, decreasing the interfacial tension between the oil droplets and the continuous aqueous phase [3]. In addition, increasing the sonication cycles promotes the disruption of the emulsion droplets into ones with smaller dimensions [4].

EFFECT OF THE EXPERIMENTAL PARAMETERS ON THE NPS' PDI
The PDI of the NPs ranged from 0.011 (formulation 24) to 0.239 (formulation 19), as shown in Table S2. In Figure  As shown in Figure S2 and       As observed in Table S9, both diameter and PDI responses of the checkpoint formulations were within the predicted range, validating the mathematical model. Although excluded from the model, the zeta potential and the encapsulation efficiency values were also evaluated. Data are represented as mean ± SD (n=3).