The glassware must be previously cleaned with a 40% nitric acid solution and Milli-Q Nanopure water (see below Nota bene, Lab-203 rules for washing glassware) and then dried in the TROX-100 hood. Before the beginning of the experiments, the glassware is placed under the germicidal lamp of the TROX-100 hood for 1 hour. Regarding storage, glassware (class A) must be protected from environmental dust in duly clean, closed, and identified containers.

The method used to clean the glassware is known as drag washing and consists of first washing the glassware with a strong stream of water and letting the tap water overflow when filling the containers. This procedure is repeated at least 5 times. Then, the glassware is immersed in 40% nitric acid for 24 hours and, thereafter, rinsed again with distilled and deionized water until all the acid is removed (at least 4 times, allowing the water to overflow).

The experiments were carried out at the Catalysis and Interfaces Science Laboratory (LACFI-203) with a controlled temperature of 23±1 °C. The preparation of solutions and the synthesis of NPs should be carried out in the TROX-100 hood (flow 41 and 34.5 m/second). Synthesis should be carried out in a silicone bath. Before starting the experimental procedure, the hood should be sanitized with alcohol, along with all the material that will be placed inside the chapel, for example, supports, micropipettes, and reagent jars.

Silicone oil should be previously heated on a heating plate (selector position at 300). In approximately 1 hour, an ideal oil temperature between 110 °C and 120 °C for the experiment is achieved.

Sodium citrate (0.1 g) should be weighed in a 5-mL beaker, completely dissolved in 2-3 mL of water, and transferred to a 10-mL volumetric flask with its volume completely adjusted with distilled water. Thereafter, the 1% sodium citrate solution should be filtered by weight in the 0.45-μm filter (Chromafil Xtra CA-45/25) using a sterile plastic syringe. The solution should be conditioned at room temperature.

HAuCl₄ (0.0050 g) should be weighed in a 5-mL beaker, dissolved completely in 2-3 mL of distilled water, and transferred to a 50-mL volumetric flask. The beaker should be washed with distilled water, and this washing water is transferred to the volumetric flask to remove all HAuCl₄ from the walls of the beaker. The volume of the volumetric flask is then completely adjusted with deionized water. The control of Au concentrations follows the methodology described by Fiedler et al [19].

AuNPs with a diameter between 160 Å and 715 Å are prepared as described by Frens [20].

Fifty milliliters of 0.01% gold solution (by mass) should be transferred to the synthesis flask together with a magnetic bar and then placed in a previously heated oil bath. A watch glass should be placed on the mouth of the synthesis flask. In approximately 20 minutes, the gold solution boils, and then the sodium citrate solution should be added as described in Table 1, with stirring (with the stirrer in position 3). Stirring should be carried out for 30 minutes. Thereafter, the heating and stirring should be turned off, and the solution should be allowed to cool to room temperature and transferred to a Teflon tube.

The solution must be conditioned in a Teflon tube previously washed (as shown in the Preparation of 10 mL of a 1% [by Weight] Sodium Citrate Solution section), duly identified at the room temperature of the LACFI-203, and protected from light.

Because it is highly hygroscopic, care should be taken when weighing HAuCl₄. The weighted masses are shown in Table 2. The HAuCl₄ reagent bottle must be stored in a desiccator [21].

The weight of sodium citrate should be 0.1004 g.

To measure the volume of sodium citrate, the micropipette aligned with the laboratory’s quality control was used. An error rate of 4% was tolerated, and the volume was 502.21 μL.

We propose coating AuNPs with polyethylene glycol to enhance their stability and biocompatibility [15,22]. Temozolomide, a chemotherapeutic agent, would be either attached to the surface of the AuNPs or could be encapsulated within them [4,12]. Additionally, the NPs’ design has to offer co-delivery of MGMT inhibitors to overcome resistance in GBM cells [7].

More data are needed to reveal the overall integrity of AuNPs post synthesis and post modification to validate successful incorporation of peptides onto AuNPs. Thus, verifying peptide attachment and assessing pH stability are fundamental steps prior to initiating in vitro experimental procedures.

After the synthesis and functionalization of the AuNPs, it is crucial to confirm the successful attachment of peptides onto the NPs. We propose a combination of Fourier transform infrared spectroscopy (FTIR) and surface plasmon resonance (SPR) for this verification.

FTIR can confirm peptide attachment by identifying the characteristic absorption peaks of the peptide-NP bond. After the peptide functionalized AuNPs, samples must be freeze-dried and mixed with potassium bromide. FTIR spectra must be collected in the range of 4000 cm^–1 to 400 cm^–1. The appearance of characteristic absorption bands in the FTIR spectrum, not present in the AuNPs without peptides, would confirm the successful attachment of peptides onto the NPs.

For SPR, we plan to coat a gold chip with a layer of the NPs and flow the peptide solution across the chip. The SPR sensor will monitor the real-time interaction between the NPs and the peptides, as reflected by changes in the resonance angle. A shift in the resonance angle upon the introduction of the peptide solution would confirm the binding of peptides to the AuNPs.

Both these methods can provide complementary information—FTIR would confirm the formation of a covalent bond between the AuNPs and peptides, while SPR will provide real-time information about the binding process and the stability of the peptide-AuNP complex.

Given the acidic nature of the tumor microenvironment, it is crucial to ensure that our NPs are stable and retain their functionality under such conditions. We propose conducting pH stability tests to tackle this problem. After verifying peptide attachment, NPs are incubated in solutions of varying pH (ranging from pH 4 to 8 to simulate physiological and tumor microenvironment conditions). We then evaluate the size, shape, and surface charge of the NPs through dynamic light scattering and zeta potential measurements, and the functionality of attached peptides is assessed using FTIR or SPR. This provides a comprehensive view of NP stability and functional integrity in a range of pH conditions.

The functionalized AuNPs can be tested in vitro using GBM cell lines to evaluate their cellular uptake, cytotoxicity, and ability to overcome drug resistance [6]. As our current proposal is at an early stage, it focuses on in vitro studies and computational modeling. Upon successful completion of these initial stages, we plan to include in vivo animal studies in future protocols.

Based on the preliminary results, constant optimization of NP design is needed to enhance their mobility within the brain’s extracellular space, their penetration from the cerebrospinal fluid into the brain parenchyma, and their targeting efficiency [8]. Future developments in NP design optimization can involve adjusting NP size and shape to improve mobility and penetration, modifying surface properties for enhanced drug delivery and stability, and potentially incorporating targeting ligands that can increase affinity for GBM cells or specific receptors on the BBB. Each step represents a critical aspect of the research process and will contribute to the development of a more effective treatment for GBM. It is important to note that this complex and challenging project requires a multidisciplinary team of researchers with expertise in NP synthesis, drug delivery, cell biology, neuroscience, and oncology.