CisPt is widely used in cancer therapy, sometimes in combination with other drugs, despite its multiple side effects. Nanotechnology provides an alternative solution that unites the effectiveness of CisPt and a reduction of its secondary effects.
In this study, we carried out the synthesis of a nanoparticle using the Turkevich et al. (1951) technique. We obtained AuNps functionalized with PEG and CisPt. The AuNps synthesized exhibits a characteristic spectrum in solution (Alcantar et al. 2000).
The first step out was to determine the composition of the nanoparticles. We used the FTIR technique that indicates the presence of the elements that make up each of the synthesized Nps by recognizing the bonds they have formed. The EDS technique was then used to determine the composition of the Nps quantitatively.
We observed two signals using the FTIR analysis: one in the range between 2900 and 3800 cm−1 and another between 1600 and 1700 cm−1. It seems that the OH signal overshadowed the methyl groups that the OH groups produce (Botha et al. 2015). When functionalizing the AuNps with PEG, the methyl groups (2960–2870 cm−1) and carboxylic acid (3400–3200 cm−1), the H2O signal overshadowed them (Botha et al. 2015). In the case of the CisPt adhered to the AuNpPEG, the bonds Pt–N (599–560 cm−1) and Pt (3458–3418, 1641–1640 and 599–560 cm−1) also showed this effect (Rajath et al. 2015). CisPt displays two signals at 1318 and 1298 cm−1. We also observed these signals in AuNpPEGCisPt at 1066 and 1046 cm−1. A possible explanation for this difference between the peaks is the existence of an alteration of the ions of the CisPt, which encapsulate inside the matrix of the AuNpPEGCisPt (Rajath et al. 2015).
To confirm directly the functionalization of the AuNps with PEG and CisPt, we used scanning electron microscopy and transmission electron microscopy. The micrographics (SEM/EDS) corresponding to the three nanoparticles are given in Fig. 4. We stress that we took the images in the backscattering electron mode to detect any visual difference in the atomic composition, with the aim of exposing the core–shell shape. The images are blurred and remained so, even using a higher resolution, because the size of the sample was very small. Therefore, to check any possible bonding between AuNps and PEG/CisPt, we carried out an EDS analysis in the punctual mode, that is, and an EDS at a point in the image. For the case of AuNps, we carried out punctual EDS measurements on several AuNPs. The results showed a high percentage of the nanoparticle is Au (Fig. 4a–c). By a similar procedure, we analyzed the samples with AuNpPEGCisPt and we detected traces of Au, Pt and O (Fig. 4c). This is what one would expect if PEG and CisPt invariably bonded to AuNps. To rule out the possibility that these components appeared all over the substrate, we carried out several punctual EDS in the interstitial regions between nanoparticles and we observed no traces of Au, Pt, Cl, Na, or O. This shows that, in between the nanoparticle areas, there is no other material. Therefore, it is possible to deduce that PEG and CisPt bond to AuNps.
Once we have found the composition of each of the synthesized Nps, we proceed to measure its size. It is important to note that the size of the Nps is influenced by the characteristics of the environment in which they are located (Moore et al. 2015; Wang et al. 2015). That is why we measured the size of the different Nps after their synthesis and then their size in conditions closer to their physiological conditions. That is, their hydrodynamic size and potential Z were determined. Both parameters gave us an idea of how Nps would behave in a more complex system such as cancer cell lines (Lowry et al. 2016).
The size of the Nps after their synthesis was measured by TEM. It was found to be 8.6 nm with a standard deviation of 1.97 nm for AuNps, and 10 nm with standard deviation of 1.76 nm for AuNpPEGCisPt. This clearly shows that there is an overall increase in diameter of 1.4 nm which can only be attributed to the functionalization of the AuNps with PEG and CisPt. Gathering all this information, we could make a rough estimate of the quantity of CisPt that adhered to the AuNps. We subtracted the volume of the sphere of AuNps from the volume of AuNpPEGCisPt, which is the volume occupied by the “shell” full of PEG and CisPt. To determine the quantity of CisPt in this shell, we divide the volume of the shell by the volume of a CisPt molecule and multiply it by 7% which is the maximum atomic concentration of Pt in AuNpPEGCisPt (according to the EDS). Taking the radius of a single CisPt molecule as 0.5 nm (Baowan et al. 2017), we found that each AuNpPEGCisPt has roughly 462 molecules of CisPt, which corresponds to a mass of 2.263 × 10−19 g.
As for the hydrodynamic size and potential Z, we have confirmed that the behavior of the different Nps is influenced by the medium in which they are resuspended (Danaei et al. 2018). We observed that the AuNp, regardless of the medium in which they are resuspended, tend to form agglomerates. This is one of the reasons why we decided to functionalize the AuNps with PEG. Previous work shows that PEG is a compound that provides solubility and stability to the AuNps (Guerrini et al. 2018). In our case, we observed that, when functionalizing the AuNps with PEG, the size of the agglomerates which formed decreases.
Finally, the behavior of the Nps was observed in an identical medium to that in which the cancer cells develop. Under these conditions, closer to their physiological conditions, we observed that the agglomerates were smaller and more stable in a medium similar to the conditions found in cellular development.
Once we had confirmed that the AuNpPEGCisPt contained CisPt and that its behavior in a physiological-like environment was acceptable, we proceeded to perform cell viability tests to determine whether the synthesized Nps had a cytotoxic effect on cancer cell lines. First, it was necessary to establish if, in carrying out the feasibility test, there was no overlap (and subsequent overestimation) of the wavelengths of the product of MTT metabolism (formazan) and the wavelengths of the cells and the different synthesized Nps. Our results show (Additional file 1) that there is no overlap between the wavelengths of the cells, the Nps and the formazan. Hence, we did not obtain overestimated figures when determining the cell viability and the IC50. It has been shown that AuNps and functionalized AuNps do not interfere with the MTT feasibility tests. (Altunbek and Culha 2017; Sanderson et al. 2014).
Under experimental conditions, we found that neither AuNps nor AuNpPEG have a cytotoxic effect on the cell lines tested here.
On the other hand, when the gold particle is the vehicle for the CisPt (AuNpPEGCisPt), the amount necessary to obtain the IC50 diminishes. In the case of the HeLa cells, it is necessary to use 69.2 ± 3.2 μg/mL of CisPt and only 104.5 ± 7.0 μg/mL of AuNpPEGCisPt. At first sight, it would seem that we use more CisPt. However, we must take into consideration that in the case of the HeLa cells, the AuNpPEGCisPt only contains approximately 7% of CisPt to obtain IC50. We used only 7.31 μg/mL of CisPt to obtain the same IC50. We observed the same tendency in the other cell lines (Table 2).
The synthesized nanoparticles must have a series of characteristics to be a valid alternative in the treatment of cancer. They must be harmless and not destroy normal cells. In our case, both AuNps and AuNpPEG comply with this requirement. AuNpPEG can therefore function as a vehicle for the CisPt. We have shown that AuNpPEGCisPt contains small amounts of CisPt. However, a smaller amount of CisPt is required, if it is attached to the AuNPEGCisPt, than the quantity required if CisPt is administered alone.
Previous work has shown that the Nps can suffer a series of inconveniences when they come into contact with a more complex system such as that of the human metabolism. Nps can be recognized by the immune system and become less effective due to a low bioavailability (Gustafson et al. 2015). Thus, we decided to functionalize our AuNps with PEG, which has been shown to help AuNps to gain solubility, evade the immune system and therefore increase their half-life (Li et al. 2014a, b). In our case, we have also observed an increase in the effectiveness of CisPt in which it diminishes the IC50, despite the fact that the AuNpPEGCisPt forms agglomerates. This phenomenon seems to support our position. Nps functionalized with PEG have the ability to bind to cell membranes and, in the case of AuNpPEGCisPt, because of its size, may permit it to cross the cell membrane. This would facilitate the arrival of CisPt inside the cell and its subsequent transportation to the nucleus, which is where this compound would take effect (Suk et al. 2016).
Reports show that cancer cells can reduce the effects of CisPt by binding to the metallothionine protein or glutathione (Huska et al. 2009). The CisPt ejects through the membrane (Ahmad 2017). By joining the CisPt to AuNpPEG, these proteins may no longer recognize the CisPt because it is inside the core–shell nanoparticle (Li et al. 2014a, b; Zrazhevskiy et al. 2010). This is the reason why, in this investigation, we used much smaller amounts of CisPt (Brown et al. 2010). The AuNp, because of its size and polarity, helps the CisPt to cross the cellular membrane (Lin et al. 2010). In addition, AuNpPEG works as a masking system for the CisPt (Babu et al. 2013). CisPt is poorly soluble in water. One must suspend it in water to reach the nucleus of the cell. It seems that AuNpPEGCisPt improves its solubility (Johnstone et al. 2016).
The size of the AuNpPEGCisPt provides a large contact surface that facilitates its interaction with the genetic material of the cell (Wang et al. 2016). It also enhances the interaction of CisPt with the nitrogenous bases of DNA, intervening in the functions of cellular transcription and replication and causes cell death by apoptosis (Hall et al. 2007).
Conclusions
We developed a new methodology for the synthesis of a Gold core–shell type nanoparticle. It contains the anticancer CisPt within the shell and may facilitate transport across the cell membrane.
This in vitro study has determined that, if we use AuNps as the nanocarriers, the amount of CisPt necessary to cause the desired cytotoxic effect is less than the amount used in conventional cancer treatments.
These results show that the synthesized AuNpPEGCisPt is a good transport of CisPt. It decreases substantially the dose and has the same effect as the CisPt, so that the undesired side effects may decrease. The synthesized nanoparticles AuNps and AuNpPEG could be potential nanovehicles for the creation of theranostic nanoparticles.