**7. Strategies for safe drug delivery using cadmium-containing quantum dots**

fraction of the QDs aggregated to larger particles and were retained in liver tissue for a long

Several studies suggest that the kidneys can remove QDs that are less than 5 nm. It has been observed that, after i.v. administration of CdSe/ZnS-QDs, only 10% and 40% of the injected dose was found in the kidney and liver respectively, suggesting that only a fraction of the total QDs dose passed through this route [117]. Another study quantitatively detected the excretion of QDs in mice feces and urine after i.v. injection of CDSe QDs. The speed of excretion was quicker via feces, and the peak (0.214 ng of Cd) occurred 6–12 h postexposure. The excretion from urine was relatively delayed, and the peak (0.174 ng of Cd) was 24–36 h post-exposure [118]. The elimination of silica-coated CdSe QDs (~5.5 nm) from the body via feces and urine occurred in its totality after 5 days. Yet many reports propose that a portion of the administered QD dose may not be excreted and remains in the tissues. The extent of excretion and the extent of persistence in tissues take on added importance when one considers the potential delivery of QD as a cancer-targeting drug. More comprehensive studies of potential excretion will

Numerous studies have addressed the cellular level, but these are often difficult to compare due to varied dosing parameters and lack of physicochemical particle characterization. In general, it appears that most QDs examined found ready uptake at the cellular level, primarily via endocytic mechanisms, which depends upon the surface ligands coated over the QD surface [119, 23]. It has been reported that CdSe QDs and CdTe QDs that enter the cell, are visible at the cell surface and in the cytoplasm after a short time. QDs are likely to bind to the cell surface due to their interaction with cell surface glycoproteins and glycolipids [66]. Employing red-and green-emitting cationic QDs, it was found that the CdTe QD distribution was in part dependent on nanoparticle size. In the murine microglial N9 cell line, red cationic QDs (5 nm) were distributed throughout the cytoplasm. In contrast, green and also positively charged QDs (2 nm) were often found in the nucleus of N9 cells upon 1 h of QD exposure [120]. It has been shown that green QDs coated with tri-metoxysilylprolyl urea and acetate groups bind with high affinity in the cell nucleus of mouse 3T3 fibroblasts [121]. In human mammary epithelial tumor cells (MDA-MB-231) green-emitting CdSe/ZnS/SiO2 QDs were packaged in large vesicles found in the perinuclear region [23]. Confocal images have showed that MPAcoated QDs were distributed inside the cytoplasmic region of cells. In contrast, GA/TOPOcoated QDs were not found inside cells. These results indicate that cellular uptake of QDs depends upon the hydrodynamic size of the QDs as well as surface coating material [122]. These results strongly suggest that surface coatings can improve cytocompatibility and, consequently, decrease toxicity. Previous studies have shown that long-term exposure of surface coated QDs to their bioenvironment can destabilize the binding strength of the surface molecules, which in turn can yield unprotected QDs inside or outside the cells [123]. Therefore, the stability and binding strength of surface molecules over the QD surface define the cyto‐ compatibility of the QDs and hence their cytotoxicity. Thus, according to previous studies: (1) the surface coating strategy could improve the cytocompatibility of QDs, (2) surface molecules could determine the intracellular uptake and consequent cytotoxicity of QDs, and (3) intra‐

therefore be critical to QD development as a nanopharmaceutical.

cellular uptake of QDs could depend on their hydrodynamic size.

time [116].

486 Application of Nanotechnology in Drug Delivery

Discussion regarding the toxicity of cadmium-containing QDs can be somewhat confusing because of the diversity QDs being synthesized. Besides and as we have mentioned before, we have to consider that not all QDs are alike. Each individual type of QD possesses its own unique physicochemical properties, which in turn determine its potential toxicity or lack thereof [124]. In general, there are discrepancies in the current literature regarding the toxicity of QDs and these can be attributed to several factors: the lack of toxicology-based studies, the variety of QD dosage/exposure concentrations reported in the literature, and the widely varying physicochemical properties of individual QDs.

Up to date, toxicity studies have been conducted on a variety of both human and non-human cells and cell lines; research has been focused on *in vitro* assays of cytotoxicity [125-131]. *In vitro* studies are very important and can serve as background data to inform the design of *in vivo* studies but, on their own, they provide an insufficient basis for a complete risk assessment. Administration of QDs in animal models has revealed that QDs induce: (1) accumulation of QDs in specific organs [23, 132, 133], (2) excretion in urine, bilis and feces [134], (3) toxicity in selective organs [135], (4) embryotoxicity [109, 111], and (5) oxidative damage [136-138]. Importantly, and a potential source of confusion in assessing QD toxicity, the latter depends on multiple factors derived from both individual QD physicochemical properties and envi‐ ronmental conditions: QD size, charge, concentration, outer coating bioactivity (capping material, functional groups), and oxidative, photolytic, and mechanical stability have each been shown to be determining toxicity factors. Therefore, all these aspects should be extended to examine alternate QD formulations, compositions, and shapes to help facilitate any future generalizations regarding size thresholds in the regulatory context. There are only a few studies specifically designed for toxicological assessment (e.g., dose, duration, frequency of exposure, mechanisms of action). Many of the studies, from which QD toxicity information is derived, it has been cited in reference to it were performed by nanotechnology researchers rather than toxicologists or health scientists. It is therefore difficult to extrapolate the results of such studies in order to reach any conclusions regarding the health and safety of QDs. Nonetheless, these studies may provide important insights that will be useful in guiding the eventual design of standardized toxicity tests and protocols.

The wealth of data accumulated from QD toxicity studies is an invaluable asset that should be exploited to design appropriate methodologies to further assess the toxicity of novel QDs. Researchers often neglect to carry out a comprehensive characterization of QDs prior to using them. In our opinion, this step is absolutely necessary, especially before any toxicity screening is started, precisely because the exact property or properties of QDs responsible for said toxicity are still poorly understood. This omission is one of the reasons behind the current state of confusion surrounding this issue. As epigenetic changes may lead to long-term reprogram‐ ming of gene expression long after the initial insult has been removed, results from "nanoe‐ pigenetic" assessments may have important implications on the future use of new nanomaterials in bioimaging and therapeutic applications. They should be evaluated early in the development of new QDs as well as QD-based devices and clinical tools. Future QD toxicity studies should be standardized and systematized because methodological variability in the current body of literature makes it difficult to compare and contrast results. We advocate the following steps for consistent, comparable toxicology data: (a) standardize dose metrics, (b) characterize QD uptake concentration, (c) identify *in vitro* models that reflect how the QDs QDs interact with cells *in vivo*, and (d) use multiple assays to determine sublethal toxicity and biocompatibility. Proceeding without careful evaluation of these critical areas will blunt the progress of nanomedicine and place human health at risk. However, judicious further research into these areas will undoubtedly contribute to development of nanopharmaceuticals for cancer treatment and drug delivery that have minimal to low risk and can highly benefit public health.

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#### **8. Conclusion**

Cadmium-containing QDs are leading the way toward new preparations that can overcome the fundamental limitations of simple free drug formulations, providing the means to change their pharmacological properties and also understand their biological fate in great detail. However, ADME properties depend on multiple factors derived from both inherent physico‐ chemical properties and environmental conditions. The findings also suggest that, under certain conditions, QDs may pose risks to human health, as determined by rodent animal models and *in vitro* cell cultures. This review outlined the unique features that make QDs an ideal platform for nanocarrier design and discussed how this model has been applied to study vehicle behavior for diverse drug delivery applications. However, it is clear that to make such a goal feasible and relatively risk-free for human beings, more extensive pharmacological and toxicological research of QDs are needed.

### **Author details**

Lourdes Rodriguez-Fragoso1\*, Ivonne Gutiérrez-Sancha1 , Patricia Rodríguez-Fragoso2 , Anahí Rodríguez-López1 and Jorge Reyes-Esparza1

\*Address all correspondence to: mrodriguezf@uaem.mx

1 Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Morelos, Mexico

2 Departamento de Física, CINVESTAV-I.P.N Apartado, México
