## 3 Results and Discussion
### Preparation of DOX-NPs (7)
The size of these nanodevices was measured after 1, 3, and 6 months at 4 \({}^{\circ}\)C and 25 \({}^{\circ}\)C, showing a constant size distribution (Figure 1a,b). DLS measurements revealed that the long-term stability of these nanoparticles was not influenced by storage temperature conditions. These results confirm that these particles are stable for a long time allowing them to be stored. This is a key property for further translation of this nanodevice. On the basis of the fluorescence properties of DOX (\(\lambda\)ex = 470 nm, \(\lambda\)em = 560 nm), the efficiency of drug conjugation onto the nanoparticles was monitored easily using fluorescence-based techniques such as flow cytometry and microscopy. Figure 1e shows a representative plot of the obtained results by flow cytometry analysis. An increase in fluorescence of the DOX-NPs (7) conjugates with respect to unloaded nanoparticles (NAKED-NPs (1)) can be observed. This result was corroborated by confocal microscopy (Figure 1f). In order to evaluate the efficiency of the conjugation of doxorubicin to DOX-NPs (7), a spectrophotometric quantification of the remained unconjugated drug in the supernatant of the reaction was measured by UV spectroscopy (A480 nm). A calibration curve, with a lineal ratio between the optic density of doxorubicin and its concentration using a set of standard samples, was generated (Supplementary Figure S3, see Supplementary Material). To determine the value of loading capacity (LC), the amount of conjugated drug with respect to the number of nanoparticles was considered instead of the nanoparticle weight (as frequently reported) [31]. For this approach, an accurate spectrophotometric method to determine the number of nanoparticles per volume previously developed by our team was carried out [26]. The concentration of nanoparticles DOX-NPs (7) was estimated as \(3.72\times 10^{8}\) NPs/\(\mu\)L. The drug loading capacity is related to the number of nanoparticles, thereafter, the loading capacity per nanoparticle can be calculated. A loading capacity of \(5.2\times 10^{-9}\) nmol DOX per NP was estimated. This parameter provides the value of drug dose with precision and accuracy; this fact being of extreme relevance for the clinical translation of nanomedicine. Taking into account the drug conjugated with respect to the total amount of the drug, the conjugation efficiency (CE) was 89%. These results shows the high efficiency of this nanodevice compared to previously reported therapeutic nanodevices based on drug encapsulation [18]. A major deficiency of the sustained release system is that the release is not specific. To ensure the release of the drug at the target site, to avoid a non-specific release, a pH-sensitive stimuli release strategy was implemented. The hydrazone is used in our approach as a cleavable bond that responds to stimuli depending on the pH; very useful in a tumor microenvironment that has a slightly acidic pH [32]. In order to release the drug in acidic conditions, doxorubicin was covalently conjugated to nanoparticles by a hydrazone bond sensitive to pH 6. Therefore, it is necessary to determine the pH-responsive drug release to evaluate this nanodevice. A high performance liquid chromatography (HPLC) assay was conducted to monitor the release process of doxorubicin in vitro. Release profiles were obtained by comparing the percentage of the released drug with respect to the amount of doxorubicin conjugated to the DOX-NPs (7) for seven days at pH 6 and pH 7.4 by HPLC analysis. As shown in Figure 1g (blackg line), in an acidic environment (pH 6 PBS), pH-sensitive cleavage of the hydrazone linker resulted in the exponential sustained release of the drug, and an accumulative release was obtained for up to one week (168 h). These results were expected as the hydrazone bond is sensitive to pH 6, and the drug is consequently released from the nanoparticles. A burst release was achieved within 24 h of incubation at pH 6 reaching a release rate of \(52\%\pm 0.26\).