## 4 Conclusions  
In conclusion, a covalent strategy for the development of a PEGylated therapeutic nanosystem for pH-sensitive release was developed. Better anti-tumor activity was shown in cells treated with the nanoparticles than free DOX. The IC50 value was reduced by half by the conjugation of doxorubicin to the nanoparticle, compared to free doxorubicin, demonstrating the therapeutic capacity of DOX-NPs (**7**). This nanodevice has a proven capability of releasing the drug in a controlled manner at acidic pH and improving the drug therapeutic index by increasing efficacy compared to doxorubicin in solution. In addition, it was demonstrated to be stable for up to 6 months at 4 \({}^{\circ}\)C and 25 \({}^{\circ}\)C. The next step will involve conjugating other drugs in clinical use to this nanodevice using the same chemical strategy; thereafter, applying this therapeutic strategy to other types of cancer; and in the long term, to other  
Figure 4: Evaluation of the DNA damage caused by the treatment with DOX-NPs (**7**) by immunostaining of \(\gamma\)-H2 Ax in A549, H520, and MDA MB 231 cancer cell lines analyzed by confocal microscopy. The untreated cells (UNT) (**a**) and cells treated with NAKED-NPs (**1**) (**b**) were used as negative controls. Cells treated with DOX-NPs (**7**) were analyzed without (**c**) and with (**d**) a primary anti-\(\gamma\)-H2 Ax antibody. Images with an increase of 63x show a composition of the four channels used: blue, DAPI for the nucleus staining; red, DOX-NPs (**7**); and green, for H2 Ax staining with Alexa488-antiphospho-H2 Ax. pathologies. Future work will be focused on in vitro and in vivo preclinical characterization to move this nanodevice closer to clinical use. The following are available online at [[http://www.mdpi.com/2073-4360/12/6/1265/s1](http://www.mdpi.com/2073-4360/12/6/1265/s1)]([http://www.mdpi.com/2073-4360/12/6/1265/s1](http://www.mdpi.com/2073-4360/12/6/1265/s1)), supplementary figures: Figure S1. Calibration standard curve of concentration of nanoparticles (OD 600) by spectrophotometry. Figure S2. Numbers of NPs per mL calculation. Figure S3. Calibration standard curve of doxorubicin solution (OD 480) by spectrophotometry. Figure S4. Calibration standard curve of doxorubicin solution by HPLC. Figure S5. Dose-response curves (percentage of cell viability versus concentration) of treatment with DOX-NPs (7) in the cell models studied, represented in NPs/Cell. Figure S6. Dose-response curves (percentage of cell viability versus concentration) of treatment with free doxorubicin in the cell models studied. General protocol for cellular nanofection. Conceptualization, [PERSON]-M. and [PERSON]-M.; Methodology, M.V.C.-C. and R.M.S.-M.; Investigation, M.V.C.-C. and J.A.L.-R.; Formal analysis, M.V.C.-C. and J.A.L.-R.; Supervision, [PERSON]-M. and [PERSON]-M.; Writing--Original draft preparation, M.V.C.-C. and R.M.S.M. ; Writing--Review and editing, [PERSON]-M. and [PERSON]-M.; Project administration, [PERSON]-M. and [PERSON]-M.; Funding acquisition, [PERSON]-M. and R.M.S.-M. All authors have read and agreed to the published version of the manuscript. This research was funded by the Instituto de Salud Carlos III (ISCIII) (grant number DTS18/00121) co-funded by European Union (ERDF/ESF, \"A way to make Europe\"//Investing in your future\"), the Spanish Ministry of Economy and Competitiveness (MINECO) (grant number BIO2016-80519) and the Andalusian Regional Government (grant number PAIDI-TC-PVT-PSETC-2.0.). The authors thank the Research Results Transfer Office (OTRI) of the University of Granada for their support for the technological development of this project. We thank the technical support of Microscopy and Molecular Image Unit and Flow Cytometry from GENYO. The authors thank the technical support of the Mass Spectrometry and Chromatography Unit of the Scientific Instrumentation Center of the University of Granada, in particular to [PERSON]. The authors are members of the network NANOCARE (RED2018-102469-T) funded by the STATE INVESTIGATION AGENCY. J.A.L.R.