ABSTRACT Cancer has emerged as a leading global cause of mortality. The management of cancer continues to provide a significant challenge. Recent advancements in nanotechnology have led to a significant interest in layered double hydroxide (LDH)-based nanosystems. These nanosystems have gained attention due to their favourable characteristics such as biocompatibility, pH-dependent biodegradability, anion exchange capacity, easy surface modification, and high chemical stability. As a result, they hold great promise for cancer therapy. Over the last ten years, researchers have developed LDH-based nanosystems for a range of cancer treatments. We examined the alterations in the crystal structure and morphology caused by hydrothermal treatment of LDH for 24, 48 hours and the non-hydrothermally treated LDH. We have shown that the time of the hydrothermal treatment may effectively regulate the nanostructures of LDHs, employing scanning electron microscopy, Zeta potential and hydrodynamic diameter measurements, and X-ray diffraction. Our investigation revealed that the hydrothermal treatment induced alteration in the structure of the LDHs. During the initial stage, crystallization took place within the first 24 hours of the hydrothermal treatment, resulting in an expansion of the interlayer width and an increase in the hydrodynamic diameter size. After that, the most prominent occurrences were the stacking and planar expansion that took place as the crystallization process neared equilibrium. Consequently, the previously formed layered structure was also arranged in a stacked manner, causing the LDH to extend in a flat direction. Furthermore, the different treated LDHs greatly affected the loading of chemotherapeutic drug, as the time of hydrothermal treatment increase the concentration of the loaded chemotherapeutic drug decrease. Our research indicates that the length of time the hydrothermal treatment is applied is a critical element in the development of the structure of LDHs. This component can be utilized to manipulate the structure of LDHs for optimal application as drug carrier.