Studying the Effect of Independent Variables on Formulation

The successful application of nanocarriers as drug delivery systems necessitates a comprehensive understanding of their interactions with Biological systems. Key factors influencing nanocarrier uptake and distribution include particle size, surface charge, and surface functionalization. Optimal nanocarrier dimensions facilitate cellular internalization, typically ranging from 50 to 100 nm. Concurrently, a balanced surface charge minimizes non-specific interactions and promotes cellular uptake. Nanocarriers can be further engineered to enhance targeting specificity and drug delivery efficiency by incorporating specific ligands onto their surface. By conjugating proteins, antibodies, or other biomolecules, nanoparticles can be directed at specific cell membrane receptors, increasing intracellular drug concentrations and reducing therapeutic dosages. This study investigates the impact of these parameters on the physicochemical properties and drug delivery potential of LDH-based nanoparticles [7]. Developing efficient drug delivery systems hinges on a comprehensive understanding of nanocarrier characteristics and their interactions with biological environments. This study investigated the impact of key parameters, including particle size, surface charge, and drug loading, on the physicochemical properties and drug delivery potential of LDH-based nanocarriers.

Particle Size

Table 1 summarizes the particle size (PS) values obtained from the fifteen experimental runs (F1-F15). As depicted in Fig. 1A, PAC concentration had negligible effects on LDH particle size.

Fig. 1

The effect of (A) PAC concentration, (B) BSA concentration, and (C) post-synthesis treatment on the hydrodynamic diameter of LDH nanoparticles. The solid Lines represent the predicted responses from a D-optimal experimental design, with dashed LInes indicating the 95% confidence intervals. Particle size was measured by dynamic light scattering (DLS). All data points represent the mean ± standard deviation (SD) of three independent experiments (n = 3)

In contrast, BSA functionalization, Fig. 1B, significantly influenced particle size (p < 0.0001). Increasing BSA concentration led to a gradual increase in particle size, reaching a maximum of 8 mg/mL BSA, followed by a subsequent decrease. BSA functionalization of the optimized formula significantly influenced particle size, leading to an increase at high BSA concentrations and subsequent stabilization at higher levels. BSA-LDH particles produced at high BSA concentrations (16 mg/mL) exhibited an average size of 100 nm, slightly larger than that of d-LDH (89 nm). This suggests effective de-aggregation of BSA-LDH hybrids, enhancing particle stability. The observed size increase is primarily attributed to the formation of a BSA hydrophilic corona around the LDH particles, rather than random aggregation [29]. These findings align with those reported by Gu et al., who demonstrated that BSA functionalization of LDH particles significantly enhanced their stability in pH 7.4 buffer and water when using a high BSA: LDH ratio (25:2) [29].

The post-synthesis process (Fig. 1C) significantly influenced LDH particle morphology and, consequently, drug loading capacity. Delamination produced nanosheets with increased surface area and accessible interlayer spaces, as evidenced by the TEM micrographs showing smaller, less porous d-LDH-PAC particles than c-LDH-PAC. This structural modification enabled improved drug entrapment. In addition, calcination resulted in larger, porous c-LDH-PAC particles, as observed in TEM images, which, despite potential benefits for drug diffusion, hindered overall drug loading due to partial pore obstruction during reconstruction, as reported by Kim et al. These findings point to the importance of LDH structural integrity in optimizing drug delivery systems, which points to the possibility of delamination as a promising approach for enhancing the performance of LDH-based drug delivery platforms.

Drug Entrapment Efficiency

As shown in Fig. 2A, %EE was highly affected by the formulation variables, with a wide range of %EE from 1.5 ± 0.2% to 51.2 ± 0.5%. In contrast, Fig. 2B illustrates that BSA functionalization minimally influenced PAC entrapment efficiency (%EE). Conversely, drug concentration (Fig. 2A) and post-synthesis treatment (Fig. 2C) significantly impacted (p < 0.0001 for both variables) the percentage of entrapped drug. An inverse relationship was observed between drug concentration and EE%, primarily attributed to the saturation of available adsorption sites on LDH particles at higher drug loadings [44]. Additionally, hydrophobic interactions between drug molecules, leading to aggregation, competed with drug-LDH interactions, hindering entrapment within the LDH interlayer spaces [44].

Fig. 2

The effect of (A) PAC concentration, (B) BSA concentration, and (C) post-synthesis treatment on the drug entrapment efficiency (EE) of LDH nanoparticles. The solid Lines represent the predicted responses from a D-optimal experimental design, with dashed LInes indicating the 95% confidence intervals. All data points represent the mean ± standard deviation (SD) of three independent experiments (n = 3)

Regarding the post-synthesis treatment described in Sect. 2.2.1, the particles were delaminated using alcohol (d-LDH) and sonication or calcinated via oven treatment (c-LDH). The delamination/calcination process increases the surface area of the particles to increase drug adsorption; hence, the percentage of drug entrapment efficiency is expected to increase. Figure 2C illustrates a significantly higher drug entrapment efficiency for delaminated LDH (d-LDH) than calcined LDH (c-LDH).

The observed high drug loading capacity aligns with the results reported by Mei et al. for monolayered LDH nanosheets (3.6 mg doxorubicin per mg LDH), further supporting the potential of delaminated LDH as a drug delivery platform [45, 46].

Zeta Potential

Figure 3A and C demonstrate negligible changes in particle zeta potential with variations in drug concentration or post-synthesis treatment. In contrast, BSA concentration significantly influenced zeta potential (p < 0.0001) as depicted in Fig. 3B.

Fig. 3

The effect of (A) PAC concentration, (B) BSA concentration, and (C) post-synthesis treatment on the drug entrapment efficiency (EE) of LDH nanoparticles. The solid Lines represent the predicted responses from a D-optimal experimental design, with the dashed LInes indicating the 95% confidence intervals. All data points represent the mean ± standard deviation (SD) of three independent experiments (n = 3)

LDH nanocarriers, characterized by a positive zeta potential of + 27 mV, exhibited an initial propensity for cellular uptake due to electrostatic interactions with cancer cell membranes. However, their aggregation in physiological conditions necessitated surface modification. BSA functionalization was the primary determinant of LDH zeta potential, significantly shifting the charge from positive to negative. In contrast, drug concentration and post-synthesis treatment had minimal effects on the zeta potential. Notably, increasing BSA concentration resulted in a more negative zeta potential, which can be associated with improved tumour accumulation for particles of approximately 150 nm [47, 48]. BSA coating enhanced stability, facilitated targeting via Gp60 receptors, and induced a negative zeta potential shift, aligning with BSA’s intrinsic charge [47, 48].

Design Validity and Optimization

Formulation variables influencing BSA-LDH-PAC nanocarrier characteristics were optimized using a D-optimal design by minimizing PS and maximizing EE% and ZP. The optimal formulation (F3) was determined using desirability function analysis, yielding a desirability value of 0.993. F3 comprises 16 mg/mL BSA, 0.5 mg/mL PAC, and d-LDH. The freshly formed aqueous dispersion of F3 was subjected to measurements of its particle size, %EE, and zeta potential, resulting in values of 95 ± 0.14 nm, 51 ± 0.54%, and − 27 ± 0.1 mV, respectively. The outcomes of the freshly produced sample were contrasted with those ascertained using the D-optimal design. The observed lower magnitude of error (9.8%, 2.7%, and 1.8%) for mean particle size, %EE, and zeta potential, respectively, suggests that there are no significant changes and/or there is a good agreement between the prior and present experimental data. These results demonstrate the robustness of the optimization model in accurately predicting real-world outcomes, as further supported by supplementary data (Table S2) [24].

Physicochemical Characterization of the Optimized Formula F3 (BSA-d-LDH-PAC)Molecular Modelling and FTIR

As shown in molecular modelling diagram of LDH (Fig. 4A and B), LDH crystals assemble in layers of Mg and Al hydroxides bonded together via hydrogen bonding which is confirmed via FTIR (Fig. 5A) of d-LDH showing a very broad band at around 3450 cm − 1 belongs to OH stretching of the hydroxide layer and water with the broadness evident of hydrogen bond formation [49]. The characteristic peaks at 1307 cm − 1 are characteristic of the NO3 − group [50], in addition to strong bands at 290 cm−1and 400 cm − 1, whichre attributed to the vibrations of Mg–O and Al–O [51]. The band at about 1009 cm − 1 can be attributed to the deformation vibration of water molecules in the interlayer domain [52]. BSA successfully functionalized LDH functionalization is due to the formation of hydrogen bonds between the OH group in LDH and BSA at multiple sites, which was also confirmed in the IR spectrum of BSA-LDH-PAC, showing characteristic peaks of BSA present in the pure BSA IR spectrum. Moreover, PAC also interacted with both BSA and LDH through hydrogen bonding, resulting in the successful loading of PAC on the surface of LDH [53]. These interactions are evident in BSA-LDH-PAC IR spectrum which retained the characteristic peaks of LDH, BSA and PAC at 2965 cm−1 (C–H), 1707 cm − 1 (CO group), 1641 cm−1 (C–C stretch), 1370 cm−1 (CH3), 1248 cm−1 (C–N), 1072 cm−1 (C–O) and 709 cm − 1 (C–H off the plane) [53] reflecting the successful strong incorporation of the drug within the particles and accounting for the high percentage of drug entrapped.

Fig. 4

Molecular modelling data illustrating interactions between different components for (A) LDH crystals as acquired from Materials Project and processed using Avogadro Software, (B-D) Molecular modelling data via Maestro software comprising (B) LDH layers attached via hydrogen bonds (yellow dotted line), (C) Pac molecule (red) and LDH(green) interactions via hydrogen bonds (yellow) and aromatic via hydrogen bonds (cyan blue), (D) LDH (green) and albumin (white) interactions via hydrogen bonds (yellow), aromatic hydrogen bonds (cyan blue) and Pi interactions (pink) and (E) PAC molecule (red) and albumin (white) interactions via hydrogen bonding (yellow), aromatic hydrogen bonding (cyan blue)

Fig. 5

Fourier-transform infrared (FTIR) spectra of individual components and the final nanocomposite. The graph shows the vibrational spectra of plain d-LDH (dotted black line), PAC (dashed red line), BSA (solid purple line), and the final BSA-d-LDH-PAC nanocomposite (dashed-dotted green line)

Transmission Electron Microscopy (TEM)

TEM micrographs (Fig. 6A-B) reveal a brucite-like (hexagonal) morphology for both d-LDH-PAC and c-LDH-PAC particles, consistent with previous reports [29]. However, c-LDH-PAC particles exhibit a larger size compared to d-LDH-PAC, aligning with dynamic light scattering data (Sect. 3.1). Furthermore, c-LDH-PAC displays a porous structure.

Fig. 6

Transmission electron microscope (TEM) images of (A) de-intercalated LDH-PAC (d-LDH-PAC) and (B) co-precipitated LDH-PAC (c-LDH-PAC) captured at 80 kV with a 200 nm scale bar

Differential Scanning Calorimetry (DSC)

Figure 7 presents the DSC thermograms of pure PAC and the optimized F3 formulation. PAC exhibited a sharp endothermic peak at 218 °C, consistent with the literature [53]. The endothermic peak shifted to 200 °C. The crystalline nature of pure paclitaxel was confirmed by the presence of a sharp endothermic peak at 218 °C in the DSC thermogram, consistent with the literature [53]. In contrast, the broader and less intense endothermic peak observed for the paclitaxel-loaded LDH formulation at 200 °C in the DSC thermogram indicated a reduction in drug crystallinity.

Fig. 7

DSC thermogram of PAC and BSA-d-LDH-PAC

X-ray Diffraction (XRD)

This decrease in crystallinity was further corroborated by the XRD diffractogram (Fig. 8), which compares the XRD diffractograms of pure PAC and PAC incorporated into LDH particles. The diffraction pattern of BSA-d-LDH-PAC exhibits broader and less intense peaks compared to pure PAC, confirming decreased drug crystallinity within LDH particles [39]. The reduced crystallinity of the drug within the LDH particles likely contributed to the enhanced drug solubility and subsequent release profile observed in Sect. 3.3.5 [54, 55].

Fig. 8

XRD diffractograms of (blue) PAC and (red) BSA-d-LDH-PAC

In Vitro Drug Release

Figure 9 illustrates the pH-dependent release profile of PAC from drug powder and BSA-LDH-PAC formulations. PAC’s poor water solubility resulted in negligible release at pH 7.4. LDH exhibited a sustained release pattern at physiological pH, while a pronounced burst release was observed at pH 5.5, with 90% drug release within 5 h [56].

Fig. 9

In vitro drug release profiles of PAC from BSA-LDH-PAC and free PAC at either pH 5.5 or pH 7.4 (n = 6)

To elucidate the release mechanism of the BSA-LDH-PAC complex, drug release kinetics were fitted to zero-order, first-order, Higuchi, and Korsmeyer-Peppas models. The Korsmeyer-Peppas model exhibited the best fit (R² = 0.961 for pH 5.5 and 0.942 for pH 7.4), with a release exponent (n) of 0.4. The pH-responsive behaviour of LDH-based nanocarriers is a key attribute for targeted drug delivery. The observed pH-dependent release profile, with a pronounced burst release at pH 5.5, suggests the potential for effective drug delivery in the acidic tumour microenvironment. This pH-responsive behaviour is attributed to the protonation of LDH surfaces at acidic pH, disrupting drug-LDH interactions and facilitating drug release. Conversely, at higher pH, electrostatic interactions between the drug and LDH inhibit rapid release, leading to a sustained profile. These findings align with previous reports on the pH-dependent drug release behaviour of LDHs [56]. The Korsmeyer-Peppas model suggests a quasi-Fickian diffusion mechanism where the drug diffuses from the LDH interlayer via ion exchange. Notably, the formulation’s pH-responsive release behaviour, with enhanced drug release in acidic environments, is particularly advantageous for cancer therapy.

Cytotoxicity MTT Assay

Figure 10 depicts the cytotoxic effects of plain LDH, PAC suspension, and BSA-LDH-PAC on KF-28 cells. Plain LDH exhibited negligible cytotoxicity, confirmed by microscopic images as shown in Fig. 11, confirming its suitability as a safe drug carrier, as confirmed by microscopic images in Fig. 11. Free PAC displayed a cytotoxic effect over the range of concentrations from 10 to 250 µg/mL with a calculated IC50 of 60.2 µg/mL. Notably, BSA-LDH-PAC displayed a sharper decrease in cytotoxicity compared to PAC, with a considerably lower IC50 of 52.7 µg/mL, indicating higher efficacy. Statistical analysis revealed that at lower concentrations (0 and 10 µg/mL), no significant difference was observed between PAC and BSA-LDH-PAC (p > 0.05). However, starting from 25 µg/mL, BSA-LDH-PAC exhibited significantly lower viability compared to PAC (p < 0.05). The superior efficacy of BSA-LDH-PAC compared to pure PAC suspension, which is evident in both higher cytotoxic effects and lower IC50, can be attributed to the nanocarriers’ ability to be internalized by cells via endocytosis due to their 95 nm size, in addition to their better release profile compared to free PAC [2]. The superior efficacy of BSA-functionalized LDH-entrapped PAC compared to the free drug highlights the potential of this system for overcoming PAC’s bioavailability limitations, improving therapeutic outcomes, and reducing systemic side effects. Moreover, BSA-LDH has proven to be a biocompatible, non-toxic carrier due to its low cytotoxicity, which paves the way for its use as a biocompatible drug delivery system. Further studies are warranted to evaluate the in vivo efficacy of this formulation and to explore its applicability to other therapeutic agents.

Fig. 10

%Cell viability percentages of KF-28 treated with different concentrations of free PAC, BSA-d-LDH-PAC, and plain BSA-LDH (n = 3)

Fig. 11

The microscopic images of KF-28 cells after treatment with the BSA-d-LDH-PAC, PAC, and BSA-d-LDH

Stability Studies

Stability studies conducted over six months demonstrated no significant changes (p > 0.05) in particle size, drug entrapment efficiency, and zeta potential compared to freshly prepared samples (Table 2), indicating satisfactory sample stability.

Table 2 Stability of the optimized formulation (F3) after 6 months of storage. On the particle size (PS), entrapment efficiency (%EE), and zeta potential (ZP). Data are presented as the mean ± standard deviation from three independent experiments (n = 3)

Chemotherapy, exemplified by paclitaxel (PAC), often induces severe systemic side effects due to indiscriminate drug distribution. To address this, a targeted drug delivery system utilizing layered double hydroxide (LDH) nanocarriers was developed. The developed BSA-functionalized LDH nanocarriers offer a promising platform for paclitaxel delivery. PAC was successfully loaded onto delaminated LDH (d-LDH) and subsequently stabilized with bovine serum albumin (BSA) to enhance stability, facilitate cellular uptake, and improve drug targeting via the GP60 pathway. Optimization of formulation parameters using D-optimal design led to the identification of an optimal formulation (F3) characterized by desirable properties including particle size (95 nm), drug entrapment efficiency (51%), and zeta potential (−27 mV). These results validate the stability of the formulation after six months, which would be beneficial in its translation from the lab to industrial applications. However, further studies under accelerated and long-term conditions, including hot temperature, humidity, and light exposure, are warranted to fully establish the long-term robustness and translational potential of the formulation.