Structural aspects of dimeric prodrug-based carrier-free nanomedicines for tumor chemotherapy

Abstract

The effect of flexible linker length in dimeric prodrugs was explained through their aggregation structures, as revealed by molecular dynamics simulations. The results indicated that the aggregation structure of the dimeric prodrug was controlled by the molecular structure (mainly flexible linkers) as an internal factor and the fabrication methods as an external factor.

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Dimeric prodrugs have been recognized as one of the most promising types of carrier-free nanomedicines, owing to their high drug content, which is comparable to that of pure drugs, while minimizing premature drug leakage.^1–5 The ability of stimuli-labile dynamic covalent linkers between two drug molecules to regulate stimuli-triggered drug release performance has been widely investigated.¹ In addition to the tremendous efforts to control drug release using various stimuli-labile dynamic covalent linkers in the molecular structure of dimeric prodrugs, stimuli-triggered drug release can also be controlled by combining two dimeric prodrugs with different stimulus sensitivities.⁶

For the first time, the effects of the aggregation structures of dimeric prodrug-based nanomedicines have been explored using a disulfide/β-carbamate-bridged doxorubicin dimeric prodrug as a model.⁷ Dimeric prodrug-based carrier-free nanomedicines were fabricated by adding a DMF solution to water (NP1) or by adding water to a DMF solution (NP2), resulting in different in vitro drug release rates and antitumor efficacies. This result was explained by the distinct aggregation structures arising from π–π stacking interactions between the conjugated anthraquinone ring on doxorubicin (DOX), as revealed by UV-vis and fluorescence analysis.

Most recently, similar results were obtained in our studies, which were based on a pH/GSH dual-triggered disulfide/α-amide-bridged dimer (DDOX_SS)⁸ and a pH-triggered ketal-mediated dimer (DDOX_ketal)⁹ (Scheme 1). However, the two dimeric prodrug-based nanomedicines exhibited distinctly different porous structures, despite having short flexible linkers between the DOX units.

Scheme 1 Molecular structures of DDOX_SS and DDOX_ketal.

To explore the mechanism, the aggregation structures of the two dimeric prodrugs were investigated via molecular dynamics simulations in the present work, by fabricating nanomedicines through precipitation and self-assembly approaches, as follows:

NP1 via precipitation: 1.0 mL of dimeric prodrug solution in DMF (1.0 mg mL⁻¹) was added to 10 mL of water with stirring at room temperature at a rate of 1 drop per 10 s.

NP2 via self-assembly: 10 mL of water was added to 1.0 mL of dimeric prodrug solution in DMF (1.0 mg mL⁻¹) with stirring at room temperature at a rate of 1 drop per 10 s.

The dimeric prodrug-based nanomedicines were dialyzed against water (MWCO of 1 kDa) for 2 days to remove DMF and were finally collected via lyophilization.

At the same concentration, water/DMF volume ratio and dropping rate, NP1 and NP2 were obtained with different hydrodynamic diameters for the disulfide-/β-carbamate-bridged doxorubicin dimeric prodrug with a longer flexible linker,⁷ whereas similar hydrodynamic diameters and distributions were obtained for DDOX_SS and DDOX_ketal with short flexible linkers (Fig. S1, ESI†).

With similar hydrodynamic diameters, the stimuli-triggered drug release from NP1 and NP2 was compared to eliminate the effect of nanomedicine diameter.¹⁰ Compared with NP2, the NP1 nanomedicine exhibited faster stimuli-triggered drug release (Fig. 1). Although non-stimulated drug release was similar for both NP1 and NP2 nanomedicines in the absence of any corresponding stimulus, drug release from DDOX_ketal NP1 was faster than from DDOX_ketal NP2 from the onset of their stimulated medium (pH 5.0). In contrast, a noticeable difference appeared after 12 h for the DDOX_SS dimer prodrugs at pH 5.0 + 10 mM GSH. Moreover, the drug release from the NP1 nanomedicines was 1.19 and 1.12 times faster than that from the NP2 nanomedicines of the DDOX_SS and DDOX_ketal dimer prodrugs, respectively, in their stimulated media during the first 48 h (Fig. 1). The results indicated that the fabrication method had a more significant influence on DDOX_SS than on DDOX_ketal. DDOX_SS NP1 and DDOX_ketal NP1 exhibited lower half-maximal inhibitory concentrations (IC₅₀ values) of 4.94 μg mL⁻¹ and 11.13 μg mL⁻¹, respectively, compared to DDOX_SS NP2 and DDOX_ketal NP2, which had values of 8.53 μg mL⁻¹ and 12.54 μg mL⁻¹, respectively, on HepG2 cells. This difference was attributed to faster stimuli-triggered DOX release, as observed in the MTT assays (Fig. 2).

Fig. 1 In vitro DOX release profiles from the DDOX_SS and DDOX_ketal nanomedicines.

Fig. 2 In vitro cytotoxicity of the DDOX_SS and DDOX_ketal nanomedicines on HepG2 cells.

This disparity could be explained by the different aggregation structures formed by the different fabrication approaches. In the precipitation method for the NP1 nanomedicines, there was not enough time for the self-assembly of the dimeric prodrugs via π–π stacking. Hence, the dimeric prodrugs were precipitated and aggregated into nanomedicines with a loose architecture, favouring the diffusion and accessibility of stimulus signals (such as H⁺ ions and GSH) and subsequent drug release. For the NP2 nanomedicines, a more compact architecture was formed via adequate π–π stacking, hindering the diffusion and accessibility of the stimulus signals. This explanation was revealed through UV-vis and fluorescence analysis of the dimeric prodrug-based carrier-free nanomedicines (Fig. 3). The significant redshift in the UV absorption and distinct fluorescence quenching in the fluorescence spectra at an excitation wavelength of 485 nm demonstrated the π–π stacking interaction between the dimeric prodrugs.^11–14 Furthermore, a more significant redshift and fluorescence quenching were observed for the NP2 nanomedicines, revealing that a more compact aggregation structure was formed with more well-defined π–π stacking interactions during the slow self-assembly.

Fig. 3 UV-vis and fluorescence spectra of the DDOX_SS and DDOX_ketal nanomedicines.

In the TEM observations, mesopores of approximately 8 nm were observed in both the DDOX_ketal NP1 and DDOX_ketal NP2 nanomedicines (Fig. 4), whereas no porous structure was observed in the DDOX_SS nanomedicines, regardless of the fabrication method. The N₂ adsorption–desorption technique was used to assess the porous structure of the DDOX_SS nanomedicines. DDOX_SS NP1 had a greater BET surface area of 0.1407 cm³ g⁻¹ and a larger average pore diameter (7.4427 nm) than DDOX_SS NP2 (0.0791 cm³ g⁻¹ and 4.9235 nm, respectively).⁸ These results demonstrated that the different porous structures, resulting from the different fabrication methods, favour the diffusion and accessibility of the stimulus signals, accelerating the stimuli-triggered cleavage of the dynamic covalent linkers and the release of the protonated DOX.

Fig. 4 TEM images of the DDOX_SS and DDOX_ketal nanomedicines.

However, different porous structures were formed in the dimeric-based prodrug nanomedicines using the same fabrication method. The results indicated that the length of the flexible linker might play an important role in the aggregation structures of dimeric prodrug-based nanomedicines, specifically in the compactness of the aggregated dimeric prodrugs. Molecular dynamics (MD) simulation is a key technology used to link the microcosm and macrocosm and can simulate the specific process of intermolecular interactions, explaining the experimental observations at the microscopic level.¹⁵ To explore the compactness of the aggregated dimeric prodrugs, their molecular interactions via π–π stacking were simulated with the Dmol³ software in bimolecular (Fig. 5a) and trimolecular (Fig. 5b) modes. In the molecular dynamics simulation at the lowest energy levels, regardless of the multimolecular mode, the DDOX_SS dimer with a longer flexible linker containing disulfide/α-amide conjugation and the two sugar moieties on DOX clearly presented a curly molecular topological structure, whereas the DDOX_ketal dimer presented an extended molecular topological structure due to its short flexible ketal linker between the rigid conjugated anthraquinone rings on the two DOX units. The higher rigidity of the DDOX_ketal dimer made it difficult to adopt a more compact stacking arrangement, resulting in the formation of mesopores. The molecular dynamics simulation results demonstrated that the flexible linker length had a significant effect on the aggregation structure of the DOX-based dimeric prodrugs via π–π stacking interactions.

Fig. 5 Molecular dynamics simulation of DDOX_SS and DDOX_ketal: bimolecular mode (a) and trimolecular mode (b).

Conclusions

In summary, the effect of flexible linker length in the dimeric prodrugs on their stimuli-triggered DOX release was investigated by comparing the in vitro drug release profiles of dimeric prodrug-based nanomedicines fabricated using different methods. This effect was explained by the aggregation structures of the dimeric prodrug-based carrier-free nanomedicines and revealed through molecular dynamics simulation. The results demonstrated that the aggregation structure of dimeric prodrugs, in addition to their molecular structures, was a decisive factor in their in vitro antitumor efficacy. However, the aggregation structure was influenced by the internal factor (i.e., molecular structure) and the external factor (i.e., the fabrication methods). This understanding is expected to inspire new ideas in the research and development of high-performance, dimeric prodrug-based carrier-free nanomedicines for tumor chemotherapy, featuring on-demand and controlled drug release.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02850c

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