A Liposomal Strategy for Dual-Action Therapy in Sarcopenia: Co-Delivery of Caffeine and HAMA

Abstract

The biological complexity of sarcopenia presents a major challenge for therapeutic intervention due to the wide range of degenerative changes it induces in skeletal muscle. This study demonstrates the potential of liposomal controlled release systems to address these challenges by combining two bioactive agents with complementary actions: caffeine (CAF), encapsulated in DMPC-based liposomes, and hyaluronic acid methacrylate (HAMA), encapsulated in DOPC-based liposomes. A hybrid system was also developed to deliver both substances simultaneously, aiming to restore tissue function through combined metabolic, anti-inflammatory, and regenerative effects. The liposomes exhibited nanoscale dimensions, spherical morphology, and intact membrane structure, as confirmed by electron microscopy. DLS analysis indicated good colloidal stability and monodisperse size distribution across all formulations, with improved stability observed in the hybrid system. Drug release studies showed a time-dependent profile, with HAMA releasing rapidly and CAF releasing gradually, supporting a dual-action therapeutic approach tailored to the multifactorial pathology of sarcopenia. The biological assays, performed in an established in vitro sarcopenia model, revealed the potential of liposomes co-delivering caffeine and HAMA to mitigate oxidative stress, preserve mitochondrial function, and reduce apoptosis in H₂O₂-damaged myotubes.

Keywords: caffeine; hyaluronic acid methacrylate; liposomal hybrid drug delivery system; sarcopenia.

Figure 1

The conditions necessary to maintain healthy skeletal muscle tissue and the factors that influence and are found in the case of sarcopenia.

Scheme 1

Schematic representation of the hybrid liposomal formulation design and therapeutic mechanism.

Figure 2

DLS results as hydrodynamic diameter (nm) registered for DMPC_Ctrl, DMPC_CAF, DOPC_Ctrl, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 3

DLS result as zeta potential (mV) registered for DMPC_Ctrl, DMPC_CAF, DOPC_Ctrl, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 4

SEM micrographs (20,000×, 40,000×) obtained for DMPC_Ctrl, DMPC_CAF, DOPC_Ctrl, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 4

SEM micrographs (20,000×, 40,000×) obtained for DMPC_Ctrl, DMPC_CAF, DOPC_Ctrl, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 5

EDS results obtained for DMPC_Ctrl, DMPC_CAF, DOPC_Ctrl, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 6

Liposome size distributions obtained for DMPC_Ctrl, DMPC_CAF, DOPC_Ctrl, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 7

Encapsulation efficiency of caffeine and HAMA, evaluated for DMPC_CAF, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA.

Figure 8

Drug release rate of caffeine and HAMA evaluated for DMPC_CAF, DOPC_HAMA, and DMPC + CAF_DOPC + HAMA depending on time (8 h = 480 min).

Figure 9

Viability of C2C12 myotubes assessed by MTT assay after 48 h of treatment with DMPC_CAF, DOPC_HAMA, and the combined DMPC + CAF_DOPC + HAMA formulation. Except for the experimental control (untreated C2C12 myotubes), all groups were exposed to H₂O₂ to induce a sarcopenia-like phenotype. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 6 software. Significance levels compared to the control group are indicated as follows: p ≤ 0.05 (*), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Figure 10

Intracellular ROS levels in C2C12 myotube cultures after 48 h of treatment with DMPC_CAF, DOPC_HAMA, and the combined DMPC + CAF_DOPC + HAMA formulation. Except for the experimental control (untreated C2C12 myotubes), all groups were exposed to H₂O₂ to induce a sarcopenia-like phenotype. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism. Significance levels compared to the control group are indicated as follows: p ≤ 0.01 (**) and p ≤ 0.0001 (****).

Figure 11

Mitochondrial membrane potential (MMP) in C2C12 myotubes cultures after 48 h of treatment with DMPC_CAF, DOPC_HAMA, and the combined DMPC + CAF_DOPC + HAMA formulation. Except for the experimental control (untreated C2C12 myotubes), all groups were exposed to H₂O₂ to induce a sarcopenia-like phenotype. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism. Significance levels compared to the control group are indicated as follows: p ≤ 0.0001 (****).

Figure 12

Detection of apoptosis by flow cytometry after labeling with Annexin V FITC/PI in C2C12 myotube cultures after 48 h of treatment with DMPC_CAF, DOPC_HAMA, and the combined DMPC + CAF_DOPC + HAMA formulation. (A) Representative flow cytometry dot-plots showing annexin V(⁻)/PI(⁻) viable cells in the lower left quadrant, annexin V(⁺)/PI(⁻) early apoptotic cells in the lower right quadrant, annexin V(⁺)/PI(⁺) late apoptotic cells in the upper right quadrant, and annexin V(⁻)/PI(⁺) necrotic cells in the upper left quadrant. (B) Graphical representation of the distribution of the cells (% cell percentage) between viable, early apoptotic, late apoptotic, and necrotic cells after 48 h of treatment. Except for the experimental control (untreated C2C12 myotubes), all groups were exposed to H₂O₂ to induce a sarcopenia-like phenotype.