Design of Electrostatic Nanocomplex of Semaglutide with Protamine and Zinc for Subcutaneous Prolonged Delivery

Abstract

The aim of this study was to design a poorly water-soluble electrostatic nanocomplex of semaglutide (SMG) with protamine sulfate (PS) and zinc ions (Zn) for prolonged subcutaneous delivery. Complexation of SMG with the cationic peptide PS increased the lipophilicity (logP) proportionally from -4.7 to 0.3, particularly in the presence of Zn. The optimized nanocomplex exhibited spherical morphology, an amorphous state, a particle size of 196.0 nm, and a zeta potential of -45.7 mV. In an in vitro dissolution test under sink conditions, native SMG showed rapid drug release with 98% dissolution within 24 h. In contrast, the nanocomplexes showed markedly delayed release, with a concentration-dependent relationship between PS/Zn contents and SMG release rate, exhibiting 19% drug release over 7 days in the optimized formula. These findings suggest that the proposed nanocomplex is a promising system for long-acting injectable delivery of SMG, potentially enhancing patient compliance in patients with obesity or type 2 diabetes.

Keywords: electrostatic interaction; lipophilicity; nanocomplex; protamine sulfate; semaglutide; sustained release.

Figure 1

Chemical structure of SMG (a) and PS (b).

Figure 2

Changes in lipophilicity and solubility of SMG in aqueous media following complexation with PS in the absence and/or presence of Zn. (a) LogP (octanol/distilled water) values of SMG–PS complexes at varying SMG:PS ratios (5.36:0–5.36:1.2 mg/mL) in the presence of Zn (0.5 mg/mL), measured at 25 °C (b) Percentage of SMG remaining in solid-state form following the addition of complexes into phosphate-buffered saline (PBS), as a function of PS concentration (0–1.2 mg/mL) and Zn ion content (0 or 0.5 mg/mL) with 5.36 mg/mL of SMG, measured at 25 °C. Notes: Data represents mean ± SD (n = 3). N.D. means ‘not detected’. In panel (a), significant differences compared with 5.36:0 (* p < 0.05), 5.36:0.4 (** p < 0.05), and 5.36:0.8 (^† p < 0.05), respectively. In panel (b), significant difference compared with Zn 0 mg/mL (* p < 0.05).

Figure 3

Effects of suspending agents on particle size, surface charge, redispersibility, and the proportion of SMG in solid-sate (%) of SMG–PS–Zn complexes in PBS at 25 °C. (a) Particle size and zeta potential of complexes formulated with varying Na.CMC concentrations (0–10 mg/mL). (b) Redispersibility (%) of the complexes after centrifugation at 13,000 rpm. (c) The proportion of SMG in solid-sate (%) in PBS following dilution of complexes prepared with different Na.CMC concentrations. (d) Particle size and zeta potential of complexes prepared with varying concentrations of Kolliphor ELP (0–10 mg/mL) and 2.5 mg/mL Na.CMC. (e) Redispersibility (%) after centrifugation, and (f) The proportion of SMG in solid-sate (%) in PBS of complexes prepared with Kolliphor ELP/Na.CMC combinations. Notes: The concentration of SMG, PS, and Zn was set to 5.36, 1.2, 0.5, mg/mL, respectively. Data represents mean ± SD (n = 3). N.D means ‘not detected’. In panel (b), significant differences compared with Na.CMC 0 mg/mL (* p < 0.05), 5 mg/mL (^† p < 0.05), 7.5 mg/mL (^†† p < 0.05), and 10 mg/mL (^‡ p < 0.05), respectively. In panel (e), significant difference compared with ELP 0 mg/mL (* p < 0.05). In panels (c,f), no significant differences (p > 0.05) between formulations.

Figure 3

Effects of suspending agents on particle size, surface charge, redispersibility, and the proportion of SMG in solid-sate (%) of SMG–PS–Zn complexes in PBS at 25 °C. (a) Particle size and zeta potential of complexes formulated with varying Na.CMC concentrations (0–10 mg/mL). (b) Redispersibility (%) of the complexes after centrifugation at 13,000 rpm. (c) The proportion of SMG in solid-sate (%) in PBS following dilution of complexes prepared with different Na.CMC concentrations. (d) Particle size and zeta potential of complexes prepared with varying concentrations of Kolliphor ELP (0–10 mg/mL) and 2.5 mg/mL Na.CMC. (e) Redispersibility (%) after centrifugation, and (f) The proportion of SMG in solid-sate (%) in PBS of complexes prepared with Kolliphor ELP/Na.CMC combinations. Notes: The concentration of SMG, PS, and Zn was set to 5.36, 1.2, 0.5, mg/mL, respectively. Data represents mean ± SD (n = 3). N.D means ‘not detected’. In panel (b), significant differences compared with Na.CMC 0 mg/mL (* p < 0.05), 5 mg/mL (^† p < 0.05), 7.5 mg/mL (^†† p < 0.05), and 10 mg/mL (^‡ p < 0.05), respectively. In panel (e), significant difference compared with ELP 0 mg/mL (* p < 0.05). In panels (c,f), no significant differences (p > 0.05) between formulations.

Figure 4

Effects of PS and Zn concentrations on particle size, surface charge, suspended drug content (%), and drug precipitation (%) of SMG–PS–Zn nanocomplexes in PBS at 25 °C. (a) Particle size and zeta potential of complexes formulated with 0.4 mg/mL PS and varying Zn concentrations (0–0.5 mg/mL). (b) Suspended drug content (%) of SMG–PS–Zn complexes in the vehicle at corresponding PS and Zn levels. (c) The proportion of SMG in solid-state (%) in PBS following dilution of the above complexes. (d–f) Same analyses as panels (a–c) for complexes prepared with 0.8 mg/mL PS and different Zn concentrations (0–0.5 mg/mL). (g–i) Same analyses for complexes prepared with 1.2 mg/mL PS and varying Zn concentrations (0–0.5 mg/mL). Notes: Data represents mean ± SD (n = 3). In panels (b,e,h), significant differences compared with ZnCl₂ 0 mg/mL (* p < 0.05), 0.1 mg/mL (** p < 0.05), 0.2 mg/mL (^† p < 0.05), 0.3 mg/mL (^†† p < 0.05), and 0.4 mg/mL (^‡ p < 0.05), respectively. In panels (c,f,i), significant difference compared with ZnCl₂ 0 mg/mL (* p < 0.05).

Figure 5

FT-IR spectra of SMG, PS, and their electrostatic complexes prepared at various weight ratios: SMG–PS (5.36:1.2), SMG–PS–Zn (5.36:1.2:0.5), and SMG–PS–Zn–CMC (5.36:1.2:0.5:2.5). (a) Full spectral range from 500 to 4000 cm⁻¹. (b) Expanded region highlighting the shifts in the Amide I and II bands. (c) Fingerprint region showing amide I and amide II bands, along with additional characteristic peaks indicating newly formed bonding interactions. (d) Expanded region highlighting hydroxyl (O–H) and protonated amine (NH₃⁺) stretching vibrations. Note: Dashed boxes mark key absorption bands of interest. Remarkable wavenumbers were denoted in the FT-IR spectrum.

Figure 6

Representative SEM images of SMG raw material and SMG–PS–Zn nanocomplexes. (a) SMG raw material; (b) SMG–PS–Zn nanocomplex prepared at a 5.36:0.4:0.5 ratio (NC1); (c) nanocomplex at a 5.36:0.8:0.5 ratio (NC2); and (d) nanocomplex at a 5.36:1.2:0.5 ratio (NC3). Physical characteristics of SMG-PS-Zn nanocomplexes. (e) XRD patterns and (f) DSC curves of SMG raw material, blank vehicle, and nanocomplexes respectively. (g) Particle size distributions and (h) zeta potential profiles of the nanocomplexes (NC1, NC2, and NC3).

Figure 6

Representative SEM images of SMG raw material and SMG–PS–Zn nanocomplexes. (a) SMG raw material; (b) SMG–PS–Zn nanocomplex prepared at a 5.36:0.4:0.5 ratio (NC1); (c) nanocomplex at a 5.36:0.8:0.5 ratio (NC2); and (d) nanocomplex at a 5.36:1.2:0.5 ratio (NC3). Physical characteristics of SMG-PS-Zn nanocomplexes. (e) XRD patterns and (f) DSC curves of SMG raw material, blank vehicle, and nanocomplexes respectively. (g) Particle size distributions and (h) zeta potential profiles of the nanocomplexes (NC1, NC2, and NC3).

Figure 7

In vitro dissolution profiles of SMG raw material (X) and SMG–PS–Zn nanocomplexes with varying SMG:PS:Zn ratios 5.36:0.4:0.5 (NC1, ♦), 5.36:0.8:0 (■), 5.36:0.8:0.3 (●), 5.36:0.8:0.5 (NC2, ○), and 5.36:1.2:0.5 (NC3, ▲), respectively in PBS (pH 7.4, 37 °C) using a dialysis bag model. Note: Data represent mean ± SD (n = 3).