Biodynamer Nano-Complexes and -Emulsions for Peptide and Protein Drug Delivery

Figures & data

Table 1 Surface Charge (ζ) and Mw of Model Cargos; ζ, Hydrodynamic Diameter (D_H), and Mw of Biodynamers

Model cargos	ζ (mV)		Mw
OVA	−15.3		44.3 kDa
IH	−16.3*		5.8 kDa
GLP-1	+8.6		4.4 kDa
Biodynamers	ζ (mV)	DH (nm)	Mw
R-biodynamer	+47.1	10.7	155.0 kDa^±
E-biodynamer	−11.6	7.5	67.3 kDa^±
RF-biodynamer	+37.5	8.5	90.5 kDa^±

Note: All of the cargo was measured in DI water (10 mg/mL), except IH. *The IH was measured in 10 mM phosphate buffer (PB, 1 mg/mL) due to its solubility. ^±The Mw of biodynamers was measured using static light scattering (SLS).

Scheme 1 Formation of PNCs through electrostatic Interaction between R-biodynamer and f-OVA.

Figure 1 D_H (left y-axis) and PDI (right y-axis) of f-OVA-PNCs at different weight ratio. White bar is D_H and red dot is PDI (A). Zeta potential of f-OVA-PNCs at different weight ratios (B). Size distribution of f-OVA-PNCs at 10: 5 (w/w), measured by DLS (C). Morphology of f-OVA-PNCs at 10: 5 (w/w), measured by SEM (D).

Figure 2 EE%, LC%, and theoretical loading capacity (TLC%) of f-OVA-PNCs at different weight ratios (A). Physical stability of 100 μg/mL f-OVA-PNCs (10: 5, w/w) for 48 h (B).

Table 2 D_H, PDI, and DCR of OVA-, IH-, and GLP-1-Loaded PNCs for 2 h

PNC Components	Incubation Time (h)	DH (nm)	PDI	DCR (kcps)
OVA-PNCs-R	0	234	0.32	6940
	2	1422	0.50	5410
IH-PNCs-R	0	356	0.33	68,655
	2	1260	0.23	54,460
GLP-1-PNCs-E	0	145	0.25	7857
	2	912	0.60	1793

Figure 3 pH-Responsive size change of 100 μg/mL f-OVA-PNCs (10: 5, w/w) in DI water, pH 7.4 (10 mM PB), and pH 5.0 (10 mM AB) for 6 h (A). Acid-responsive degradation of f-OVA-PNCs (10: 5, w/w) in DI water, pH 7.4 (10 mM PB), and pH 5.0 (10 mM AB) for 48 h (B). The comparison of mean size changes in PNCs at 0 and 6 h under pH 7.4 and pH 5.0, respectively, was conducted using a T-test. P** ˂ 0.05 was considered significant, and p^ns > 0.05 were no statistical difference.

Scheme 2 Preparation of Protein-loaded DE-NPs via DE method.

Table 3 D_H (SD ≤ ± 50), PDI (SD ≤ ± 0.05), ζ (SD ≤ ± 0.7), and DCR (SD ≤ ± 3000) of f-OVA-DE-NPs

f-OVA-DE-NPs	DH (nm)	PDI	ζ (mV)	DCR (kcps)
10% f-OVA	166	0.28	+4.8	18345
20% f-OVA	268	0.29	+4.7	9001
30% f-OVA	283	0.46	+4.4	4297

Figure 4 Size distribution of DE-NPs (R-biodynamer: f-OVA, 10: 1, w/w) in aqueous solution (A). Morphology of DE-NPs (R-biodynamer: f-OVA, 10: 1, w/w), measured by SEM (B).

Figure 5 EE%, LC% and TLC% of f-OVA loaded DE-NPs with various amounts of PLGA (R-biodynamer: f-OVA: PLGA, 10: 1: x, w/w/w) (A). Stability of f-OVA-DE-NPs-0% PLGA (B) and f-OVA-DE-NPs-10% PLGA (C) in DI water for 48 h.

Table 4 D_H and PDI of OVA (or Insulin, or GLP-1)-DE-NPs-R Measured After 48 h at R.t

	With X% PLGA	DH (nm)	PDI
OVA-DE-NPs-R	0%	214 ± 49	0.48 ± 0.11
	10%	189± 48	0.26 ± 0.02
IH-DE-NPs-R	0%	200 ± 11	0.45 ± 0.07
	10%	183± 34	0.30 ± 0.09
GLP-1-DE-NPs-R	0%	199 ± 9	0.3 ± 0.09
	10%	189± 62	0.30 ± 0.02

Figure 6 pH-responsive size change of 100 μg/mL f-OVA-DE-NPs-0% PLGA and DE-NPs-10% PLGA in DI water, pH 7.4 (10 mM PB), and pH 5.0 (10 mM AB) for 6 h (A). Acid-responsive degradation of f-OVA-DE-NPs-0% PLGA (B) and DE-NPs-10% PLGA (C) in DI water, pH 7.4 (10 mM PB), and pH 5.0 (10 mM AB) for 48 h. The comparison of mean size changes in PNCs at 0 and 6 h under pH 7.4 and pH 5.0, respectively, was conducted using a T-test. P** ˂ 0.05 was considered significant, and p^ns > 0.05 were no statistical difference.

Figure 7 Release dynamic f-OVA in PNCs (A), DE-NPs-0% PLGA (B), and DE-NPs-10% PLGA (C) in pH 5.0 (10 mM acetate buffer) and pH 7.4 (10 mM PB)₂.

Figure 8 Cell viability of f-OVA-loaded NPs on A549 cells analyzed by MTT assay. White bar is f-OVA-PNCs-R (10: 5, w/w); light gray bar is f-OVA-DE-NPs-0% PLGA; dark gray bar is f-OVA-DE-NPs-10% PLGA. (n=6). A one-way ANOVA was employed to assess the statistical differences in cell viability among PNCs, DE-NPs-0% PLGA, and DE-NPs-10% PLGA. The red lines represent the standard values of the control group.

Figure 9 A549 cellular uptake of f-OVA-loaded NPs after 6 h incubation. DAPI stained nucleus in blue, LysoTracker™ Red DND-99 stained endosome/lysosome in red, and f-OVA in green.