Delivering amoxicillin at the infection site – a rational design through lipid nanoparticles

Figures & data

Figure 1 AMX-loaded LNPs, which were designed to release AMX near H. pylori. The double-emulsion LNPs are composed of cetyl palmitate, Tween 80, linolenic acid, and DOPE.

Abbreviations: AMX, amoxicillin; DOPE, dioleoylphosphatidylethanolamine; H. pylori, Helicobacter pylori; LNPs, lipid nanoparticles.

Table 1 Composition and physical characterization (size, PDI, zeta potential, and LC) of the four AMX-loaded LNPs and the corresponding unloaded LNPs

Formulation	P1	F1	P2	F2	P3	F3	P4	F4
mg
Cetyl palmitate	178.5	178.5	178.5	178.5	178.5	178.5	178.5	178.5
Tween 80	130	130	125	125	110	110	105	105
Linolenic acid	–	–	5	5	–	–	5	5
DOPE	–	–	–	–	20	20	20	20
AMX	–	19.9	–	19.9	–	19.9	–	19.9
Size (nm)	291±5	284±11	243±17	240±16	237±12	236±18	178±9	197±2
PDI (±0.02)	0.14	0.14	0.12	0.12	0.12	0.14	0.11	0.12
ζ (mV)	-48±1	-40±1	-39±5	-46±4	-42±4	-37±4	-42±2	-43±3
LC (%)	–	6.3±0.1	–	6.8±0.4	–	5.7±0.6	–	7.5±0.2

Abbreviations: AMX, amoxicillin; DOPE, dioleoylphosphatidylethanolamine; LC, loading capacity; LNPs, lipid nanoparticles; PDI, polydispersity.

Figure 2 TEM images of the AMX-loaded LNPs and the corresponding unloaded LNPs.

Notes: P1, F1, P2, F2, F3, P4A, and F4A are at a magnification of 50,000×. P3 is at a magnification of 25,000×. P4B and F4B are at a magnification of 100,000×.

Abbreviations: AMX, amoxicillin; LNPs, lipid nanoparticles; TEM, transmission electron microscopy.

Figure 3 Characterization of the LNPs suspensions. (A) LNPs size and PDI. Bars represent the size (left y-axis) and dots represent the PDI (right y-axis). (B) LNPs zeta potential. (C) LNPs LC. The parameters were evaluated over time (0 months [dark grey], 3 months [intermediate grey] and 6 months [light grey]).

Notes: Values represent the mean±SD of three independently produced formulations. *P<0.05, **P<0.01, ****P<0.0001 relative to 0 month.

Abbreviations: LC, loading capacity; LNPs, lipid nanoparticles; PDI, polydispersity.

Figure 4 In vitro AMX release profiles from the LNPs in three simulated conditions, namely (i) pH 1.6, (ii) pH 5.0, and (iii) pH 7.4.

Notes: Vertical lines represent media changes. Values represent the mean±SD of three independently produced formulations.

Abbreviations: AMX, amoxicillin; LNPs, lipid nanoparticles.

Figure 5 In vitro cell viability studies. (A) L929 cell viability study. (B) MKN-74 cell viability study. Different formulations in different solid lipid concentrations, from 0 (black) to 8 (light gray) mg/mL of solid lipid were evaluated. For free AMX, the same amount of AMX existent in those concentrations of LNPs was used, with the exception of 1 and 4 mg/mL.

Notes: Values represent mean±SD of three independently produced formulations. *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001 relative to 0 mg/mL.

Abbreviations: AMX, amoxicillin; LNPs, lipid nanoparticles.

Table 2 Percentage of permeability and apparent permeability (P_app) of the different AMX-loaded LNPs after 3 hours of incubation in different setups: without cells/with mucins; with cells/without mucins; and with cells/with mucins

	%Permeability			Papp × 10^−5 (cm/s)
	Without cells	With cells		Without cells	With cells
Mucins	X	–	X	X	–	X
F1	30±1	<LOD	<LOD	3.35±0.06	<LOD	<LOD
F2	31±5	<LOD	<LOD	3.4±0.5	<LOD	<LOD
F3	31±5	<LOD	<LOD	3.5±0.6	<LOD	<LOD
F4	31±4	<LOD	<LOD	3.4±0.4	<LOD	<LOD

Abbreviations: AMX, amoxicillin; LNPs, lipid nanoparticles; P_app, apparent permeability; LOD, limit of detection.

Figure 6 Characterization of the AMX-loaded LNPs suspensions before (dark gray) and after (light gray) the incubation with mucins. (A) LNPs size and PDI. Bars represent the size (left y-axis) and dots represent the PDI (right y-axis). (B) LNPs zeta potential.

Notes: Values represent the mean±SD of three independently produced formulations. *P<0.05, **P<0.01, ****P<0.0001 relative to the LNPs without mucins.

Abbreviations: AMX, amoxicillin; LNPs, lipid nanoparticles; PDI, polydispersity.