Example 2

Peptide colloids were prepared with either Ac-FLIVI-KKKKK-CO-NH₂(SEQ ID NO:1, where X is F, “h₅F-L”) or Ac-VLIVI-KKKKK-CO-NH₂(SEQ ID NO:1, where X is V, “h₅V-L”), in high purity soy oil were tested for average size and zeta potential (surface charge) using light scattering on a ZetaPlus particle sizing instrument. The results for the two peptides are shown in the table below. A control was prepared using high purity soy oil mixed with water and sonicated.

TABLE

h₅F-L

Elapsed Time 00:02:30

Median Diam. 363.8 nm

Mean Diam. 408.4 nm

Polydispersity 0.260

GSD 1.618

d(nm)
G(d)
C(d)

164.8
26
5

196.3
44
10

221.0
58
15

242.6
70
20

263.0
80
25

282.7
87
30

302.3
93
35

322.1
97
40

342.4
99
45

363.8
100
50

386.5
99
55

410.8
97
60

437.8
93
65

468.1
87
70

503.1
80
75

545.5
70
80

598.8
58
85

674.1
44
90

802.7
26
95

h₅V-L

Elapsed Time 00:02:30

Median Diam. 216.5 nm

Mean Diam. 236.8 nm

Polydispersity 0.197

GSD 1.528

d(nm)
G(d)
C(d)

107.8
26
5

125.7
44
10

139.6
58
15

151.5
70
20

162.7
80
25

173.4
87
30

183.9
93
35

194.5
97
40

205.2
99
45

216.5
100
50

228.4
99
55

241.0
97
60

254.8
93
65

270.3
87
70

288.1
80
75

309.3
70
80

335.8
58
85

372.7
44
90

434.7
26
95

The h₅F-L peptide yielded a mean diameter of around 410 nm with a polydispersity index (PDI) of 0.23 (upper panel) while the h₅V-L peptide (lower panel) gave a smaller mean diameter of around 240 nm with a PDI of 0.2. PDI values of less than 0.3 are considered monodispersed in the pharmaceutical industry. The sizes of the suspended lipid droplets in the control sample were larger than that observed for the peptide colloids, and increased over time as the lipid droplets further coalesced, ultimately separating from the water to form an oil layer on top of the water. The polydispersity index for the control was higher than 0.3.

Regarding zetal potential, the larger h₅F-L particle had a value of 46±2.5 mV while the smaller h₅V-L particle had a value of 43±1.6 mV. These very positive values are the result of the highly cationic surface of the particles that are imparted by the presence of the oligo-lysine segments that are solvent exposed. These values are also an indicator of the stability of the particles in terms of remaining mono-dispersed. Positive zeta potentials are also associated with efficient cellular uptake of nanoparticles. The values observed for these new colloids are similar to those observed for of patented branched amphipathic peptide capsules that are readily take up by different cell types.

Given that particle sizes for both peptides were above the nanoscale cutoff of 100 nm, the samples were subjected to resizing by extruding them through 100 nm polycarbonate filters. The h₅F-L particles were resized to about 80 nm after extrusion (data not shown). The h₅V-L particles could be resized to about 90 nm.

Example 3

The ability of LEAPs to deliver a lipid soluble active ingredient to an organism was tested using the antifungal agent Captan: (3aR,7aS)-2-[(Trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione. This compound is readily soluble in solvents such as acetone and xylene but 4000-fold less in water. The compound also has good solubility in the C₃organic acid, propionic acid. It should be noted that propionic acid by itself has anti-fungal activity. For this experiment two different peptides were tested: h₅F-L and h₅V-L.

A 0.5% solution of Captan was prepared in a 1:1 mixture of propionic acid and soy oil (final volume of 10 μL for each peptide). The solution was added to 1 mg of dried peptide. After incubating for 15 min, water (490 μL, pH=3) was added dropwise, then sonicated in a bath sonicator for 5 min at 37° C. and allowed to stand for 20 min. The sample was then centrifuged at room temperature for 10 min at 14K×g. At this point each peptide-lipid complex formed a whitish film on the surface of the water. The volume of water was reduced using a Pipetman leaving a final volume of 150 μL. The solution was again vortexed to resuspend the colloids. An illustration of the colloidal particle with the active ingredient distributed throughout the colloidal body is shown in FIG. 3. A number of different controls were tested along with the peptides prepared with all of the components: soy oil only, soy oil+propionic acid, soy oil+propionic acid+Captan. Volumes of from 5 to 40 μL were spotted on spore seeded plates of Aspergillus nidulans grown on 1% skim milk agar gel) yielding 0.33 and 1.33 μg of active agent delivered, respectively (FIG. 4).

In FIG. 4, lawns of A. were seeded just before testing. The actively growing fungus appears greenish yellow in color. For controls (FIG. 5A) soy oil and soy oil+propionic acid (1:1, v/v) were applied directly to the plates at the indicated volumes. The 40 μL soy oil (FIG. 5A, column 1) shows some antifungal activity however when it is diluted in half in the 1:1 soy plus propionic acid (FIG. 5A, column 2) this effect is greatly diminished. When the 1:1 soy plus propionic acid was separately encased using the two peptides (FIG. 5B, columns 1 and 2) there was minimal antifungal activity. In FIG. 5C, column 1, Captan was dissolved in the propionic acid prior to mixing with the soy oil. The topical application of this mixture was not very effective as an anti-fungal agent. When this mixture was encased in the h5V-L peptide (FIG. 5C, column 2) a small amount of antifungal activity was observed. However, when the mixture was encased in the h₅F-L peptide (FIG. 5C, column 3) complete growth inhibition of the fungus was observe as judged by the reappearance of the white milk-agar surface. This result indicates that the Captan is most effective at killing when complexed with oil and internalized by the organism using the LEAP system.

Example 4

In this example, the effectiveness of Captan alone or encased in a LEAP colloidal particle on A. nidulans growth was compared. Different concentrations of Captan were tested for growth inhibition when applied directly to freshly seeded fungi growing on 1% milk plates or after encapsulation as a colloidal particle with a LEAP (h₅F-L). Captan was dissolved in a formulated (1:1:2) solution containing propionic acid, soy oil, and a pesticide synergist, Piperonyl butoxide (PBO). The resulting active solution was diluted to the following concentrations: 1%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%. A volume of 50 μL of the 1:1:2 solution containing the fungicide was combined with 1 mmole of dry peptide and then allowed to stand for 20 minutes. Deionized water was then added to the colloidal mixture to raise the total volume to 1 mL, followed by sonicating the mixture for 5 minutes, and then incubating for 60-90 minutes. A volume of 50 μL for each test solution was added to the center of each plate (FIG. 6).

The LEAP colloidal particles containing the actives were significantly more efficient in delivering inhibitory concentrations compared to applying the compound alone. They exhibited near complete growth inhibition, even at the lowest doses of active ingredient. The Captan by itself showed strong growth inhibition only at the highest concentration (left column). There was only slight inhibition observed with the 0.1% concentration. Using LEAPs to prepare colloidal particles containing the active agents showed growth inhibition at all concentrations tested (right column). These results are attributable to to the improved cellular uptake of the LEAP colloidal particles. It will be appreciated that employing this technology could permit growers to use greatly reduced amounts of active ingredients without affecting the effectiveness of the treatment.

Example 5

In this study, we looked at the effects of the Captan fungicide formulation with and without the LEAP peptide when applied to the leaves of Vicia faba (Fava beans). Three formulations were prepared for this experiment:

Soy oil
Proponic acid
Captan
LEAP (h₅F-L)

1
50 μL
50 μL
—
—

2
50 μL
50 μL
5 mg/mL
—

3
50 μL
50 μL
5 mg/mL
1 mM final

concentraion

Control
—
50 μL in water
—
—

Control
50 μL in water
—
—
—

Deionized water was added to each formulation to a final volume 1 mL and allowed to stand at room temperature for 60-90 min. These formulations were each used to spray leaves of V. faba with effects registered 10 min after spraying the plants and 48 h after treatment.

As expected, the leaves treated with water alone or soy oil in water showed no deleterious effects. However, propionic acid in water caused black spots on the leaves after 24 hr (data not shown). The leaves sprayed with formulations containing soy oil combined to propionic acid also exhibited extreme damage after 24 h with leaf death around 48 h (FIG. 7A). Nearly identical results were observed on leaves treated with the solution containing soy oil, propionic acid, and Captan (FIG. 7B), with leaves showing a visible damage observed as black spots and leaf death around 96 h (not shown). Meanwhile, the same components when encapsulated in colloidal particles via the LEAPs exhibited only healthy leaves (FIG. 7C) with no signs of damage, even after 5 days (not shown).

When formulated solutions requiring propionic acid to dissolve the Captan came in contact with the leaves, it caused visible damage, observed as black spots or in some cases total damage of the leaf. When analyzing each component of the formulation isolated, it was confirmed that propionic acid was the ingredient responsible for the toxic effect on the leaves. Interestingly, when combined in the LEAP formulation though, the damage caused by propionic acid was suppressed, suggesting that the LEAP colloidal particles are able to promote a protective result.