Skin penetration enhancing composition and use thereof in preparation of skin delivery formulation

Abstract

The present disclosure discloses a skin penetration enhancing composition and a use thereof in preparation of a skin delivery formulation. The skin penetration enhancing composition comprises sponge spicules and nanoparticles. The nanoparticles comprise at least one of one or more drugs or one or more cosmetic active ingredients.

Claims

1. A skin penetration enhancing composition, comprising: sponge spicules, and nanoparticles, wherein: the sponge spicules are derived from Haliclona sp., and the nanoparticles comprise flexible nanoliposomes and at least one of: one or more drugs, or one or more cosmetic active ingredients.

2. The skin penetration enhancing composition according to claim 1, wherein: the nanoparticles further comprise nanocarriers comprising at least one of the one or more drugs or the one or more cosmetic active ingredients, or the nanoparticles further comprise nanosized particles of the at least one of the one or more drugs or the one or more cosmetic active ingredients.

3. The skin penetration enhancing composition according to claim 2, wherein the nanocarriers comprise at least one of solid lipid nanoparticles, nanocapsules, nanospheres, polymer micelle, or nanosuspensions.

4. The skin penetration enhancing composition according to claim 1, wherein a purity of the sponge spicules is not less than 90%.

5. The skin penetration enhancing composition according to claim 1, wherein: the sponge spicules are a sponge spicules suspension, the sponge spicules suspension is utilized in combination with the nanoparticles, the sponge spicules suspension is prepared by at least one of a buffer solution, deionized water, double distilled water, or physiological saline, and a mass concentration of the sponge spicules is from 0.01% to 100%.

6. The skin penetration enhancing composition according to claim 1, wherein the flexible nanoliposomes comprise a surfactant.

7. A method of treatment comprising: using the skin penetration enhancing composition according to claim 1 for preparing a skin delivery formulation.

8. The method of treatment according to claim 7, comprising: applying the sponge spicules of the skin delivery formulation to skin after the skin has been cleaned, and applying the nanoparticles of the skin delivery formulation to the skin after applying the sponge spicules to the skin.

9. The method of treatment according to claim 8, comprising: massaging the skin while applying the nanoparticles.

10. The method of treatment according to claim 9, comprising: cleaning to remove residual sponge spicules of the skin delivery formulation on a surface of the skin before applying the nanoparticles.

11. The method of treatment according to claim 7, comprising: directly applying the skin delivery formulation on the skin after the skin has been cleaned.

12. The method of treatment according to claim 11, comprising: massaging the skin while applying the skin delivery formulation.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 illustrates appearances and structures of the sponge spicules under a microscope.

(2) FIGS. 2A-2C illustrate a comparative view of skin penetration and deposition of ANTS-Fucoidan from a control group and from a group using a sponge spicules suspension in Embodiment 1. The concentration of ANTS-Fucoidan is 10 mg/mL, the concentration of the sponge spicules suspension is 100 mg/mL (equivalent to a mass concentration of 10%), a solvent involved is 100 μL of PBS 0.2 M (Mol/L) PBS (pH 7.4), and these components and conditions are the same across embodiments. FIG. 2A illustrates a distribution profile of ANTS-Fucoidan in different skin layers. FIG. 2B illustrates a fluorescence image of ANTS-Fucoidan from a skin section of the control group. FIG. 2C illustrates a fluorescence image of ANTS-Fucoidan from a skin section of the group using the sponge spicules suspension. The scale of FIGS. 2B and 2C is 100 μm, and these components are the same across embodiments.

(3) FIGS. 3A-3C illustrate a comparative view of skin penetration and deposition of ANTS-Fucoidan from the control group and a group using the sponge spicules suspension and flexible nanoliposomes in Embodiment 1. A concentration of phospholipid (90G) is 4%, a concentration of polyoxyethylene (20) oleyl ether (surfactant) is 1.2%, and these components are the same across embodiments. FIG. 3A illustrates a skin distribution profile of ANTS-Fucoidan in different skin layers. FIG. 3B illustrates a fluorescence image of ANTS-Fucoidan from a skin section of the control group. FIG. 3C illustrates a fluorescence image of ANTS-Fucoidan from a skin section of the group using the sponge spicules suspension and the flexible nanoliposomes.

(4) FIG. 4 illustrates a comparative view of a total skin absorption of ANTS-Fucoidan from the control group, the group using the sponge spicules suspension, and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 1.

(5) FIGS. 5A-5C illustrate a comparative view of skin penetration and deposition of FITC-Hyaluronic acid from a control group and a group using the sponge spicules suspension in Embodiment 2. A concentration of FITC-Hyaluronic acid is 1 mg/mL, the concentration of the sponge spicules suspension is 100 mg/mL (equivalent to a mass concentration of 10%), a solvent involved is 100 μL of PBS (0.2 M, pH 7.4), and these components and conditions are the same across embodiments. FIG. 5A illustrates a distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 5B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the control group. FIG. 5C illustrates a fluorescence view of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension.

(6) FIGS. 6A-6C illustrate a comparative view of skin penetration and deposition of FITC-Hyaluronic acid from the control group and a group using the sponge spicules suspension and the conventional nanoliposomes in Embodiment 2. The concentration of phospholipid (90G) is 4%, and these components are the same across embodiments. FIG. 6A illustrates a distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 6B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the control group. FIG. 6C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension and the conventional nanoliposomes.

(7) FIGS. 7A-7C illustrate a comparative view of skin penetration and deposition of FITC-Hyaluronic acid from the control group and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2. The concentration of phospholipid (90G) is 4%, and the concentration of polyoxyethylene (20) oleyl ether (surfactant) is 1.2%, and these components are the same across embodiments. FIG. 7A illustrates a distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 7B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the control group. FIG. 7C illustrates a fluorescence view of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension and the flexible nanoliposomes.

(8) FIG. 8 illustrates a comparative view of a total skin absorption of FITC-Hyaluronic acid from the control group, the group using the sponge spicules suspension, the group using the sponge spicules suspension and the conventional nanoliposomes, and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2.

(9) FIGS. 9A-9C illustrate a comparative view of a total skin absorption of FITC-Hyaluronic acid from a group using the conventional nanoliposomes and the group using the sponge spicules suspension and the conventional nanoliposomes in Embodiment 2. FIG. 9A illustrates a skin distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 9B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the conventional nanoliposomes. FIG. 9C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension and the conventional nanoliposomes.

(10) FIGS. 10A-10C illustrate a comparative view of a total skin absorption of FITC-Hyaluronic acid from a group using the flexible nanoliposomes and a group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2. FIG. 10A illustrates a skin distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 10B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the flexible nanoliposomes. FIG. 10C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension and the flexible nanoliposomes.

(11) FIGS. 11A-11C illustrate a comparative view of a total skin absorption of FITC-Hyaluronic acid from a group using microneedles and a group using the sponge spicules suspension in Embodiment 2. FIG. 11A illustrates a skin distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 11B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the microneedles. FIG. 11C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension.

(12) FIGS. 12A-12C illustrate a comparative view of a total skin absorption of FITC-Hyaluronic acid from a group using microneedles and the flexible nanoliposomes and a group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2. FIG. 12A illustrates a distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 12B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the microneedles and the flexible nanoliposomes. FIG. 12C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using the sponge spicules suspension and the flexible nanoliposomes.

(13) FIG. 13 illustrates a comparative view of a transepidermal water loss over 360 hours of a control group, a group using the microneedles, a group using sponge spicules suspension and massaging by hand, and a group using sponge spicules suspension and massaging by an electric massager in Embodiment 2.

(14) FIGS. 14A-14E illustrate a comparative view of morphologies of sponge spicules derived from Haliclona sp. with a high purity, sponge spicules derived from Haliclona sp. with a low purity, sponge spicules derived from Calcareous sponge, sponge spicules derived from Tethya sp., and sponge spicules derived from Mycale sp. The scale of FIGS. 14A-14E is 100 μm. FIG. 14A illustrates the sponge spicules derived from Haliclona sp. with the high purity. FIG. 14B illustrates the sponge spicules derived from Haliclona sp. with the low purity. FIG. 14C illustrates the sponge spicules derived from Calcareous sponge. FIG. 14D illustrates the sponge spicules derived from Tethya sp. FIG. 14E illustrates the sponge spicules derived from Mycale sp.

(15) FIG. 15 illustrates a comparative view of effects on skin barrier of different species and different morphologies of the sponge spicules.

(16) FIGS. 16A-16C illustrate a comparative view of a skin absorption of FITC-Hyaluronic acid in different skin layers after applied with the sponge spicules derived from Tethya sp. and the sponge spicules derived from Haliclona sp. with a high purity in Embodiment 10. FIG. 16A illustrates a distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 16B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the sponge spicules derived from Tethya sp. FIG. 16C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the sponge spicules derived from Haliclona sp. with a high purity.

(17) FIGS. 17A-17C illustrate a comparative view of a skin absorption of FITC-Hyaluronic acid in different skin layers from a group using Tethya sp. and the flexible nanoliposomes and a group with using Haliclona sp. with a high purity and the flexible nanoliposomes in Embodiment 10. FIG. 17A illustrates a distribution profile of FITC-Hyaluronic acid in different skin layers. FIG. 17B illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using Tethya sp. and the flexible nanoliposomes. FIG. 17C illustrates a fluorescence image of FITC-Hyaluronic acid from a skin section of the group using Haliclona sp. with a high purity and the flexible nanoliposomes.

(18) FIGS. 18A-18L illustrate results of a skin irritation test by using the sponge spicules derived from Haliclona sp. with a high purity and the sponge spicules derived from Haliclona sp. with a low purity. FIG. 18A illustrates a picture of skin immediately (0 h) after being treated by the sponge spicules derived from the Haliclona sp. with a high purity. FIG. 18B illustrates a picture of skin immediately (0 h) after being treated by the sponge spicules derived from the Haliclona sp. with a low purity. FIG. 18C illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a high purity for 24 h. FIG. 18D illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a low purity for 24 h. FIG. 18E illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a high purity for 48 h. FIG. 18F illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a low purity for 48 h. FIG. 18G illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a high purity for 96 h. FIG. 18H illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a low purity for 96 h. FIG. 18I illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a high purity for 168 h. FIG. 18J illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a low purity for 168 h. FIG. 18K illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a high purity for 240 h. FIG. 18L illustrates a picture of skin after being treated by the sponge spicules derived from the Haliclona sp. with a low purity for 240 h.

(19) FIG. 19 illustrates a comparative view of a time-recovery curve of a barrier function of the skin after the skin being treated by the sponge spicules derived from Haliclona sp. with a high purity (purified spicules) and the sponge spicules derived from Haliclona sp. with a low purity (unpurified spicules).

DETAILED DESCRIPTION OF THE EMBODIMENTS

(20) The present disclosure will be further described below with the combination of the accompanying drawings together with the embodiments.

Embodiment 1

(21) (1) Pre-treatment of porcine skin: fresh porcine skin were obtained and the subcutaneous adipose tissue was removed. The porcine hair shaft was cut off to no more than 2 mm. The obtained skin was used immediately or stored at −20° C.-−80° C. for use.

(22) (2) Preparation of flexible nanoliposomes: a preset ratio of phospholipid 90 g (100 mg/mL), polyoxyethylene (20) oleoyl ether (100 mg/mL) as a surfactant, and methanol/chloroform at a 1:1 ratio was added into a round flask. A uniform film was formed on a bottom surface of the round flask by evaporating to dryness in a rotary evaporator. A solution of 1-5 mL comprising ANTS-Fucoidan was added into the round flask and a liposomal solution was obtained by film hydrating by flask shaking or ultrasonic dispersion, etc. A liposome extrusion device was used to squeeze the liposomal solution for 21 times, and it was finally transferred into an Eppendorf (EP) tube to obtain a solution of flexible nanoliposomes comprising ANTS-Fucoidan with a concentration of 1-100 mg/mL.

(23) (3) Preparation of a sponge spicules suspension: a preset amount of PBS buffer with a molar concentration of 0.01-0.2M (mol/L) and a pH of 6.5-7.5, deionized water, physiological saline, double distilled water, or any solvent without any risk of skin irritation was mixed with the sponge spicules to obtain the sponge spicules suspension with a mass concentration of 0.01-100%.

(24) (4) Skin penetration experiment in vitro: porcine skins with the same sizes were punched out with a round puncher. The porcine skins were mounted on a Franz diffusion cell. Transcutaneous electrical resistance of the porcine skins was measured in vitro to evaluate conductivity variation of the porcine skins before and after an application of the sponge spicules suspension. Before the application of the sponge spicules suspension, the porcine skins with the conductivity less than 10 μA were considered a good stratum corneum barrier of skin and can be used in following experiments. The conductivity of the porcine skins was increased to 40-150 μA after the application of the sponge spicules suspension, indicating that stratum corneum barrier of skin was disrupted by sponge spicules with varying degrees. A solution of 200 μL comprising ANTS-Fucoidan (an average molecular weight is 60 kDa) with a concentration of 1-100 mg/mL (a group using the sponge spicules suspension) or a solution comprising the flexible nanoliposomes encapsulating ANTS-Fucoidan with a concentration of 1-100 mg/mL (a group using the sponge spicule suspension and the flexible nanoliposomes) were applied on the skin evenly. The skin were incubated for 16 hours at 37° C. with stirring (600 r/min), and then the skin was taken out. The penetration enhancing effect of the sponge spicules on ANTS-Fucoidan was determined by both a quantitative method and a qualitative method.

(25) (5) The quantitative method: ten layers of stratum corneum (SC) and active epidermis were separated from the skin by using a tape stripping method (Ten SC layers were collected according to the following scheme: SC 1=first strip, SC 2=second-fifth strips, and SC 3=sixth-tenth strips). After tape-stripping, the stratum corneum and the active epidermis was separated from dermis of the skin. The dermis was then cut into small pieces. A mixture (4 mL) of methanol and PBS (1:1, V/V) was used to extract ANTS-Fucoidan from separated skin layers at room temperature (i.e., 20-25° C.) with a speed of 200 r/min for 8-24 hours. The concentration of ANTS-Fucoidan in the stratum corneum, the active epidermis, the dermis, and receptor phase were determined by a full-wavelength microplate reader at the detection wavelength of ANTS (excitation wavelength: 350 nm, and emission wavelength: 520 nm). Referring to FIGS. 2A-2C, as a result, compared with the control group, a total skin absorption in skin layers of ANTS-Fucoidan from the group using the sponge spicules suspension increased from 0.01% to 4.83%. Referring to FIGS. 3A-3C, a total skin absorption of ANTS-Fucoidan in skin layers from the group using the sponge spicules suspension and the flexible nanoliposomes greatly increased to 24.76%.

(26) (6) The qualitative method: porcine skin sections with the same sizes were punched out by a round puncher with a diameter of 5 mm and then immediately frozen in optimal cutting temperature (OCT) compound and sectioned at a thickness of 20 μm to obtain skin section slices. The skin section slices were sealed by neutral resin. Referring to FIGS. 2-4, skin distributions of ANTS-Fucoidan in different skin layers from the control group, the group using the sponge spicules suspension, and the group using the sponge spicules suspension and the flexible nanoliposomes were observed by a confocal microscope.

(27) FIG. 1 illustrates appearances and structures of the sponge spicules under a microscope. FIGS. 2A-2C illustrate comparative results of skin absorption of ANTS-Fucoidan from the control group and the group using the sponge spicules suspension in Embodiment 1 (comprising a distribution profile of ANTS-Fucoidan in the skin layers and a fluorescence image of ANTS-Fucoidan from the skin section slices). FIGS. 3A-3C illustrate comparative results of skin absorption of ANTS-Fucoidan from the control group and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 1. FIG. 4 illustrates a comparative view of a total skin absorption of ANTS-Fucoidan from the control group, the group using the sponge spicules suspension, and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 1.

Embodiment 2

(28) (1) Pre-treatment of a porcine skin: the same as that in Embodiment 1.

(29) (2) Preparation of conventional nanoliposomes: a preset ratio of phospholipid 90 g (100 mg/mL) and methanol/chloroform at a ratio of 1:1 was added into a round flask. A uniform film was formed on a bottom surface of the round flask by evaporating to dryness in a rotary evaporator. A solution of 1-5 mL comprising FITC-Hyaluronic acid was added into the round flask and a liposomal solution was obtained by film hydrating by flask shaking or ultrasonic dispersion, etc. A liposome extrusion device was used to squeeze the liposomal solution for 21 times, and it was finally transferred into an Eppendorf (EP) tube to obtain a solution of conventional nanoliposomes comprising FITC-Hyaluronic acid with a concentration of 1-100 mg/mL.

(30) (3) Preparation of flexible nanoliposomes: similar with that in Embodiment 1.

(31) (4) Preparation of a sponge spicules suspension: the same as that in Embodiment 1.

(32) (5) Skin penetration experiment in vitro: similar with Embodiment 1, a control group (a solution comprising FITC-Hyaluronic acid), a conventional nanoliposomes group (a solution comprising conventional nanoliposomes and FITC-Hyaluronic acid), and a flexible nanoliposomes group (a solution comprising flexible nanoliposomes and FITC-Hyaluronic acid). The solution of 150 μL comprising FITC-Hyaluronic acid (an average molecular weight of 250 KDa) with a concentration of 1-100 mg/mL (a group using the sponge spicules suspension), the solution comprising the conventional nanoliposomes and FITC-Hyaluronic acid with a concentration of 1-100 mg/mL (a group using the sponge spicules suspension and the conventional nanoliposomes), or the solution comprising the flexible nanoliposomes and FITC-Hyaluronic acid with a concentration of 1-100 mg/mL (a group using the sponge spicules suspension and the flexible nanoliposomes) were applied on the skin evenly. The skins were incubated for 16 hours at 37° C. with stirring (600 r/min), and the skins ware taken out. The penetration enhancing effect of the sponge spicules suspension on skin penetration of ANTS-Fucoidan was determined by both a quantitative method and a qualitative method.

(33) (6) The quantitative method: similar with that in Embodiment 1. Skin deposition of FITC-Hyaluronic acid in stratum corneum, active epidermis, dermis, and receptor phase were determined by a full-wavelength microplate reader at the detection wavelength of FITC (excitation wavelength: 490 nm, and emission wavelength: 530 nm). Referring to FIG. 5, as a result, compared with the control group, a total skin absorption of FITC-Hyaluronic acid in skin layers from the group using the sponge spicules suspension increased from 1.2% to 6.92%. Referring to FIG. 6, a total skin absorption of FITC-Hyaluronic acid in skin layers from the group using the sponge spicules suspension and the conventional nanoliposomes group increased to 9.01%. Referring to FIG. 7, a total skin absorption of FITC-Hyaluronic acid in skin layers from the group using the sponge spicules suspension and the flexible nanoliposomes greatly increased to 37.89%.

(34) (7) The qualitative method: similar with that in Embodiment 1. Referring to FIGS. 6-8, skin distributions of FITC-Hyaluronic acid in different skin layers from the control group, the group using the sponge spicules suspension, the group using the sponge spicules suspension and the conventional nanoliposomes, and the group using the sponge spicules suspension and the flexible nanoliposomes were observed by a confocal microscope.

(35) FIGS. 5A-5C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from the control group and the group using the sponge spicules suspension in Embodiment 2. FIGS. 6A-6C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from the control group and the group using the sponge spicules suspension and the conventional nanoliposomes in Embodiment 2. FIGS. 7A-7C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from the control group and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2. FIG. 8 illustrates a comparative view of a total skin absorption of FITC-Hyaluronic acid from the control group, the group using the sponge spicules suspension, the group using the sponge spicules suspension and the conventional nanoliposomes, and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2. FIGS. 9A-9C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from the group using of the sponge spicules suspension and the conventional nanoliposomes, and a group using the conventional nanoliposomes in Embodiment 2. FIGS. 10A-10C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from the group using the sponge spicules suspension and the flexible nanoliposomes, and a group using the flexible nanoliposomes in Embodiment 2.

(36) From the results, the skin penetration enhancing effect of the group using the sponge spicules suspension and the conventional nanoliposomes is better than those of the control group, the group using the sponge spicules suspension, and the group using the conventional nanoliposomes, which indicates that a combination use of the sponge spicules and the conventional nanoliposomes had synergistic effect on improving skin drug delivery and therefore significantly increased the skin absorption of drug. In addition, the group using the sponge spicules and the flexible nanoliposomes show much better skin penetration enhancing effect than the control group, the group using the sponge spicules suspension, and the group using the flexible nanoliposomes, which indicates that a combination use of the sponge spicules and flexible nanoliposomes had synergistic effect on improving skin drug delivery and therefore significantly increased the skin absorption of drug. Moreover, the synergy effect induced by the combination use of the sponge spicules and the flexible nanoliposomes is much better than that induced by the combination use of the sponge spicules and the conventional nanoliposomes.

(37) Further, the sponge spicules (i.e., the group using the sponge spicules suspension or the group using the sponge spicules suspension and the flexible nanoliposomes) of the present disclosure are compared with a traditional microneedle (Dermaroller). FIGS. 11A-11C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from a group using microneedles and the group using the sponge spicules suspension in Embodiment 2. FIGS. 12A-12C illustrate comparative results of skin penetration of FITC-Hyaluronic acid from a group using the microneedles and the flexible nanoliposomes and the group using the sponge spicules suspension and the flexible nanoliposomes in Embodiment 2. As illustrated, skin penetration enhancing effects of the sponge spicules (i.e., the group using the sponge spicules suspension or the group using the sponge spicules suspension and the flexible nanoliposomes) are significantly better than that of the microneedles (i.e., the group using the microneedles or the group using the microneedles and the flexible nanoliposomes).

(38) FIG. 13 illustrates comparative results of transepidermal water loss over 360 hours of the control group, the group using the microneedles, the group using the sponge spicules suspension with massaging by hand, and the group with the sponge spicules suspension with massaging by an electric massager in Embodiment 2. Compared with the microneedles, the sponge spicules can retain in the stratum corneum of the skin for a long time and create a plenty of long-lasting microchannels, thereby increasing the skin absorption of biomacromolecular drugs or cosmetic active ingredients.

Embodiment 3

(39) (1) Pre-treatment of a porcine skin: the same as Embodiment 1.

(40) (2) Preparation of solid lipid nanoparticles: prepared according to existing techniques.

(41) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(42) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, the group using the sponge spicules suspension and the solid lipid nanoparticles has a significant skin penetrating enhancing effect compared to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 4

(43) (1) Pre-treatment of a porcine skin: the same as Embodiment 1.

(44) (2) Preparation of nanocapsules: prepared according to existing techniques.

(45) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(46) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, a group using the sponge spicules suspension and the nanocapsules has a significant skin penetrating enhancing effect compared to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 5

(47) (1) Pre-treatment of a porcine skin: the same as Embodiment 1.

(48) (2) Preparation of nanospheres: prepared according to existing techniques.

(49) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(50) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, a group using the sponge spicules suspension and the nanospheres has a significant skin penetrating enhancing effect relative to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 6

(51) (1) Pre-treatment of a porcine skin: the same as Embodiment 1.

(52) (2) Preparation of polymer micelle: prepared according to existing techniques.

(53) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(54) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, a group using the sponge spicules suspension and the polymer micelle has a significant skin penetrating enhancing effect relative to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 7

(55) (1) Pre-treatment of porcine skin: the same as Embodiment 1.

(56) (2) Preparation of nanosuspensions: prepared according to existing techniques.

(57) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(58) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, a group using the sponge spicules suspension and the nanosuspensions has a significant skin penetrating enhancing effect relative to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 8

(59) (1) Pre-treatment of a porcine skin: the same as Embodiment 1.

(60) (2) Preparation of ethosomes: prepared according to existing techniques.

(61) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(62) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, a group using the sponge spicules suspension and the ethosomes has a significant skin penetrating enhancing effect relative to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 9

(63) (1) Pre-treatment of a porcine skin: the same as Embodiment 1.

(64) (2) Preparation of nano-sized particles: prepared according to existing techniques.

(65) (3) Preparation of the sponge spicules suspension: the same as Embodiment 1.

(66) (4) Skin penetration experiment in vitro: prepared according to Embodiment 1. As a result, a group of the sponge spicules suspension and the nano-sized particles has a significant skin penetrating enhancing effect relative to the other groups (i.e., the control group and the group using the sponge spicules suspension).

Embodiment 10

(67) FIGS. 14A-14E illustrate a morphological comparison view of sponge spicules derived from Haliclona sp. with a high purity, sponge spicules derived from Haliclona sp. with a low purity, sponge spicules derived from Calcareous sponge, sponge spicules derived from Tethya sp., and sponge spicules derived from Mycale sp. FIG. 14A illustrates a morphology of oxea spicules derived from Haliclona sp. with a high purity. FIG. 14B illustrates a morphology of oxea spicules derived from Haliclona sp. with a low purity. The sponge spicules derived from Haliclona sp. with high purity have more uniform shapes, more integrated structures, and less impurities. FIG. 14C illustrates a morphology of triactine spicules derived from Calcareous sponge. FIG. 14D illustrates a morphology of acanthostyle spicules derived from Tethya sp. FIG. 14E illustrates a morphology of style spicules derived from Mycale sp.

(68) Referring to the skin penetration experiment in vitro of Embodiment 1 (step 4 of Embodiment 1), penetration-enhancing effects of the sponge spicules derived from different species and with different morphology were investigated. The results are shown in FIG. 15. While skin were treated with different sponge spicules derived from different species but at the same concentration, the sponge spicules derived from Haliclona sp. with a high purity (a purity of 99.5%) led to a much significantly higher enhancement ratio of skin conductivity than the sponge spicules derived from Calcareous sponge (a purity of 95.1%), the sponge spicules derived from Tethya sp. (a purity of 93.2%), the sponge spicules derived from Mycale sp. (a purity of 96.7%), and the sponge spicules derived from Haliclona sp. with a low purity (a purity of 69.9%), which indicates that penetration-enhancing effect of the sponge spicules derived from Haliclona sp. with a high purity are much more efficient than those of the sponge spicules derived from Calcareous sponge, the sponge spicules derived from Tethya sp., the sponge spicules derived from Mycale sp., and the sponge spicules derived from Haliclona sp. with a low purity.

(69) Referring to the method of Embodiment 2, the skin penetration and deposition of FITC-Hyaluronic acid induced by a group using the sponge spicules derived from Tethya sp. and induced by a group using the sponge spicules derived from Haliclona sp. with a high purity were compared. As shown in FIGS. 16A-16C, the sponge spicules derived from Haliclona sp. with a high purity resulted in a better penetration-enhancing effect.

(70) Referring to the method of Embodiment 2, the skin penetration and deposition of FITC-Hyaluronic acid induced by the group using the sponge spicules derived from Tethya sp. and the flexible nanoliposomes and induced by the group using the sponge spicules derived from Haliclona sp. with a high purity and the flexible nanoliposomes were compared. As shown in FIGS. 17A-17C, a total skin absorption of FITC-Hyaluronic acid from the group using the sponge spicules derived from Haliclona sp. with the high purity and the flexible nanoliposomes was 37.89%, while a total skin absorption of FITC-Hyaluronic acid from the group using the sponge spicules derived from Tethya sp. and the flexible nanoliposomes group is 4.76%, which indicates that the group using the sponge spicules derived from Haliclona sp. with a high purity and the flexible nanoliposomes group has a much better penetration-enhancing effect.

Embodiment 11

(71) Sponge spicules derived from Haliclona sp. with a high purity and sponge spicules derived from Haliclona sp. with a low purity were used in skin irritation experiments.

(72) Experimental method: the hair shaft of guinea pig was shaved. A suspension of 100 μL comprising 100 mg/mL (equivalent to 10% mass concentration) sponge spicules derived from Haliclona sp. with a high purity (purity of 99.5%) and a suspension of 100 μL comprising 100 mg/mL (equivalent to 10% mass concentration) sponge spicules derived from Haliclona sp with a low purity (purity of 69.9%) were applied on two symmetrical sides of the back of the guinea pig with massaging for two minutes. After massaging, transepidermal water loss of the treated skin area were measured and recorded by photos at 24.sup.th hour, 48.sup.th hour, 96.sup.th hour, 168.sup.th hour, and 240.sup.th hour.

(73) The results are shown in FIGS. 18A-18L and 19. The guinea pig skin treated by the sponge spicules derived from Haliclona sp. with a high purity were basically recovered after 96 hours and without infection. In contrast, the guinea pig skin treated by the sponge spicules derived from Haliclona sp. with a low purity appeared to be red with the signs of infection. Further, the speed of skin recovery was slowed down, and the wounds were basically recovered after 168 hours. Considering skin immunity, the faster skin recovers, the lower the risk to be infected by external pathogens. When treated with the sponge spicules derived from Haliclona sp. with a high purity, skin recovered with a faster speed, indicating that the sponge spicules derived from Haliclona sp. with a high purity leads to a better safety of its application.

Embodiment 12

(74) Flexible nanoliposomes were extruded through a membrane with a pore size of 200 nm, an average particle size (i.e., particle sizes) of the flexible nanoliposomes was 168.13 nm±2.99 nm. Flexible nanoliposomes were extruded through a membrane with a pore size of 100 nm, an average particle size (i.e., particle sizes) of the flexible nanoliposomes 130.57 nm±0.65 nm.

(75) Test method: 1 mL of different testing samples respectively comprising pure water, conventional nanoliposomes, or the flexible nanoliposomes was added into 1 mL syringe, a pressure of 500 g (measured by a balance) was applied, and a time for different testing samples to completely pass through the membrane with the pore size of 100 nm was recorded. Pure water is taken as a control group, and a ratio of a membrane-through time of other testing samples to a membrane-through time of pure water was calculated to determine the deformability of the nanoliposomes (i.e., the conventional nanoliposomes and the flexible nanoliposomes).

(76) Measurement results: the ratio of the membrane-through time of the conventional nanoliposomes to the membrane-time of the pure water is 156.71%±4.32% (to pass through the membrane with the pore size of 100 nm), and the ratio of the membrane-through time of the flexible nonoliposomes to the membrane-time of the pure water is 139.27%±2.06% (to pass through the membrane with the pore size of 100 nm), which indicates that the deformability of the flexible nanoliposomes is much better, and therefore the penetration-enhancing effect is better. When flexible nanoliposomes with an average particle size (i.e., particle sizes) of 80-150 nm is utilized in combination with the sponge spicules, and a combination use of the sponge spicules and the flexible nanoliposomes lead to a better penetration-enhancing effect.

(77) Referring to FIGS. 6A-10C, a ratio of total skin absorption of drugs induced by the group using the sponge spicules and the flexible nanoliposomes group to that induced by the group using the sponge spicules and the conventional nanoliposome is much higher than a ratio of total skin absorption induced the flexible nanoliposomes to that induced by the conventional nanoliposomes. This also indicates there is a synergistic action induced by the combination use of the sponge spicules and the flexible nanoliposomes.

(78) The aforementioned embodiments are merely some embodiments of the present disclosure, and the scope of the disclosure of is not limited thereto. Thus, it is intended that the present disclosure cover any modifications and variations of the presently presented embodiments provided they are made without departing from the appended claims and the specification of the present disclosure.