TWI748476B - Dissolvable ulvan polysaccharide microneedles - Google Patents

Abstract

The invention relates to a dissolvable ulvan polysaccharide microneedle patch, which comprises a microneedle part and a substrate part. The microneedle part is made of Ulvan polysaccharide.

Description

Soluble Ulva Polysaccharide Microneedles

The present invention relates to a microneedle patch for transdermal delivery of active substances, especially a microneedle patch containing Ulva polysaccharide.

Transdermal drug delivery (TDD) uses the skin as a gateway to deliver biologically active substances or drugs to the systemic circulation. In addition to being convenient for users, it also avoids degradation or first crossing caused by the gastrointestinal tract due to oral medication. Effects (first pass liver effects). However, the transdermal drug delivery system needs to overcome the stratum corneum barrier of the outermost layer of the skin in order to deliver biologically active substances or drugs into the body.

The microneedle technology uses a needle with a tiny size to form a reversible tiny channel in the skin to transport various macromolecular drugs or active substances that cannot penetrate the skin into the body, so it can effectively promote the delivery efficiency. However, microneedles made of metal or silicon may not only irritate the nerves in the subject’s skin and cause discomfort to the subject, but also may fall off in the skin and become sharp biohazard waste, so it is safe doubt.

Dissolving microneedles are microneedles made of water-soluble or biodegradable polymers. This type of microneedle coats the drug or active substance in it, and the microneedle After piercing the stratum corneum of the skin, the microneedles will melt to release drugs or active substances, so there will be no safety problems caused by the microneedles made of metal or silicon falling off the skin.

However, soluble microneedles often suffer from insufficient mechanical strength and cannot effectively penetrate the stratum corneum of the skin. In addition, the dissolution rate of the soluble microneedles will also affect the time and efficiency of drug delivery. If the dissolution rate of the microneedles is too slow, it may not be conducive to drug delivery. Furthermore, most of the soluble microneedles are only carriers, and need to be coated with active ingredients to be effective, which increases the complexity and uncertainty of the manufacturing process. Therefore, there is still a need to develop soluble microneedles that can improve the above shortcomings.

In one aspect, the present invention relates to the use of Ulva polysaccharide for preparing microneedles for skin penetration.

In another aspect, the present invention relates to a soluble Ulva polysaccharide microneedle, which is made of Ulva polysaccharide.

In yet another aspect, the present invention relates to a method for preparing soluble Ulva polysaccharide microneedles.

In another aspect, the present invention relates to a soluble Ulva polysaccharide microneedle patch, comprising a microneedle part and a backing part, and the microneedle part is made of Ulva polysaccharide.

In yet another aspect, the present invention relates to a method for preparing a soluble Ulva polysaccharide microneedle patch.

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples.

1: Mother template

20: Liquid polydimethylsiloxane (PDMS)

21: Polydimethylsiloxane (PDMS) negative mold

211: Micro-needle mold

2111: Needle tip mold

2112: Needle bottom mold

212: Back plate mold

3: Ulva polysaccharide solution

4: Polyvinylpyrrolidone (PVP) / sodium carboxymethyl cellulose (CMC) mixed liquid

5: Ulva polysaccharide microneedle patch

51: Microneedle

52: Backboard

Figure 1 shows the preparation process of the Ulva polysaccharide microneedle patch.

Figure 2 shows the results of observing the appearance of microneedle patches prepared with different concentrations of Ulva polysaccharide solution under a dissecting microscope from different angles; Figure 2A-1, Figure 2A-2, and Figure 2A-3 show the results with 0 Observe the appearance of the microneedle patch prepared with 4% (w/v) Ulva polysaccharide solution at angles of degrees, 45 degrees, and 90 degrees; Figure 2B-1, Figure 2B-2, and Figure 2B-3 respectively are Observe the appearance of the microneedle patch prepared with 5% (w/v) Ulva polysaccharide solution at 0 degree angle, 45 degree angle and 90 degree angle; shown in Figure 2C-1, Figure 2C-2, and Figure 2C-3 The appearance of the microneedle patch prepared with 6% (w/v) Ulva polysaccharide solution was observed at an angle of 0 degrees, 45 degrees, and 90 degrees, respectively.

Figure 3 shows the results of observing the appearance of the microneedle patch prepared with different concentrations of Ulva polysaccharide solution with a scanning electron microscope from different angles; Figure 3A-1 and Figure 3A-2 show the results at a 0 degree angle, Observe the appearance of the microneedle patch prepared with 4% (w/v) Ulva polysaccharide solution at a 90 degree angle; Fig. 3B-1 and Fig. 3B-2 show the observation at a 0 degree angle and a 90 degree angle with 5% (w/v) The appearance of the microneedle patch prepared from the Ulva polysaccharide solution; Figure 3C-1 and Figure 3C-2 show the observation at a 0 degree angle and a 90 degree angle with 6% (w/v) Ulva The appearance of the microneedle patch prepared from the polysaccharide solution.

Figure 4 shows the mechanical strength curve of Ulva polysaccharide microneedles.

Figure 5 shows the results of a 10-second pig skin puncture test with Ulva polysaccharide microneedles; Figure 5A shows the appearance of the pig skin after the test; Figure 5B is an enlarged view of Figure 5A; Figure 5C shows the Ulva polysaccharide after the test The appearance of the microneedle; Figure 5D is an enlarged view of Figure 5C.

Figure 6 shows the appearance of Ulva polysaccharide microneedles after puncturing pig skin with Ulva polysaccharide microneedles for different times; Figure 6A shows the appearance of Ulva polysaccharide microneedles 10 seconds after the puncture test; Figure 6B shows Figure 6C shows the appearance of the Ulva polysaccharide microneedles after 30 seconds of the puncture test; Figure 6C shows the appearance of the Ulva polysaccharide microneedles after 60 seconds of the puncture test; Figure 6D shows the appearance of the Ulva polysaccharide microneedles after the puncture test for 120 seconds The appearance of Ulva polysaccharide microneedles.

Figure 7 shows the dissolution rate of Ulva polysaccharides with different concentrations of Ulva polysaccharide microneedles in water within 60 minutes.

Figure 8 shows the observation of Ulva polysaccharide microneedles coated with the fluorescent dye Rhodamine 6G (Rhodamine 6G, R6G) under a laser scanning conjugate focus microscope for 10 minutes. Ulva polysaccharide microneedles are on the pig skin after 10 minutes Figure 8A shows the 3D reconstructed image of pig skin; Figure 8B shows the results of fluorescent dye diffusion displayed by scanning pig skin at different z-axis heights; 1-27 respectively Z axis depth is 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520μm.

Figure 9 shows the results of puncturing pig skin with Ulva polysaccharide microneedles for 3 hours, and analyzing the results of Ulva polysaccharide infiltration into pig skin at different time points; Figure 9A shows the cumulative release content curve of Ulva polysaccharide; Figure 9B shows Shown as the cumulative release rate curve of Ulva polysaccharides.

The present invention uses Ulva polysaccharide as the material of the soluble microneedle, which not only has sufficient mechanical strength, can effectively penetrate the stratum corneum of the skin, and the dissolution rate of the soluble Ulva polysaccharide microneedle of the present invention is fast, which is beneficial to drug delivery. More importantly, Ulva polysaccharide itself has anti-oxidation, anti-inflammatory, anti-tumor and other biological activities. It not only acts as a carrier, but also acts as an active ingredient. It can be effective without coating other active ingredients.

Therefore, the present invention provides a use of Ulva polysaccharide for preparing microneedles for skin penetration.

The invention also provides a soluble ulva polysaccharide microneedle, which is made of ulva polysaccharide.

The present invention also provides a soluble Ulva polysaccharide microneedle patch, which comprises a microneedle part and a backing part, and the microneedle part is made of Ulva polysaccharide.

In some embodiments, the soluble ulva polysaccharide microneedles are made of about 3.5~6.5% (w/v) ulva polysaccharide solution, preferably about 3.5%, about 3.6%, about 3.7% , About 3.8%, about 3.9%, About 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1%, about 5.2 %, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6.0%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, About 6.5% (w/v), or about 3.5~6.5% (w/v) any concentration, not limited to an integer concentration, for example, but not limited to 4.56% (w/v); in some preferred In a specific embodiment, the soluble Ulva polysaccharide microneedles are made with about 4%, about 5%, or about 6% Ulva polysaccharide.

In some specific embodiments, the back plate may be made of, but not limited to, one of the following materials: a combination of starch (starch) and gelatin (gelatin), and a polyvinyl alcohol (PVA) Combination material with sucrose, a chitosan (chitosan), and a combination material of polyvinylpyrrolidone (PVP) and sodium carboxymethyl cellulose (CMC).

In some embodiments, the back plate is made of a combination material (PVP/CMC) of the polyvinylpyrrolidone (PVP) and sodium carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) can enhance the hardness of the backplane, and sodium carboxymethyl cellulose (CMC) can enhance the flexibility of the backplane; the ratio of the two can be adjusted according to actual needs. In some embodiments, the ratio of PVP/CMC is about 1:10 to about 10:1 (w/w), preferably about 1:1, about 1:2, about 1:3, about 1. :4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:1, about 3:1, about 4:1, about 5 :1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 (w/w), or about 1:10 to about 10:1 (w/w) A ratio, not limited to an integer ratio, for example, but not limited to 1:3.56 (w/w); in some preferred embodiments, the PVP/CMC ratio is about 1:4 (w/w) .

In some embodiments, the solution also contains other biologically active substances.

The present invention also provides a method for preparing soluble Ulva polysaccharide microneedles, including the following steps: (i) providing a microneedle mold, the microneedle mold including a microneedle mold, the microneedle mold including a needle tip mold, and A needle bottom mold; (ii) Add a solution containing about 3.5~6.5% (w/v) Ulva polysaccharide Into the microneedle mold, the solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is dried to form a microneedle portion; and (iii) the microneedle portion is separated from the microneedle mold to The soluble Ulva polysaccharide microneedles are obtained.

The present invention also provides a method for preparing a soluble Ulva polysaccharide microneedle patch, comprising the following steps: (i) providing a microneedle mold, the microneedle mold comprising a microneedle part mold and a back plate part mold, the microneedle The mold includes a needle tip mold and a needle bottom mold, and the back plate mold is connected to the needle bottom mold; (ii) a solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is added to the micro In the needle mold, the solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is dried to form a microneedle; (iii) a solution of backing material is added to the backing of the microneedle mold In the part model, drying the backing material solution to form a backing part; and (iv) separating the microneedle part together with the backing part from the microneedle mold to obtain the soluble ulva polysaccharide microneedle patch .

In some embodiments, the concentration of Ulva polysaccharide in the solution is about 3.5~6.5% (w/v), preferably about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9% , About 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6.0%, about 6.1%, about 6.2%, about 6.3%, about 6.4% , About 6.5% (w/v), or about 3.5~6.5% (w/v) any concentration, not limited to an integer concentration, for example, but not limited to 4.56% (w/v); in some relatively In a preferred embodiment, the concentration of Ulva polysaccharide in the solution is about 4%, about 5%, or about 6%.

In some embodiments, the backing material is one of the following: a combination material of starch and gelatin, a combination material of polyvinyl alcohol (PVA) and sucrose, a chitosan, and a polyvinylpyrrolidone (PVP) and carboxymethyl cellulose sodium (CMC) combination material.

In some embodiments, the backing material is a combination material of the polyvinylpyrrolidone (PVP) and sodium carboxymethylcellulose (CMC), and the polyvinylpyrrolidone (PVP) and sodium carboxymethylcellulose The ratio of the composition solution of (CMC) is about 1:10 to about 10:1 (w/w), preferably about 1:1, about 1:2, about 1:3, about 1:4, about 1. :5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 (w/w), or about 1:10 to about 10:1 (w/w) Any ratio is not limited to an integer ratio, for example, but not limited to 1:3.56 (w/w); in some preferred embodiments, the ratio of PVP/CMC is about 1: 4(w/w).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

As used herein, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural references. Thus, for example, reference to "a component" includes a plurality of such components and their equivalents known to those skilled in the art.

As used herein, "about", "about" or "approximately" generally means within 20% of a given value or range, preferably within 10%, more preferably within 5%. The quantities given in this article are approximate, and unless explicitly stated, they can be inferred to refer to the terms "approximately", "approximately" or "approximately".

A：[->4-β-D-Glcp-(1->4)-α-L-Rhap3S-(1->]_n；B：[->4-α-L-Idop-(1->4)-α-L-Rhap3S-(1->]_n。石蓴多醣具有抗氧化(Li等人(2018)Antioxidant and antihyperlipidemic activities of purified polysaccharides from Ulva pertusa.Journal of Applied Phycology 30,2619-2627.)、抗凝劑(Adrien等人(2019)，Anticoagulant Activity of Sulfated Ulvan Isolated from the Green Macroalga Ulva rigida.Mar Drugs 17,291-329.)、抗腫瘤(Abd-Ellatef等人(2017)，Ulva lactuca polysaccharides prevent Wistar rat breast carcinogenesis through the augmentation of apoptosis,enhancement of antioxidant defense system,and suppression of inflammation.Breast Cancer(Dove Med Press) 9,67-83.)，以及免疫調節(Tabarsa等人(2018)，Water-soluble polysaccharides from Ulva intestinalis：Molecular properties,structural elucidation and immunomodulatory activities.J Food Drug Anal 26,599-608)等生理活性，此外，富含鼠李醣的多醣類表現出抗發炎、降低細菌貼附、防止紫外線損傷、刺激細胞增殖以及膠原蛋白合成等生理活性(Adrien等人(2016)，Pilot production of ulvans from Ulva sp.and their effects on hyaluronan and collagen production in cultured dermal fibroblasts.Carbohydrate Polymers 157,1306-1314.；de Araujo等人(2016)，Analgesic and anti-inflammatory actions on bradykinin route of a polysulfated fraction from alga Ulva lactuca.Int JBiol Macromol 92,820-830.)。石蓴多醣可從石蓴屬(Ulva)藻類中萃取或分離，石蓴屬(Ulva)藻類包含，但不限於，魚棲台石蓴(Ulva acanthophora)、阿南德氏石蓴(Ulva anandii)、窄葉石蓴(Ulva angusta)、阿氏石蓴(Ulva arasakii)、阿莫里加石蓴(Ulva armicalicana)、綠石蓴(Ulva atroviridis)、薄葉石蓴(Ulva attenuata)、貝特石蓴(Ulva beytensis)、雙面石蓴(Ulva bifrons)、短柄石蓴(Ulva brevistipitata)、球石蓴(Ulva bulbosa)、緬甸石蓴(Ulva burmanica)、柔石蓴(Ulva byssoides)、加州石蓴(Ulva californica)、蝶形石蓴(Ulva chaetomorphoides)、格石蓴(Ulva clathrata)、渥紅石蓴(Ulva coccinea)、扁石蓴(Ulva compressa)、族石蓴(Ulva conglobata)、羊角石蓴(Ulva cornucopiae)、角石蓴(Ulva cornuta)、科夫龍石蓴(Ulva covelongensis)、厚石蓴(Ulva crassa)、厚膜石蓴(Ulva crassimembrana)、拱石蓴(Ulva curvata)、椰棗石蓴(U.dactylifera)、鋸齒石蓴(Ulva denticulata)、秀剛石蓴(Ulva elegans)、艾米氏石蓴(Ulva elminthoides)、腸形石蓴(Ulva enteromorpha)、直石蓴(Ulva erecta)、寬石蓴(Ulva expansa)、裂片石蓴(U.fasciata)、窗石蓴(Ulva fenestrata)、曲石蓴(Ulva fiexuosa)、膠石蓴(Ulva gelatinosa)、雙生石蓴(Ulva geminoidea)、巨石蓴(U.gigantea)、大石蓴(Ulva grandis)、亨德石蓴(Ulva hendayensis)、勾石蓴(Ulva hookeriana)、霍克石蓴(Ulva hopkirkii)、腸石蓴(Ulva intestinalis)、類口服石蓴(Ulva intestinoides)、絞石蓴(Ulva intricata)、管狀石蓴(Ulva intybacea)、加唾石蓴(Ulva javanica)、科林石蓴(Ulva kylinii)、萬石蓴(U.lactuca)、萬葉石蓴(Ulva lactucaefolia)、亮綠石蓴(Ulva laetevirens)、朗吉石蓴(Ulva laingii)、線石蓴(Ulva linearis)、舌石蓴(Ulva lingulata)、鏈石蓴(Ulva linkiana)、緣管石蓴(Ulva linza)、里皮石蓴(Ulva lippii)、濱海石蓴(Ulva litoralis)、海岸石蓴(Ulva littorea)、裂石蓴(Ulva lobata)、滑石蓴(Ulva lubrica)、替代石蓴(Ulva marginata)、微球石蓴(Ulva micrococca)、米亞塔石蓴(Ulva myriotrema)、那不勒斯石蓴(Ulva neapolitana)、線蟲石蓴(Ulva nematoidea)、歐尼石蓴(Ulva ohnoi)、撤石蓴(Ulva olivacea)、撤除石蓴(Ulva olivaceum)、太平洋石蓴(Ulva pacifica)、匙形石蓴(Ulva papenfussii)、奇形石蓴(Ulva paradoxa)、小石蓴(Ulva parva)、微小石蓴(Ulva parvula)、潘騰石蓴(Ulva patengensis)、狹帶石蓴(Ulva percursa)、孔石蓴(Ulva pertusa)、菲氏石蓴(Ulva phyllosa)、拍石蓴(Ulva popenguinensis)、韭葉石蓴(Ulva porrifolia)、闊石蓴(Ulva procera)、深石蓴(Ulva profunda)、游石蓴(Ulva prolifera)、假曲石蓴(Ulva pseudocurvata)、假林氏石蓴(Ulva pseudolinza)、剛石蓴(Ulva pulchra)、紫絲石蓴 (Ulva purpurascens)、奎隆石蓴(Ulva quilonensis)、福狀石蓴(Ulva radiata)、拉爾夫石蓴(Ulva ralfsii)、毛茛石蓴(Ulva ranunculata)、網紋石蓴(Ulva reticulata)、拉科石蓴(Ulva rhacodes)、硬石蓴(Ulva hardica)、切石蓴(Ulva rotundata)、魯本斯石蓴(Ulva rubens)、塞弗拉石蓴(Ulva saifullahii)、斯凱戈爾石蓴(Ulva scagelii)、斯堪的納維亞石蓴(Ulva scandinavica)、賽瑞石蓴(Ulva sericea)、齒葉石蓴(Ulva serrata)、單葉石蓴(Ulva simplex)、索倫森石蓴(Ulva sorensenii)、紡石蓴(Ulva spinulosa)、狹葉石蓴(Ulva stenophylla)、柄石蓴(Ulva stipitata)、海邊石蓴(Ulva sublittoralis)、針葉石蓴(Ulva subulata)、帶狀石蓴(Ulva taeniata)、細葉石蓴(Ulva tenera)、四角石蓴(Ulva quadgona)、絞線石蓴(Ulva torta)、荸養石蓴(Ulva tuberosa)、臍狀石蓴(Ulva umbilicata)、芒石蓴(Ulva uncialis)、鉤刺石蓴(Ulva uncinata)、松蘿石蓴(Ulva usneoides)、囊狀石蓴(Ulva utricularis)、革質石蓴(Ulva utriculosa)、卵形石蓴(Ulva uvoides)，以及紫花瓣石蓴(Ulva ventricosa)。 As used herein, the term "Ulvan polysaccharide" means a water-soluble compound acidic sulfuric polysaccharide extracted with hot water (100~121℃) from the cell wall of Ulva algae. Its main components are Sulfate, Rhamnose, Xylose, Glucuronic acid, and Iduronic acid; the main structure of Ulva polysaccharide is a repetitive disaccharide Type A (i.e., Glucurorhamnose 3-sulphate, A3s) and Type B (i.e. Iduronorhamnose 3-sulphate, B3s) are composed in a linear manner, Type A The chemical structure with Type B is as follows:

A: [->4-β-D-Glc p -(1->4)-α-L-Rha p 3S-(1->] _n ; B: [->4-α-L-Ido p- (1->4)-α-L-Rha p 3S-(1->] _n . Ulva polysaccharides have antioxidant properties (Li et al. (2018) Antioxidant and antihyperlipidemic activities of purified polysaccharides from Ulva pertusa. Journal of Applied Phycology 30 , 2619-2627.), anticoagulant (Adrien et al. (2019), Anticoagulant Activity of Sulfated Ulvan Isolated from the Green Macroalga Ulva rigida. Mar Drugs 17 , 291-329.), anti-tumor (Abd-Ellatef et al. (2017), Ulva lactuca polysaccharides prevent Wistar rat breast carcinogenesis through the augmentation of apoptosis, enhancement of antioxidant defense system, and suppression of inflammation. Breast Cancer (Dove Med Press) 9 ,67-83.), and immune regulation (Tabarsa et al. Human (2018), Water-soluble polysaccharides from Ulva intestinalis: Molecular properties, structural elucidation and immunomodulatory activities. J Food Drug Anal 26 , 599-608) and other physiological activities. In addition, rhamnose-rich polysaccharides exhibit anti- Inflammation, reducing bacterial adhesion, preventing UV damage, stimulating cell proliferation and collagen synthesis and other physiological activities (Adrien et al. (2016), Pilot production of ulvans from Ulva sp. and their effects on hyaluronan and collagen production in cultured derm al fibroblasts. Carbohydrate Polymers 157 , 1306-1314.; de Araujo et al. (2016), Analgesic and anti-inflammatory actions on bradykinin route of a polysulfated fraction from alga Ulva lactuca. Int JBiol Macromol 92 , 820-830.). Ulva polysaccharides can be extracted or separated from Ulva (Ulva ) algae. Ulva ( Ulva ) algae include, but are not limited to, Ulva acanthophora (Ulva acanthophora), Ulva anandii (Ulva anandii), narrow-leaved Ulva (Ulva angusta), Apgar Ulva (Ulva arasakii), amoxicillin Riga Ulva (Ulva armicalicana), Greenstone water shield (Ulva atroviridis), thin leaves Ulva (Ulva attenuata), Beit Ulva (Ulva beytensis , Ulva bifrons , Ulva brevistipitata , Ulva bulbosa , Ulva burmanica , Ulva byssoides , Ulva byssoides, Ulva californica , Ulva chaetomorphoides , Ulva clathrata , Ulva coccinea , Ulva compressa , Ulva conglobata , Ulva cornuia ), Ulva cornuta (Ulva cornuta), Ulva covelongensis (Ulva covelongensis), Ulva crassa (Ulva crassa), Ulva crassimembrana (Ulva crassimembrana), Ulva curvata (Ulva curvata), Ulva palma (U. dactylifera , Ulva denticulata , Ulva elegans , Ulva elminthoides , Ulva enteromorpha , Ulva erecta , Ulva erecta Ulva expansa ), Ulva lobata (U.fasciata), Ulva fenestrata (Ulva fenestrata), Ulva fiexuosa (Ulva fiexuosa), Ulva gelatinosa (Ulva gelatinosa), Ulva gemini (Ulvainoidea), U. gigantea), water shield stone (Ulva grandis), Hendrick Ulva (Ulva hendayensis), hook Ulva (Ulva hookeriana), Hawke Ulva (Ul va hopkirkii ), Ulva intestinalis (Ulva intestinalis), Oral Ulva intestinoides (Ulva intestinoides), Ulva intricata (Ulva intricata), Ulva intybacea (Ulva intybacea), Ulva intestinalis, Ulva javanica Ulva kylinii , U.lactuca , Ulva lactucaefolia , Ulva laetevirens , Ulva laingii , Ulva linearis , Tongue Ulva lingulata , Ulva linkiana , Ulva linza , Ulva lippii , Ulva litoralis , Ulva littorea , Ulva littorea Ulva lobata , Ulva lubrica , Ulva marginata , Ulva micrococca , Ulva myriotrema , Ulva neapolitana , Ulva neapolitana Ulva nematoidea , Ulva ohnoi , Ulva olivacea , Ulva olivaceum , Ulva pacifica , Ulva papenfussii (Ulva paradoxa), stone water shield (Ulva parva), small Ulva (Ulva parvula), Pan Teng Ulva (Ulva patengensis), tape Ulva (Ulva percursa), hole Ulva (Ulva pertusa), the Philippine's Ulva (Ulva phyllosa), beat Ulva (Ulva popenguinensis), leek leaf Ulva (Ulva porrifolia), wide Ulva (Ulva procera), deep Ulva (Ulva profunda), visit Ulva (Ulva prolifera), fake song Ulva (Ulva pseudocurvata , Ulva pseudolinza , Ulva pulchr a ), Ulva purpurascens (Ulva purpurascens), Ulva quilonensis (Ulva quilonensis), Ulva radiata (Ulva radiata), Ulva ralfsii (Ulva ralfsii), Ulva ranunculata (Ulva ranunculata), net Ulva reticulata , Ulva rhacodes , Ulva hardica , Ulva rotundata , Ulva rubens , Ulva rubens, Ulva saifullahii , Ulva scagelii (Ulva scagelii), Ulva scandinavica (Ulva scandinavica), Ulva sericea (Ulva sericea), Ulva serrata (Ulva serrata), Ulva simplex (Ulva simplex) ), Ulva sorensenii (Ulva sorensenii), Ulva spinulosa (Ulva spinulosa), Ulva stenophylla (Ulva stenophylla), Ulva stipitata , Ulva stipitata, Ulva sublittoralis , Ulva conifer ( Ulva subulata , Ulva taeniata , Ulva tenera , Ulva quadgona , Ulva torta , Ulva tuberosa , Ulva Ulva ( Ulva umbilicata ), Ulva uncialis (Ulva uncialis), Ulva uncinata (Ulva uncinata), Ulva usneoides (Ulva usneoides), Ulva cystic (Ulva utricularis ), Ulva Ulva, Ulva utriculosa Oval Ulva ( Ulva uvoides ) and Purple Petal Ulva ( Ulva ventricosa ).

As used herein, the term "back plate" refers to the base connected to the bottom of the microneedles, so that the microneedles are fixedly arranged to form a patch form, which is convenient for the user to use.

As used herein, the term "permeable skin" means that it can penetrate the stratum corneum (thickness of about 10-40 μm). The Ulva polysaccharide microneedle of the present invention can penetrate the stratum corneum of the skin and deliver the active substance to the epidermal layer (about 50-150 μm in thickness) and the dermis layer (about 1.5-4 mm in thickness).

As used herein, the term "microneedle height" or "microneedle portion height" means the vertical distance from the tip of a microneedle to the bottom of the microneedle. The Ulva polysaccharide microneedle of the present invention can penetrate the skin stratum corneum and deliver active substances to the epidermis and dermis. Therefore, the height of the Ulva polysaccharide microneedle of the present invention is greater than the thickness of the skin stratum corneum plus the epidermal layer. In some embodiments, the height of the Ulva polysaccharide microneedles of the present invention is not less than 200 microns (μm), preferably about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, About 550μm, about 600μm, about 650μm, about 700μm, Or any height not less than 200 μm, not limited to an integer height, for example, but not limited to 652.67 μm; in some preferred embodiments, the height of the microneedle is about 650 μm.

The invention is described in more detail in the following illustrative examples. Although the embodiments may only represent selected specific embodiments of the present invention, it should be understood that the following embodiments are illustrative and not restrictive.

The present invention is further illustrated through the following examples, which should not be construed as a further limitation in any way. The entire contents of all documents cited in this application (including references, approved patents, published patent applications, and patent applications that are also in the application) are hereby expressly incorporated into this case by reference.

Example

Example 1 Extraction of Ulva Polysaccharide

Ulva powder (Taiwan Fertilizer Co., Ltd., Taiwan) was dissolved in deionized water, extracted at high temperature (about 100°C to about 121°C), filtered and concentrated under reduced pressure, and then freeze-dried to obtain Ulva polysaccharide powder.

Example 2 Preparation of Ulva Polysaccharide Microneedle Patch

The Ulva polysaccharide microneedle patch was prepared by a two-layer casting method, and the preparation steps are shown in Figure 1. ^{First, a 3M TM} microneedle plate (St. Paul, Minnesota, USA) with a pyramid-like shape was used as the master template 1, and liquid polydimethylsiloxane (PDMS) 20 was cast on the master template 1. After the liquid polydimethylsiloxane (PDMS) 20 is cured, it is separated from the master template 1 to obtain a PDMS negative mold 21. The PDMS negative mold 21 includes a microneedle mold 211 and a The back plate mold 212 includes a needle tip mold 2111 and a needle bottom mold 2112. Next, the Ulva polysaccharide powder obtained in the above-mentioned Example 1 was dissolved in deionized water to obtain Ulva polysaccharide solution 3. The Ulva polysaccharide solution 3 of different concentrations (4%, 5%, 6%(w/v)) was added to the PDMS negative mold 21, centrifuged for 15 minutes, and left standing at room temperature overnight to dry to form Ulva The microneedle portion 51 of the polysaccharide microneedle patch. Next, the mixed solution (PVP/CMC, ratio 1:4 (w/w)) of polyvinylpyrrolidone (PVP) and sodium carboxymethylcellulose (CMC) 4 was added to the PDMS negative mold 21, and centrifuged After 15 minutes, it was allowed to stand at room temperature overnight to dry to form the back plate portion 52 of the Ulva polysaccharide microneedle patch. After the PVP/CMC mixture 4 is completely dried, the microneedle portion 51 and the back plate portion 52 are separated from the PDMS negative mold 21 to obtain the Ulva polysaccharide microneedle patch 5.

Example 3 Observation of the appearance of Ulva polysaccharide microneedle patch

The appearance of the Ulva polysaccharide microneedle patch obtained in Example 2 above was observed with a dissecting microscope (Olympus, model SZX16, Japan). Observation angles are 0 degrees (the angle between the microneedle patch and the microscope stage is 0 degrees, that is, the microneedle patch lies flat on the microscope stage), 45 degrees (the microneedle The angle between the patch and the stage of the microscope is 45 degrees), and the angle of 90 degrees (the angle between the microneedle patch and the stage of the microscope is 90 degrees).

The 0-degree observation results of microneedle patches prepared with different concentrations (4%, 5%, 6% (w/v)) of Ulva polysaccharide solutions are shown in Figure 2A-1, Figure 2B-1, and Figure 2C- As shown in 1, the 45-degree observation results are shown in Figure 2A-2, Figure 2B-2, and Figure 2C-2, and the 90-degree observation results are shown in Figure 2A-3, Figure 2B-3, and Figure 2C-3, respectively. Show. Whether 4% (w/v), 5% (w/v), or 6% (w/v) Ulva polysaccharide solution prepared microneedle patches, they all form microneedles with a pyramid-like shape (as shown in the figure) 2A-2, Figure 2B-2, Figure 2C-2), and the microneedles are evenly distributed on the patch (as shown in Figure 2A-1, Figure 2B-1, Figure 2C-1) and have complete The tip of the needle (as shown in Figure 2A-3, Figure 2B-3, and Figure 2C-3). These results are consistent with the appearance ^{of the 3M TM} microneedle plate as the master template.

In addition, using a scanning electron microscope (Scanning electron microscope, SEM) (Hitachi, Model S-3400N, Japan) to observe the detailed appearance of the Ulva polysaccharide microneedles from two different angles (0 degree angle and 90 degree angle), And use ImageJ software to measure the height of each Ulva polysaccharide microneedle (the vertical distance from the tip to the bottom of the needle), the width of the bottom of the needle, the distance between the tips of the two microneedles, and the distance between the bottom edges of the two microneedles. This test was repeated three times (n=3), and the average value and standard deviation of each concentration of microneedle samples were calculated.

The observation results of the scanning electron microscope are shown in Figure 3A-1 to Figure 3C-2. Observed by a scanning electron microscope at an angle of 0 degrees, it can be seen that whether it is a microneedle patch prepared with 4% (w/v), 5% (w/v), or 6% (w/v) Ulva polysaccharide solution The sheets all form microneedles with a pyramid-like shape (as shown in Fig. 3A-1, Fig. 3B-1, and Fig. 3C-1). Observed by a scanning electron microscope at a 90 degree angle, it can be seen that whether it is a microneedle prepared with 4% (w/v), 5% (w/v), or 6% (w/v) Ulva polysaccharide solution The patch and the microneedles on it all have complete needle tips (as shown in Fig. 3A-2, Fig. 3B-2, and Fig. 3C-2).

The results of the microneedle size measured by ImageJ software are shown in Table 1, and compared with the microneedle size ^{of the 3M TM microneedle plate as a master template.} Overall, whether it is 4% (w/v), 5% (w/v), or 6% (w/v) Ulva polysaccharide microneedles, the height, width, and aspect ratio are quite close to those of the master template. The height, width, and aspect ratio of the microneedle, and the difference between the height and width of the microneedle of the mother template, are all within 10% of the height and width of the microneedle of the mother template. It means that the Ulva polysaccharide microneedle patch of the present invention has similar appearance properties to ^{the 3M TM microneedle plate as the mother template.}

Example 4 Mechanical property test of Ulva polysaccharide microneedle patch

A universal material testing machine (model: H1KS, Tinius Olsen, USA) was used to confirm the mechanical properties of the microneedles. The microneedle patch prepared with the Ulva polysaccharide solution of different concentrations (4%, 5%, 6% (w/v)) obtained in the above example 2 was placed on the stainless steel substrate stage of the universal material testing machine After fixing the patch, move the pressure tester to a distance of 10mm from the patch, and test the mechanical strength of the Ulva polysaccharide microneedle patch of the present invention at a constant moving speed of 10mm/min, and record the pressure tester’s The force changes with the displacement of the microneedle, and the mechanical strength curve (force-displacement curve) is drawn.

The mechanical strength curve of Ulva polysaccharide microneedles is shown in Figure 4. The breaking force of the microneedle patch prepared with different concentrations (4%, 5%, 6%(w/v)) of Ulva polysaccharide solution was 2.73, 2.64, and 2.74 N; that is, the breakage of each microneedle The force is about 0.0075~0.0078 N/needle. The breaking force of the microneedle represents the force required to depress the entire height of the microneedle. According to the previous study of Lee et al. (Lee et al. (2015). Fabrication of a novel partially dissolving polymer microneedle patch for transdermal drug delivery. Journal of Materials Chemistry B 3 , 276-285), the breaking force is about 0.005 N/needle Soluble microneedles have sufficient mechanical properties for piercing the skin. The breaking force of the Ulva polysaccharide microneedles of the present invention is higher than that of the microneedles of Lee et al., indicating that the Ulva polysaccharide microneedles of the present invention have good mechanical properties.

Example 5 Puncture test of Ulva polysaccharide microneedle patch

In order to confirm whether the Ulva polysaccharide microneedle of the present invention has the ability to puncture the skin, this embodiment uses pig skin to simulate human skin to perform a microneedle puncture test. First, the Ulva polysaccharide microneedle patch containing Lissamine Green B (LGB) dye was prepared according to the method of Example 2. The only difference from the method described in Example 2 is that the preparation of the microneedle part At the time, add LGB dye with a final concentration of 6g/L to the 4% (w/v) Ulva polysaccharide solution.

Before the puncture test, treat the pigskin. Thaw the frozen skin of the pig’s ears and soak it in double distilled water (ddH ₂ O); dry the pigskin with a paper towel, then apply the hair removal Apply the cream for 5 minutes to remove the micro-hairs. If there is still unremoved hair on the pig skin after removing the cream, shave it with a razor. Next, place the processed pigskin on a self-assembled applicator with the cutin surface facing upwards, and place the Ulva polysaccharide microneedles containing the LGB dye with the cut back plate on the surface of the pigskin. Using the applicator, the microneedles were pierced through the pig skin for 10 seconds. Finally, use a dissecting microscope to observe the state of the surface of the pigskin and calculate the number of puncture holes, and calculate the success rate of the puncture with the following formula: [(number of holes on the pigskin/total number of microneedles) x 100%].

The results of the pigskin puncture test are shown in Figures 5A to 5D. After 10 seconds of puncturing pig skin with the Ulva polysaccharide microneedle of the present invention, blue spots produced by the dye released by the microneedle puncture were observed on the pig skin (as shown in Figure 5A and Figure 5B), indicating the present The invented Ulva polysaccharide microneedle has the ability to pierce pig skin, and the number of blue spots represents the number of holes for the microneedle to pierce pig skin. After calculation, the success rate of puncture is as high as 80%. In addition, the appearance of the microneedle after the puncture test was observed with a dissecting microscope. The results are shown in Figure 5C and Figure 5D. The loss of the microneedle tip was observed, indicating that the Ulva polysaccharide microneedle of the present invention can quickly dissolve after puncturing pig skin . It can be seen from the experiment of this embodiment that the Ulva polysaccharide microneedles of the present invention have sufficient mechanical strength to penetrate the stratum corneum barrier of pigskin.

Example 6 Dissolution test of Ulva polysaccharide microneedles 1

The Ulva polysaccharide microneedle patch containing LGB dye and the porcine skin puncture test were performed as described in Example 5, but with different microneedle puncture times (10, 30, 60, and 120 seconds). After the pigskin puncture test, the appearance changes of the microneedles were observed with a dissecting microscope to understand the dissolution of the microneedle tips. Then, use the Image J software to measure the height change of the microneedle, and calculate the loss rate of the microneedle height with the following formula: [(original microneedle height-microneedle height after puncture)/original microneedle height x 100%]; this test is repeated Three times (n=3), and calculate the average and standard deviation of each sample.

After the pigskin puncture test was carried out at different times (10, 30, 60, and 120 seconds), the appearance of the microneedles changed as shown in Figure 6A to Figure 6D. It was clearly observed that the height of the microneedles increased with the increase in puncture time. Become shorter. Compared with the original height of the microneedle, the calculated microneedle height loss rate is shown in Table 2. After 120 seconds of pigskin puncture, the height of the microneedle tip has been reduced by 90%, indicating that the Ulva polysaccharide microneedle of the present invention is in After penetrating the stratum corneum barrier of the skin, it quickly dissolves in contact with the interstitial fluid in the skin, and releases the active ingredients in the skin and is absorbed by the skin.

Example 7 Dissolution test of Ulva polysaccharide microneedles 2

First prepare the Ulva polysaccharide standard: prepare a 10mg/mL Ulva polysaccharide solution, and then serially dilute it for 3 times. Use an ELISA reader to determine the OD ₆₁₀ value of the Ulva polysaccharide standard at each dilution, and draw the Ulva polysaccharide concentration standard Curve to calculate the concentration of Ulva polysaccharide in each sample. Next, the Ulva polysaccharide microneedle patch obtained in Example 2 above was immersed in 2 mL of double distilled water (ddH ₂ O) at different time points (1, 3, 5, 10, 20, 30, 40, and 60 minutes) Take out 300 μL of the aqueous solution as an analysis sample, and make up the same volume of ddH ₂ O. 200 μL of Azure A was added to 100 μL of samples, and after standing for 5 minutes, the OD ₆₁₀ value of each sample was measured with an ELISA reader, and the concentration of Ulva polysaccharide in each sample was calculated using the standard curve of Ulva polysaccharide concentration. Finally, the following formula calculates the dissolution rate of the Ulva polysaccharide content of the microneedle patch: [(Ulva polysaccharide content of samples taken at different time points/Maximum Ulva polysaccharide content (that is, the dissolution rate in the sample after 60 minutes of dissolution) Ulva polysaccharide content) x 100%)]; This test was repeated three times (n=3), and the average and standard deviation of microneedle samples of various concentrations were calculated.

The dissolution rate of Ulva polysaccharide content is shown in Figure 7. Within 1 minute, the dissolution rate of Ulva polysaccharide microneedle patch of various concentrations (4%, 5%, 6%(w/v)) reached 50%, and it was completely dissolved within 30 minutes ( The dissolution rate is 100%), which means that the Ulva polysaccharide microneedle of the present invention can be quickly and completely dissolved, and is suitable for use as an effective and safe transdermal delivery device for human use.

Example 8 In vitro diffusion depth test of active substance of Ulva polysaccharide microneedle patch

In order to confirm the depth of the active substance and/or drug diffusion in the skin after the microneedle puncture of Ulva polysaccharide of the present invention dissolves the skin, this embodiment also uses pig skin to simulate human skin to perform a microneedle puncture test. First, prepare the Ulva polysaccharide microneedle patch containing the fluorescent dye Rhodamine 6G (Rhodamine 6G, R6G) according to the method of Example 2. The only difference from the method described in Example 2 is that the preparation of the microneedle part At the time, add R6G dye with a final concentration of 500 mg/L to the 4% (w/v) Ulva polysaccharide solution.

The pigskin puncture test method is the same as that described in Example 5. The microneedles are removed after puncturing the pigskin for 10 minutes. Next, observe the diffusion depth of the fluorescent dye R6G in pig skin with a laser scanning conjugation microscope (CLSM, Nikon C2+, Nikon Company, Japan) at an excitation wavelength of 561nm. Scan under the axis height, and obtain a 3D reconstructed image by superimposing the x-axis and y-axis plane images at different z-axis heights.

The 3D reconstructed image of R6G fluorescent dye in pig skin is shown in Figure 8A. The 3D reconstructed image shows that the maximum skin penetration depth of the ulva polysaccharide microneedle of the present invention is about 500 μm, which corresponds to the superficial dermal layer of human skin. In addition, the results of scanning pig skin at different z-axis heights are shown in Figure 8B. Ulva polysaccharide microneedles puncture pig skin for 10 minutes. Due to the soluble nature of Ulva polysaccharide microneedles, the active substance/drug (in this example, Ulva polysaccharide and R6G fluorescent dye) will be released in the skin. Diffusion, the maximum diffusion depth is about 520um (as shown in Figure 6B). It can be seen that the Ulva polysaccharide of the present invention In addition to being an active substance by itself, the microneedle can also assist the delivery of other active substances and/or drugs to the dermis, and can enhance the efficiency of the active substances and/or drugs to diffuse into the deeper layers of the skin.

Example 9 In vitro transdermal penetration test of Ulva polysaccharide microneedle patch

In order to confirm whether the active substance and/or the drug penetrated into the skin after the microneedle puncture of Ulva polysaccharide of the present invention is dissolved, this embodiment also uses pig skin to simulate human skin to perform a microneedle puncture test, and use a vertical diffusion groove ( Vertical diffusion cell, also known as Franz Cell) analysis.

Using the microneedle patch prepared with the 4% (w/v) Ulva polysaccharide solution obtained in Example 2, the pig skin puncture test was performed by the method described in Example 5. After the microneedle punctured the pig skin for 30 seconds, the pig skin was moved to _{the receiver tank of Franz Cell containing 17 ml of double distilled water (ddH 2} O), and the temperature was controlled at 37° C., and stirring was continued. At different time points (2, 5, 10, 20, 30, 40, 60, 90, 120, 150, 180 minutes) 400 μL of samples were taken out, and the same volume of ddH ₂ O was replaced. The azure A (azure A) spectrophotometric method was used to determine the content of Ulva polysaccharides released from the microneedles. Add 100 μL of Azure A to a 200 μL sample, let it stand for 5 minutes, and measure the absorbance (OD ₆₁₀ value) at a wavelength of 610 nm with a disc enzyme immunoassay (ELISA reader). In addition, prepare the Ulva polysaccharide standard: prepare a 10 mg/mL Ulva polysaccharide solution, and then serially dilute it for 3 times. Use an ELISA reader to determine the OD ₆₁₀ value of the Ulva polysaccharide standard at each dilution, and plot the Ulva polysaccharide concentration Standard curve to calculate the concentration of Ulva polysaccharide in each sample. The following formula calculates the cumulative release rate of Ulva polysaccharides: [(Ulva polysaccharide content at different time points/maximum Ulva polysaccharide content) x 100%]; this test was repeated three times (n=3), and the sample’s Mean and standard deviation.

The cumulative release amount of Ulva polysaccharide microneedle in vitro percutaneous penetration at different time points is shown in Figure 9A, and the cumulative release rate is shown in Figure 9B. It can be seen from Fig. 9A and Fig. 9B that the microneedle releases Ulva polysaccharides rapidly in the first 60 minutes, and slowly releases to 3 hours later. The microneedles had released 109.33±19.08μg (cumulative release rate of about 50%) of Ulva polysaccharide at 20 minutes, and the cumulative release of Ulva polysaccharide after 3 hours was 201.05±21.09μg (cumulative release rate of 100%). %). The results of this example show that the Ulva polysaccharide microneedles of the present invention can quickly dissolve in the skin and successfully penetrate the Ulva polysaccharide into the skin.

The above detailed description is a specific description of a possible embodiment of the present invention, but this embodiment is not intended to limit the scope of the patent of the present invention. Any equivalent implementation or modification that does not deviate from the technical spirit of the present invention shall be included in In the scope of the patent in this case.

In summary, the technical features disclosed in this case have fully met the statutory invention patent requirements for novelty and advancement. You file an application in accordance with the law, and I implore your office to approve this invention patent application to encourage invention and make it convenient.

Claims (9)

Ulva polysaccharide is used to prepare microneedles for skin penetration. A soluble ulva polysaccharide microneedle is made of ulva polysaccharide extracted at a temperature above 100°C. The soluble Ulva polysaccharide microneedles according to claim 2, wherein the soluble Ulva polysaccharide microneedles are made of about 3.5-6.5% (w/v) Ulva polysaccharide solution. A method for preparing soluble Ulva polysaccharide microneedles, comprising the following steps: (i) providing a microneedle mold, the microneedle mold includes a microneedle mold, the microneedle mold includes a needle tip mold and a needle bottom mold (Ii) A solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is added to the microneedle mold, and the solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is dried Forming a microneedle part; and (iii) separating the microneedle part from the microneedle mold to obtain the soluble Ulva polysaccharide microneedles. A soluble ulva polysaccharide microneedle patch comprises a microneedle part and a back plate part, and the microneedle part is made of ulva polysaccharide. The soluble Ulva polysaccharide microneedle patch according to claim 5, wherein the microneedle part is made of about 3.5-6.5% (w/v) Ulva polysaccharide solution. The soluble Ulva polysaccharide microneedle patch according to claim 5, wherein the back plate part is made of a group consisting of the following materials: a combination material of starch and gelatin, a polyvinyl alcohol (polyvinyl alcohol) , PVA) and sucrose combination material, a chitosan, and a polyvinylpyrrolidone (polyvinylpyrrolidone, PVP) and sodium carboxymethyl cellulose (sodium carboxymethyl cellulose, CMC) combination material. The soluble Ulva polysaccharide microneedle patch according to claim 7, wherein the back plate part is made of a polyvinylpyrrolidone (PVP) and carboxymethyl group comprising about 1:10 to about 10:1 (w/w) Made of composite material of sodium cellulose (CMC). A method for preparing a soluble Ulva polysaccharide microneedle patch, comprising the following steps: (i) providing a microneedle mold, the microneedle mold comprising a microneedle part mold and a back plate part mold, the microneedle part mold comprising a The needle tip mold and a needle bottom mold, the back plate mold is connected to the needle bottom mold; (ii) a solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is added to the microneedle mold , The solution containing about 3.5~6.5% (w/v) Ulva polysaccharide is dried to form a microneedle part; (iii) a polyvinylpyrrolidone containing about 1:10 to about 10:1 (w/w) is dried (PVP) and sodium carboxymethyl cellulose (CMC) composition solution is added to the back plate model of the microneedle mold, so that the containing polyvinylpyrrolidone (PVP) and sodium carboxymethyl cellulose (CMC) The composition solution of is dried to form a backing part; and (iv) separating the microneedle part and the backing part from the microneedle mold to obtain the soluble ulva polysaccharide microneedle patch.

Images