WO2020073706A1

WO2020073706A1 - Surface-modified sponge spicule, preparation method therefor and use thereof

Info

Publication number: WO2020073706A1
Application number: PCT/CN2019/096166
Authority: WO
Inventors: 陈铭; 张柯达; 王德祥; 赵婧华; 张驰; 欧徽龙; 张赛曼
Original assignee: 厦门大学; 深圳市德汉荣生物医药科技有限公司
Priority date: 2018-10-09
Filing date: 2019-07-16
Publication date: 2020-04-16
Also published as: CN111012916B; CN111012916A

Abstract

A sponge spicule, a surface of which is modified with an aliphatic group, an aromatic group, an arylated alkyl or derived groups thereof, wherein a method for preparing the sponge spicule comprises: cleaning the sponge spicule to remove impurities; activating a silanol group on the surface of the sponge spicule; and then reacting same with a silane coupling agent. The sponge spicule can physically absorb one or more polypeptides or proteins, and thus can promote the skin or transdermal administration of the polypeptides and proteins through the action of microneedles.

Description

Surface modified sponge bone needle, preparation method and application thereof

Technical field

The invention belongs to a sponge bone needle penetration-promoting technology, and in particular relates to a surface modified sponge bone needle and a preparation method and application thereof.

Background technique

Sponge bone needles (Sponge spicules) are the siliceous or calcareous fibrous materials that support the skeleton and resist external invasion in the sponge. They have various structural forms, including uniaxial, biaxial, triaxial and multiaxial. The study found that the sponge bone needle has good optical properties and mechanical properties. At present, the research on the sponge bone needle focuses on the optical properties, nanostructure, bionics, and medical transdermal agents of the sponge bone needle. Some natural spongy bone needles with high mechanical strength and sharp ends have a role similar to microneedles, which can stay in the stratum corneum of the skin for a long time and form a large number of persistent microchannels. The spongy bone needles are used alone or with some The combined application of carriers can effectively promote the transdermal absorption of drugs or active ingredients, especially suitable for active ingredients of drugs or cosmetics with small molecular weight and good stability, but for some large molecules, it is necessary to develop some more effective ways to promote penetration .

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art, and to provide a surface modified sponge bone needle and its preparation method and use.

One of the technical solutions adopted by the present invention to solve its technical problems is:

A surface-modified sponge bone needle, the surface of the sponge bone needle is modified with an aliphatic hydrocarbon group, an aromatic hydrocarbon group, an aramid group or other organic groups derived based on these three types of groups.

In an embodiment: the aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl fat group or derivative group thereof is combined with the sponge bone needle through a -Si-O-Si- bond, and the aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl fat group or derivative thereof The groups can be connected to each other through a -Si-O-Si- bond, thereby forming a thin film of aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl fatty group or derivative group on the surface of the sponge bone needle.

In an embodiment: the structural formula of the surface-modified sponge bone needle is shown in the following formula I:

Formula I

Wherein, R is the aforementioned aliphatic hydrocarbon group, aromatic hydrocarbon group, arylaliphatic group or a derivative group thereof.

Wherein, the aromatic hydrocarbon group represents that the aromatic ring in R is directly connected to Si, and there may be other substituents on the aromatic ring. The arylaliphatic group represents that the aromatic ring in R is not directly connected to Si, and there are groups such as aliphatic hydrocarbon groups between the aromatic ring and Si, and there may be other substituents on the aromatic ring.

In one embodiment: the sponge bone needle is derived from the bee sponge Haliclona sp ..

In an embodiment: the aliphatic hydrocarbon group is a C3-C18 alkyl group.

In an embodiment: the aromatic hydrocarbon group is phenyl.

In an embodiment: the arylaliphatic group is benzyl or phenethyl.

The second technical solution adopted by the present invention to solve its technical problems is:

A preparation method of surface modified sponge bone needles includes:

1) Add sponge bone needle to 1 ～ 10mol / L NaOH solution, ultrasonic for 10 ～ 60min; after filtering, washing and drying, add sponge bone needle into 5 ～ 20% hydrochloric acid solution, sonicate for 10 ～ 60min, filter , Washing and drying to obtain sponge bone needle activated by silanol group on the surface;

2) Add the surface silanol group-activated sponge bone needle obtained in step 1) to an anhydrous aprotic solvent, add a silane coupling agent at 80 to 100 ° C, the sponge bone needle and the silane coupling agent The mass ratio is 1: 0.5 to 2, the concentration of the silane coupling agent in the anhydrous aprotic solvent is 1 to 50 mM; after the reaction for 2 to 12 hours, a surface-modified sponge bone needle is prepared.

In an embodiment, the method further includes: ultrasonically cleaning and drying the sponge bone needle with water, ethanol, and acetone in sequence to obtain a sponge bone needle with a clean surface; and then adding the NaOH solution and adding the sponge bone needle with the clean surface Silanol groups on the surface of hydrochloric acid solution, ultrasound, filtration, washing and drying are activated.

In one embodiment: the purity of the sponge bone needle is not less than 90%. Preferably, the sponge bone needle has a uniform shape and a complete structure.

In an embodiment: the anhydrous aprotic solvent is anhydrous toluene.

In an embodiment: the silane coupling agent is of the general formula R-SiX ₃ , R-Si (R ′) ₂ -X or R-Si (R ′)-X ₂ ; X is an alkoxy group (eg Methoxy, ethoxy, isopropoxy, etc.), acyloxy (such as acetoxy, etc.) or halo (such as chlorine substitution, etc.); the R is the above aliphatic hydrocarbon group (such as C3-C18 Alkyl, etc.), aromatic hydrocarbon groups (such as phenyl, etc.), araliphatic groups (such as benzyl, phenethyl, etc.) or derivatives thereof; the R ′ is a short-chain aliphatic hydrocarbon group (short-chain aliphatic hydrocarbon group generally refers to It is a C1-C3 hydrocarbon group, such as methyl, ethyl, etc.).

The principle of the preparation method is: the sponge bone needle itself has a silanol group (-SiOH), after the silanol group is activated, -Si-OH reacts with the R-Si-X of the silane coupling agent to form -Si (formula (Not shown in I) -O-Si-R, R (that is, aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl aliphatic group or derivative group thereof) is bonded to the sponge bone needle through a -Si-O-Si- bond. R-Si- can also form -Si-O-Si- bonds to connect with each other.

In an embodiment: the specific silane coupling agent is shown in the following table:

The third technical solution adopted by the present invention to solve its technical problems is:

Use of surface modified sponge bone needle in preparing percutaneous absorption composition or skin absorption composition, the surface modified sponge bone needle can absorb drugs, fluorescent molecules, polymers, proteins, peptides, nanoparticles through physical action In addition, the microneedle-like action of the sponge bone needle itself can be used to promote the percutaneous absorption or skin absorption of the above substances, and can be used to promote the absorption of drugs, cosmetics and the like.

Wherein, the transdermal absorption composition refers to the medicine absorbed from the skin into the systemic blood circulation and reaching the effective blood drug concentration to achieve disease treatment or prevention, that is, systemic administration and systemic administration; the skin absorption The composition refers to the way in which the drug reaches an effective concentration locally on the skin to prevent or treat disease, that is, a local administration method.

Compared with the background technology, this technical solution has the following advantages:

The synthesis process of the invention is simple, the reaction conditions are easy to control, and the physical and chemical properties of the product are stable. The product can physically adsorb one or more polypeptides or proteins, and then promote the skin or transdermal administration of the polypeptides and proteins through microneedle-like action The penetration of macromolecular substances into the skin provides an effective way, with broad application prospects and huge potential market value.

BRIEF DESCRIPTION

The present invention will be further described below with reference to the drawings and embodiments.

Fig. 1 is a fluorescence micrograph of GFP (green fluorescent protein) adsorbed by a bee sponge bone needle after silane modification. A: Fluorescence image of unmodified bone needle in the control group, B: fluorescence image of butyl trimethoxysilane modified bee sponge bone needle adsorbing GFP, C: fluorescence image of octyl trimethoxysilane modified bee sponge bone needle adsorbing GFP. The scale in the figure is 100 μm.

Figure 2 is a quantitative comparison of transdermal results of GFP direct transdermal, unmodified bone needles after transdermal and silane-modified bee sponge bone needles adsorbing the same amount of GFP after transdermal massage.

Fig. 3 is a comparison chart of fluorescent slices after GFP direct transdermal, unmodified bone needle massage after transdermal and silane-modified bee sponge bone needles adsorbing the same amount of GFP massage. A: Fluorescence of tissue section of GFP direct transdermal control group, B: fluorescence of tissue section of unmodified bone needle group, C: fluorescence of tissue section of butyltrimethoxysilane modification group, D: modification of octyltrimethoxysilane modification group Fluorescence image of tissue section. The scale in the figure is 100 μm.

detailed description

The following describes the content of the present invention through examples:

Example 1: Preparation of butyltrimethoxysilane modified bee sponge bone needle

(1) Ultrasonic cleaning and drying of the bee sponge bone needle with water, ethanol and acetone in sequence to obtain a bee sponge bone needle with a clean surface.

(2) Add the cleaned bee sponge bone needle obtained in step (1) to a 5 mol / L NaOH solution and sonicate for 30 min; after filtering, washing and drying, add the sponge bone needle to a 10% hydrochloric acid solution and sonicate After 30 minutes, filter, wash and dry to obtain the bee sponge bone needle activated by silanol groups on the surface;

(3) Add the surface silanol group-activated bee sponge bone needle obtained in step (2) to anhydrous toluene, add butyltrimethoxysilane at 90 ° C, the sponge bone needle and butyltrimethoxy group The mass ratio of silane is 1: 1, and the concentration of butyltrimethoxysilane in anhydrous toluene is 10mM; after 8 hours of reaction, a butyltrimethoxysilane modified bee sponge bone needle is prepared.

Example 2: Preparation of octyltrimethoxysilane modified bee sponge bone needle

(2) Add the cleaned bee sponge bone needle obtained in step (1) to a 10 mol / L NaOH solution and sonicate for 10 min; after filtering, washing and drying, add the sponge bone needle to a 20% hydrochloric acid solution and sonicate 10min, filter, wash and dry to obtain bee sponge bone needle activated by silanol group on the surface;

(3) Add the surface silanol group-activated bee sponge bone needle obtained in step (2) to anhydrous toluene, add octyltrimethoxysilane at 95 ° C, the sponge bone needle and octyltrimethoxy group The mass ratio of silane is 1: 2, and the concentration of octyltrimethoxysilane in anhydrous toluene is 5mM; after reaction for 12h, octyltrimethoxysilane modified bee sponge bone needles are prepared.

Example 3: Confirmation of butyl trimethoxysilane modified bee sponge bone needle and octyl trimethoxysilane modified bee sponge bone needle

The X-ray photoelectron spectroscopy (XPS) elemental analysis results of bee sponge bone needles modified with butyl trimethoxysilane and octyltrimethoxysilane are as follows:

As can be seen from the table above, after surface modification of bee sponge bone needles with butyltrimethoxysilane and octyltrimethoxysilane, respectively, the proportion of C element increased compared with unmodified bone needles, indicating that butyl and The octyl group has been connected to the surface of the bee sponge bone needle, that is, the butyl trimethoxysilane modified bee sponge bone needle and the octyl trimethoxysilane modified bee sponge bone needle are successfully prepared.

Example 4: Application of butyltrimethoxysilane modified bee sponge bone needle and octyltrimethoxysilane modified bee sponge bone needle

(1) Adsorption of GFP on unmodified and surface-modified bee sponge bone

Mix the butyltrimethoxysilane modified bee sponge bone needle prepared in Example 1 with the green fluorescent protein solution, and perform protein adsorption through physical action; the octyltrimethoxysilane modified bee sponge bone needle prepared in Example 2 Mix with the green fluorescent protein solution to perform protein adsorption through physical action; at the same time, use a bee sponge bone needle without surface modification to mix with the same green fluorescent protein solution. After mixing for 1 hour, filtering and washing, the bone needles of each group were placed under a fluorescence microscope and the results were shown in FIG. 1.

It can be seen from Figure 1 that when the bee sponge bone needle without surface modification is filtered and washed and then placed under a fluorescence microscope (see Figure 1A), there is almost no fluorescence display, indicating that the bee sponge bone needle without surface modification is almost Cannot adsorb GFP or the adsorption capacity is very low; but butyl trimethoxysilane modified bee sponge bone needle and octyl trimethoxysilane modified bee sponge bone needle can adsorb GFP, after filtering and washing, it can be seen under fluorescent microscope It showed obvious green fluorescence (as shown in Figure 1B and Figure 1C), indicating that the butyl trimethoxysilane modified bee sponge bone needle and the octyl trimethoxysilane modified bee sponge bone needle had adsorbed a large amount of GFP. After further calculation, the mass of GFP adsorbed by butyl trimethoxysilane modified bee sponge bone needle and octyl trimethoxysilane modified bee sponge bone needle were 1.1 μg / mg and 1.4 μg / mg, respectively.

(2) Promoting the transdermal effect of unmodified and surface-modified bee sponge bone against GFP

The bone needles of each group in (1) mixed with GFP solution, filtered and washed were directly smeared on the isolated pig skin, and the GFP solution was directly smeared on the isolated pig skin as a control. Each group cooperated with massage to assist absorption. After 8 hours, the distribution of green fluorescent protein in each layer of the skin is shown in Figure 3, and the quantitative comparison is shown in Figure 2.

Combined with Figure 2 and Figure 3, it can be seen that after the GFP solution is directly applied and assisted with massage absorption, the GFP fluorescence value in the skin is less than 400 (control group in Figure 2), and the tissue section is placed under a fluorescence microscope to observe that there is almost no green fluorescence in the skin (Figure 3A ), Indicating that direct application of GFP massage can hardly achieve transdermal absorption. After the surface-modified bee sponge bone needle was mixed with the same green fluorescent protein solution for a period of time, filtered, washed, directly applied and assisted with massage absorption, the GFP fluorescence value in the skin was about 700 (bone needle group in Figure 2), It indicates that the bee sponge bone needle without surface modification can also promote the transdermal absorption of GFP to a certain extent, which may be caused by the microneedle effect of the bee sponge bone needle itself, but the GFP molecular weight is large, and the tissue section is placed Observed under a fluorescence microscope, it can be seen that GFP is basically located in the superficial layer of the stratum corneum of the skin, which is difficult to absorb deeply (Figure 3B). But after butyl trimethoxysilane modified bee sponge bone needle and octyl trimethoxysilane modified bee sponge bone needle are applied directly and assisted with massage absorption, the GFP fluorescence value in the skin can reach more than 1200 (Figure 2 butyl modified group And octyl-modified group), tissue sections were observed under a fluorescent microscope, and the epidermal layer and dermal layer of the skin had green fluorescence (Figure 3B and Figure 3D). After the surface modification of the bee sponge bone needle, it can adsorb GFP and It penetrates into the skin and releases it to promote the transdermal absorption and depth of GFP.

The above are only preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention Within range.

Industrial applicability

The invention provides a surface-modified sponge bone needle and its preparation method and use. The synthesis process of the invention is simple, the reaction conditions are easy to control, and the physical and chemical properties of the product are stable. The product can physically adsorb one or more polypeptides or proteins. Furthermore, the microneedle-like action promotes the skin or transdermal administration of peptides and proteins, and provides an effective way for the penetration of macromolecular substances into the skin. It has broad application prospects, huge potential market value, and good industrial practicality.

Claims

A surface-modified sponge bone needle, characterized in that the surface of the sponge bone needle is modified with an aliphatic hydrocarbon group, an aromatic hydrocarbon group, an aramid group or other organic groups derived from these three types of groups.
The surface-modified sponge bone needle according to claim 1, characterized in that: the aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl fat group or derivative group thereof is combined with the sponge bone needle through a -Si-O-Si- bond; The aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl aliphatic group or derivative group thereof is connected to each other through a -Si-O-Si- bond.
The surface-modified sponge bone needle according to claim 1, wherein the structural formula of the surface-modified sponge bone needle is as follows:

Wherein, R is the aforementioned aliphatic hydrocarbon group, aromatic hydrocarbon group, arylaliphatic group or a derivative group thereof.
The surface-modified sponge bone needle according to claim 1, characterized in that: the sponge bone needle is derived from a bee sponge Haliclona sp .; the aliphatic hydrocarbon group is a C3-C18 alkyl group; the aromatic hydrocarbon group is a phenyl group ; The aryl group is benzyl or phenethyl.
The surface-modified sponge bone needle according to claim 1, wherein the surface of the sponge bone needle is modified with a group of a silane coupling agent.
A method for preparing a surface-modified sponge bone needle according to any one of claims 1 to 5, characterized in that it comprises:

1) Add sponge bone needle to 1 ～ 10mol / L NaOH solution, ultrasonic for 10 ～ 60min; after filtering, washing and drying, add sponge bone needle into 5 ～ 20% hydrochloric acid solution, sonicate for 10 ～ 60min, filter , Washing and drying to obtain sponge bone needle activated by silanol group on the surface;

2) Add the surface silanol group-activated sponge bone needle obtained in step 1) to an anhydrous aprotic solvent, add a silane coupling agent at 80 to 100 ° C, the sponge bone needle and the silane coupling agent The mass ratio is 1: 0.5 to 2, the concentration of the silane coupling agent in the anhydrous aprotic solvent is 1 to 50 mM; after the reaction for 2 to 12 hours, a surface-modified sponge bone needle is prepared.
The preparation method according to claim 6, further comprising: sequentially cleaning and drying the sponge bone needle with water, ethanol, and acetone to obtain a sponge bone needle with a clean surface; and then cleaning the sponge bone with the clean surface The needle performs activation of the surface silanol group.
The preparation method according to claim 6, wherein the anhydrous aprotic solvent is anhydrous toluene.
The preparation method according to claim 6, characterized in that the silane coupling agent is of the general formula R-SiX 3 , R-Si (R ') 2 -X or R-Si (R')-X 2 ; The X is an alkoxy group, an acyloxy group or a halogenated group; the R is the aliphatic hydrocarbon group, aromatic hydrocarbon group, aryl aliphatic group or a derivative group thereof; and the R ′ is a short-chain aliphatic hydrocarbon group.
The preparation method according to claim 9, wherein the alkoxy group is methoxy, ethoxy, or isopropoxy; the acyloxy group is acetoxy; and the halo group is chlorine Substitution; the short-chain aliphatic hydrocarbon group is methyl or ethyl.
The preparation method according to claim 6, wherein the silane coupling agent is butyltrimethoxysilane, butyltriethoxysilane, octyltrimethoxysilane, benzyltrichlorosilane, Phenyltriethoxysilane, phenyltrimethoxysilane, dodecylmethyldiethoxysilane, octylmethyldimethoxysilane, decylmethyldichlorosilane, hexyldimethyl Acetoxysilane, octadecyldimethylchlorosilane, octyldimethylmethoxysilane.
The preparation method according to claim 6, wherein the purity of the sponge bone needle is not less than 90%.
Use of the surface-modified sponge bone needle according to any one of claims 1 to 5 in the preparation of a percutaneous absorption composition or a skin absorption composition.