TWI863309B - Devices and methods for producing near-infrared-ii contrast agent - Google Patents

Abstract

Disclosed herein is a device for producing NIR-II contrast agents from fluorescent substances and serum albumin. The device comprises a first container, a mixing vessel, a first tube, and a first flow adjusting valve. According to the embodiments of the present disclosure, the first container and the mixing vessel are connected through the first tube, and the first flow adjusting valve is coupled to the first tube. Also disclosed herein are methods for producing the NIR-II contrast agents by using the present device.

Description

Device and method for preparing near-infrared second-zone developer

This disclosure is related to the field of preparing near-infrared fluorescent developers. Specifically, this disclosure is related to an apparatus and method for preparing near-infrared zone II fluorescent developers.

Currently, developers that can emit near-infrared-I (NIR-I) fluorescence are widely used in the fields of biosensing and medical detection. However, the development effect of NIR-I developers is often disturbed by tissue autofluorescence and photon scattering, which in turn affects the image quality and the depth of tissue penetration.

Recent reports indicate that near-infrared II (NIR-II) has a longer wavelength than NIR-I fluorescence, so NIR-II fluorescence contrast agents can overcome the defects of existing NIR-I fluorescence contrast agents. Specifically, NIR-II contrast agents can penetrate deep tissues and have micron-level spatial resolution and high signal-to-background ratio. Therefore, NIR-II contrast agents are considered to be the most promising contrast agents in the field of in vivo fluorescence imaging technology.

Despite this, these NIR-II developers still face the same problem as traditional developers; that is, they need to be freshly prepared before use to achieve better development effects. Developers used in clinical practice must be prepared in a sterile environment to avoid contamination, which greatly increases the time spent on preparatory work.

In view of this, a novel device is urgently needed in this field to prepare NIR-II developers more efficiently.

The content of the invention is intended to provide a simplified summary of the disclosure so that readers can have a basic understanding of the disclosure. This content of the invention is not a complete overview of the disclosure, and it is not intended to point out the important/key elements of the embodiments of the invention or to define the scope of the invention.

The first aspect of the present disclosure is related to a device for preparing a NIR-II developer from a fluorescent substance and serum albumin. According to certain embodiments of the present disclosure, the device includes a first container for accommodating the fluorescent substance; a mixing tank for receiving the serum albumin; a first tube connected to the first container and the mixing tank; and a first flow regulating valve, which is operably coupled to the first tube to control the flow rate of the fluorescent substance flowing out of the first container. In these embodiments, the first container is vertically arranged above the mixing tank, so that the fluorescent substance can flow out of the first container and enter the mixing tank through the first tube, and then mix with the serum albumin therein to produce the NIR-II developer. When irradiated with light with an excitation wavelength of 500-830nm, the NIR-II developer prepared by mixing according to the above device will significantly enhance the NIR-II fluorescence emitted in the wavelength range of 900-2,000nm.

According to certain embodiments of the present disclosure, the device further includes a mixing element disposed in the mixing tank. Optionally or alternatively, the device further includes a first and a second pressure regulating valve, respectively coupled to the first container and the mixing tank, for respectively controlling the pressure in the first container and the mixing tank.

According to alternative embodiments of the present disclosure, the device further includes a second container for accommodating serum albumin; a second tube connected to the second container and the mixing tank; and a second flow regulating valve, which is operably coupled to the second tube to control the flow rate of serum albumin flowing out of the second container. In these embodiments, the second container is vertically arranged above the mixing tank so that serum albumin can flow out of the second container and enter the mixing tank through the second tube. In some preferred embodiments, the device further includes a third pressure regulating valve, which is coupled to the second container to control the pressure in the second container.

According to certain embodiments of the present disclosure, the fluorescent substance is a cyanine dye or a fluorescent nanoparticle. In certain preferred embodiments, the fluorescent substance is a cyanine dye. In a specific embodiment, the cyanine dye is a clinically recognized near-infrared dye indocyanine green (ICG). According to an alternative embodiment of the present disclosure, the fluorescent substance is a fluorescent nanoparticle. Exemplary fluorescent nanoparticles are fluorescent gold nanoclusters or silver sulfide quantum dots.

According to certain embodiments, the serum albumin is human serum albumin (HSA) or bovine serum albumin (BSA).

The second aspect of the present disclosure is a method for preparing a NIR-II developer using the device described in an embodiment of the present disclosure. According to certain embodiments of the present disclosure, the method comprises the following steps: (a) adding serum albumin to a mixing tank; (b) allowing a fluorescent substance to flow out of a first container, enter the mixing tank through a first tube, and mix with the serum albumin in the mixing tank; (c) adjusting a first flow regulating valve to control the flow rate of the fluorescent substance flowing out of the first container; and (d) mixing the contents of the mixing tank in step (c) at a speed of 120-1,200 revolutions per minute to prepare a NIR-II developer.

According to certain embodiments of the present disclosure, the molar concentration ratio of the fluorescent substance to serum albumin in the NIR-II developer is 1:1 to 1:500. Preferably, the molar concentration ratio of the fluorescent substance to serum albumin in the NIR-II developer is 1:1 to 1:256. In a specific embodiment, the molar concentration ratio of the fluorescent substance to serum albumin in the NIR-II developer is 1:2.

In certain embodiments, the fluorescent substance is a cyanine dye, a fluorescent gold nanocluster, or a silver sulfide quantum dot, and the serum albumin is HSA or BSA.

The third aspect of the present disclosure is a method for preparing NIR-II developer using the device described in another embodiment of the present disclosure. According to certain embodiments of the present disclosure, the method includes adjusting the first and second flow regulating valves respectively to control the flow rate of the fluorescent substance and serum albumin flowing out of the first and second containers and entering the mixing tank, thereby preparing the NIR-II developer.

According to some preferred embodiments of the present disclosure, the molar concentration ratio of the fluorescent substance to serum albumin in the NIR-II developer is 1:1 to 1:500. In some preferred embodiments, the molar concentration ratio of the fluorescent substance to serum albumin in the NIR-II developer is 1:1 to 1:256. According to a specific embodiment, the molar concentration ratio of the fluorescent substance to serum albumin in the NIR-II developer is 1:2.

According to an exemplary embodiment of the present disclosure, the fluorescent substance is a cyanine dye, a fluorescent gold nanocluster or a silver sulfide quantum dot, and the serum albumin is HSA or BSA.

After reading the implementation method below, a person with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation methods adopted by the present invention.

100, 200: Device

110, 210: First container

120, 220: Mixing tank

130, 230: first tube

140, 240: First flow regulating valve

250: Second container

260: Second tube

270: Second flow regulating valve

In order to make the above and other purposes, features, advantages and implementation methods of the present invention more clearly understood, the attached drawings are described as follows.

FIG. 1 is a schematic diagram of a device 100 according to one embodiment of the present disclosure; and FIG. 2 is a schematic diagram of a device 200 according to another embodiment of the present disclosure.

According to the usual practice, the various features and components in the figure are not drawn to scale. The drawing method is to present the specific features and components related to the new invention in the best way. In addition, the same or similar component symbols are used to refer to similar components/parts between different figures.

In order to make the description of the disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as those understood and used by persons of ordinary skill in the art to which the present invention belongs. Furthermore, where not inconsistent with the context, singular terms used in this specification include the plural form of the term, and plural terms used also include the singular form of the term. Specifically, in this specification and the scope of the patent application, the singular forms "a" and "an" include plural references, unless otherwise indicated by the context. In addition, in this specification and the scope of the patent application, the expressions "at least one" and "one or more" have the same meaning, both of which represent one, two, three or more.

Specific implementation methods

The present disclosure is intended to provide an apparatus and method for preparing NIR-II developer. Compared with existing apparatus and processes that need to be operated in a sterile environment, the apparatus of the present disclosure is simple and easy to operate, wherein the NIR-II developer is prepared in a sealed system to avoid the possibility of contamination and will not adversely affect the purity and/or quality of the developer.

Device for preparing NIR-II developer

The first aspect of the present disclosure is a device for preparing a NIR-II developer from a fluorescent substance and serum albumin. According to certain embodiments of the present disclosure, when irradiated with light having an excitation wavelength of 500-830 nanometers, the developer of the present disclosure will enhance the emission of NIR-II fluorescence in the wavelength range of 900-2,000 nanometers.

FIG. 1 is a schematic diagram of a device 100 according to an embodiment of the present disclosure. As shown in the figure, the device 100 structurally includes a first container 110, a mixing tank 120, a first tube 130 and a first flow regulating valve 140.

The first container 110 is configured to contain the first reactant (i.e., fluorescent substance). Depending on the desired purpose, the fluorescent substance may be a cyanine dye or a fluorescent nanoparticle. In some preferred embodiments, the fluorescent substance is a cyanine dye or a derivative thereof, for example, cyanine dye 3 (Cy3), Cy5, Cy7, indocyanine green (ICG), IR-783, IRDye800, IR830, IR-E1050 or other similar substances. Preferably, the cyanine dye is ICG. Alternatively, the fluorescent substance is a fluorescent nanoparticle, for example, a fluorescent gold nanocluster or a silver sulfide quantum dot.

The mixing tank 120 is configured to receive a second reactant (i.e., serum albumin). According to certain embodiments of the present disclosure, the serum albumin may be derived from cows, humans, monkeys, mice, or rats. Preferably, the serum albumin is HSA or BSA.

Additionally or alternatively, the device 100 further includes first and second pressure regulating valves, which are coupled to the first container and the mixing tank (110, 120), respectively, to control the pressure in the first container and the mixing tank.

According to other embodiments of the present disclosure, the fluorescent substance in the first container 110 is sent to the mixing tank 120 through the first tube 130 and mixed with the serum albumin in the mixing tank 120. To this end, a first flow regulating valve 140 is operably coupled to the first tube 130 to control the flow rate of the fluorescent substance flowing out of the first container 110. Preferably, the first container 110 is vertically arranged above the mixing tank 120, so that the fluorescent substance in the first container 110 flows through the first tube 130 by gravity and enters the mixing tank 120. In addition or alternatively, a stirring member (e.g., a stirrer, a stirring blade, etc.) is configured in the mixing tank 120 to mix the contents in the mixing tank 120 (e.g., fluorescent substance and serum albumin).

The first container 110 and the mixing tank 120 can be any container known in the art for containing liquid, gas or solid reactants. According to a preferred embodiment, the fluorescent substance is in liquid form in the first container 110. According to the purpose of use, the first container 110 and the mixing tank 120 can be made of translucent or opaque materials. In some embodiments of the present disclosure, the first container 110 is made of opaque material to protect the fluorescent substance from being exposed to light. Exemplary materials suitable for manufacturing the first container 110 and the mixing tank 120 include, but are not limited to, glass, polymer materials (e.g., polyethylene, polyvinyl chloride, polypropylene, polystyrene and acrylonitrile butadiene styrene) or other materials that do not react with the first and second reactants in the first container 110 and the mixing tank 120. The prepared NIR-II developer is a complex of a fluorescent substance and serum albumin, wherein the fluorescent substance is coated in the serum albumin.

FIG. 2 is a schematic diagram of a device 200 according to another embodiment of the present disclosure. The structure of the device 200 is similar to the structure of the device 100 in FIG. 1, and the only difference is that the device 200 further includes a second container 250, a second tube 260, and a second flow regulating valve 270.

In this embodiment, the second container 250 is used to accommodate serum albumin, wherein the second container 250 is vertically arranged above the mixing tank 220. In this case, serum albumin flows out of the second container 250 by gravity and flows through the second tube 260 into the mixing tank 220. Optionally, the flow rate of serum albumin flowing out of the second container 250 is controlled by a second flow regulating valve 270, wherein the second flow regulating valve 270 is operably coupled to the second tube 260. In addition or alternatively, the device 200 further includes a third pressure regulating valve coupled to the second container 250 to control the pressure in the second container 250.

Similar to the first container 110 and the mixing tank 120, the second container 250 can be any container known in the art for containing liquid, gas or solid reactants, and can be made of translucent or opaque materials.

Method for preparing NIR-II developer using the device disclosed herein

The second aspect of the present disclosure is a method for preparing a NIR-II developer using the device 100 shown in FIG. 1 or the device 200 shown in FIG. 2. According to a preferred embodiment of the present disclosure, the method comprises the following steps: (a) adding serum albumin to a mixing tank; (b) allowing a fluorescent substance to flow out of a first container 110, enter a mixing tank 120 through a first tube 130, and mix with the serum albumin in the mixing tank 120; (c) adjusting a first flow regulating valve 140 to control the flow rate of the fluorescent substance flowing out of the first container 110; and (d) mixing the contents of the mixing tank 120 in step (c) at a rotation speed of 120 to 1,200 rpm to prepare the NIR-II developer.

In step (a), serum albumin is first added to the mixing tank 120. According to some embodiments, the serum albumin can be derived from cows, humans, monkeys, mice or rats. In some operational embodiments, the serum albumin is HSA or BSA.

In step (b), the fluorescent substance in the first container 110 is dripped into the mixing tank 120 by opening the first flow regulating valve 140 to mix with the serum albumin in the mixing tank 120. According to some embodiments of the present disclosure, the fluorescent substance can be a cyanine dye or a fluorescent nanoparticle. In some preferred embodiments, the fluorescent substance is a cyanine dye or a derivative thereof; for example, the cyanine dye includes, but is not limited to, Cy3, Cy5, Cy7, ICG, IR-783, IRDye800, IR830, IR-E1050 or the like. Alternatively, the fluorescent substance is a fluorescent nanoparticle. In some embodiments, the fluorescent nanoparticle is a fluorescent gold nanocluster or a silver sulfide quantum dot.

In a preferred case, in order to form the desired structure of the complex, the molar concentration of the serum albumin is higher than the molar concentration of the fluorescent substance during the reaction. Therefore, in step (c), the flow rate of the fluorescent substance from the first container 110 to the mixing tank 120 is controlled by adjusting the first flow regulating valve 140 to ensure that the fluorescent substance and serum albumin are mixed with each other at the desired molar concentration ratio.

In step (d), the fluorescent substance and serum albumin in the mixing tank 120 of step (c) are uniformly mixed to form the NIR-II developer enhanced by the present disclosure. According to certain embodiments of the present disclosure, the fluorescent substance and serum albumin are mixed at a rotation speed of 120-1,200 rpm. In an operating embodiment, a rotor is placed in the mixing tank to achieve the purpose of stirring.

According to some preferred embodiments, the molar concentration ratio of the fluorescent substance to the serum albumin in the NIR-II developer is 1:1 to 1:500. For example, the molar concentration ratio of the fluorescent substance to the serum albumin in the NIR-II developer is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85 , 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:250, 255, 1:256, 1:257, 1:258, 1:259, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:350, 1:410, 1:450 or 1:500. Preferably, the molar concentration ratio of the fluorescent substance and serum albumin in the NIR-II developer is 1:1 to 1:256. In one operating embodiment, the molar concentration ratio of the fluorescent substance and serum albumin in the NIR-II developer is 1:2.

The third aspect of the present disclosure is a method for preparing NIR-II developer using the device 200 shown in FIG. 2. The method includes adjusting the first and second flow regulating valves (240, 270) to control the flow rate of the fluorescent substance and serum albumin flowing out of the first and second containers (210, 250) and into the mixing tank 220, respectively, to prepare the NIR-II developer.

According to some preferred embodiments, the fluorescent substance flows from the first container 210 into the mixing tank 220, and the serum albumin flows from the second container 250 into the mixing tank 220.

Implementation example

Example 1 Preparation of NIR-II developer

In this embodiment, the device shown in FIG. 1 or FIG. 2 is used to prepare the NIR-II developer.

1.1 Prepare NIR-II developer using the apparatus shown in Figure 1

ICG with a concentration of 322.7 μM and HSA protein with a concentration of 301.2 μM were added to the first container and mixing tank of the device in Figure 1, respectively. ICG was dripped into the mixing tank at a flow rate of about 0.35 to 0.41 ml per minute and mixed with the HSA solution. Five NIR-II developers were prepared according to the formula in Table 1, and were named IH-1, IH-2, IH-3, IH-4 and IH-5.

1.2 Prepare NIR-II developer using the device shown in Figure 2

Add the fluorescent substance ICG with a concentration of 322.7μM and the HSA protein with a concentration of 301.2μM to the first and second containers of the device in Figure 2 respectively. Drop the ICG and HSA solutions into the mixing tank. Five NIR-II developers were prepared according to the formula in Table 2, and were named IA-1, IA-2, IA-3, IA-4 and IA-5.

Example 2 NIR-II developer confirming the contents of this disclosure

The NIR-II developer prepared in Example 1 (i.e., IH-1, IH-2, IH-3, IH-4, IH-5, IA-1, IA-2, IA-3, IA-4, and IA-5) was irradiated with light having an excitation wavelength of 780 nm, and the fluorescent signal emitted by the NIR-II developer was detected using a NIR spectrometer. In this experiment, an ICG solution not mixed with HSA was used as a control group.

From the results shown in Table 3, it can be seen that the NIR-II fluorescence intensity of the control group (i.e., ICG solution) is 214.9, while the NIR-II fluorescence intensity of IH-1, IH-2, IH-3, IH-4, IH-5, IA-1, IA-2, IA-3, IA-4, and IA-5 are 576.0, 709.5, 657.5, 886.6, 852.5, 751.2, 619.7, 806.3, 766.0, and 857.9, respectively.

It should be understood that the above description of the embodiments is given only in the form of embodiments, and those with ordinary knowledge in the art to which this invention belongs can make various modifications. The above specification, embodiments and experimental results provide a complete description of the structure and use of the exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not used to limit the present invention. Those with ordinary knowledge in the art to which this invention belongs can make various changes and modifications to it without deviating from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the attached patent application.

100:Device

110: First container

120: Mixing tank

130: First tube

140: First flow regulating valve

Claims (20)

A device for preparing a near-infrared-II (NIR-II) developer from a fluorescent substance and serum albumin comprises a first container for containing the fluorescent substance; a mixing tank for receiving the serum albumin; a first tube body connected to the first container and the mixing tank; and a first flow regulating valve operably coupled to the first tube body for controlling the flow of the fluorescent substance from the first tube body to the mixing tank. The flow rate of the container outflow; wherein the first container is vertically arranged above the mixing tank, so that the fluorescent substance can flow out of the first container and enter the mixing tank through the first tube to mix with the serum albumin to prepare the NIR-II developer; and when irradiated with light with an excitation wavelength of 500-830nm, the NIR-II developer will emit NIR-II fluorescence with a wavelength range of 900-2,000nm. The device as described in claim 1 further comprises a second container for accommodating the serum albumin; a second tube body connected to the second container and the mixing tank; and a second flow regulating valve operably coupled to the second tube body for controlling the flow rate of the serum albumin flowing out of the second container; wherein the second container is vertically arranged above the mixing tank, so that the serum albumin can flow out of the second container and enter the mixing tank through the tube body. The device as described in claim 1 further includes a mixing element disposed in the mixing tank. The device as described in claim 1 further includes a first pressure regulating valve and a second pressure regulating valve, which are respectively coupled to the first container and the mixing tank to control the pressure in the first container and the mixing tank. The device as described in claim 2 further includes a third pressure regulating valve, which is coupled to the second container to control the pressure in the second container. The device as described in claim 1, wherein the fluorescent substance is a cyanine dye or a fluorescent nanoparticle. The device as described in claim 6, wherein the fluorescent substance is the cyanine dye. The device as described in claim 7, wherein the cyanine dye is indocyanine green (ICG). The device as described in claim 8, wherein the fluorescent substance is the fluorescent nanoparticle, and the fluorescent nanoparticle is a fluorescent gold nanocluster or a silver sulfide quantum dot. The device as described in claim 1, wherein the serum albumin is human serum albumin (HSA) or bovine serum albumin (BSA). A method for preparing a near-infrared-II (NIR-II) developer using the apparatus as described in claim 1, comprising: (a) adding the serum albumin to the mixing tank; (b) allowing the fluorescent substance to flow out of the first container, enter the mixing tank through the first tube, and mix with the serum albumin in the mixing tank; (c) adjusting the first flow regulating valve to control the flow rate of the fluorescent substance flowing out of the first container; and (d) mixing the contents of the mixing tank in step (c) at a speed of 120-1,200 revolutions per minute to prepare the NIR-II developer. The method as described in claim 11, wherein the molar concentration ratio of the fluorescent substance and the serum albumin in the NIR-II developer is 1:1 to 1:500. The method as described in claim 12, wherein the molar concentration ratio of the fluorescent substance to the serum albumin is 1:1 to 1:256. The method as described in claim 13, wherein the molar concentration ratio of the fluorescent substance and the serum albumin is 1:2. The method as described in claim 11, wherein the fluorescent substance is a cyanine dye, a fluorescent gold nanocluster or a silver sulfide quantum dot, and the serum albumin is human serum albumin or bovine serum albumin. A method for preparing a near-infrared-II (NIR-II) developer using the device as described in claim 2, comprising adjusting the first and second flow regulating valves to control the flow rates of the fluorescent substance and the serum albumin flowing out of the first and second containers and into the mixing tank, respectively, to prepare the NIR-II developer. The method as described in claim 16, wherein the molar concentration ratio of the fluorescent substance and the serum albumin in the NIR-II developer is 1:1 to 1:500. The method as described in claim 17, wherein the molar concentration ratio of the fluorescent substance to the serum albumin is 1:1 to 1:256. The method as described in claim 18, wherein the molar concentration ratio of the fluorescent substance and the serum albumin is 1:2. The method as described in claim 16, wherein the fluorescent substance is a cyanine dye, a fluorescent gold nanocluster or a silver sulfide quantum dot, and the serum albumin is human serum albumin or bovine serum albumin.

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