Bovine serum albumin functionalized pine-soot ink carbon nanoparticles as well as preparation method and application thereof

Abstract

The invention provides bovine serum albumin functionalized pine-soot ink carbon nanoparticles as well as a preparation method and application thereof. The preparation method of the bovine serum albumin functionalized pine-soot ink carbon nanoparticles comprises the following steps: dispersing pine-soot ink carbon nanoparticles in water, adding a bovine serum albumin solution, and uniformly stirring to obtain a mixed solution; and centrifugally removing the uncombined bovine serum albumin in the mixed solution to obtain the bovine serum albumin functionalized pine-soot ink carbon nanoparticles. The pine-soot ink carbon nanoparticles which are naturally sourced are creatively utilized, material selection of a drug delivery system is enriched, and a new thought and possibility are provided for further optimizing a drug delivery technology.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to bovine serum albumin-functionalized pine soot ink carbon nanoparticles, a preparation method and application thereof. Background Art

[0002] Ink is both a physical carrier of Chinese culture and a medicine capable of treating illness. Records of its use, alone or in combination with other medicinal herbs, can be found in medical texts throughout the dynasties. Pine soot and tung oil soot are the primary ingredients in traditional Chinese writing ink and were among the earliest carbon materials used. Ash (carbon nanoparticles) from burning pinewood and pine branches is the primary raw material for making pine soot ink. The ancients often added smoke, glue, and traditional Chinese medicine to the ink-making process, creating medicinal ink. Medicinal ink refers to any ink used as a medicine for treating illnesses and possessing medicinal value. The properties, flavors, meridians, and functions of medicinal inks are clearly documented in herbal remedies and prescription books throughout the ages. For example, the eighth volume of the Chinese Materia Medica records that ink is made from pine soot and is "spicy in taste and neutral in nature. It enters the heart, liver, and kidney meridians," has the effects of "dispersing blood stasis, stopping bleeding, and reducing swelling," and is mainly used to treat "vomiting blood, epistaxis, metrorrhagia, bloody diarrhea, carbuncle and back swelling." The Dictionary of Chinese Materia Medica records that ink is "spicy and neutral. It enters the heart, liver, and kidney meridians," has the effects of "stopping bleeding and reducing swelling," and is mainly used to treat "vomiting blood, epistaxis, metrorrhagia, bloody diarrhea, carbuncle and back swelling." In current clinical practice, medicinal inks have proven effective and are highly praised by doctors and patients, such as the Eight Treasures and Five Gallbladders Medicinal Ink and the Wanying Tablet.

[0003] Currently, many bioactive compounds in the existing technology have low water solubility and insufficient chemical stability, resulting in low bioavailability, which limits their effectiveness in clinical treatment.

[0004] To address this problem, a variety of nanostructured delivery systems have been developed in recent years, such as liposomes, synthetic polymers, polysaccharides, mesoporous silica nanoparticles, and protein carriers. However, these organic carriers generally have limitations such as single function, low drug loading capacity, and poor biocompatibility. Specifically:

[0005] (1) Insufficient biocompatibility: Traditional drug delivery systems may induce immune responses or toxicity in the body, limiting their application in the biomedical field. However, the functionalized natural source pine soot carbon nanoparticle delivery system is biocompatible.

[0006] (2) Stability issues: Under physiological conditions, carbon nanomaterials may aggregate, resulting in decreased stability and affecting drug delivery efficiency and accuracy. We functionalized them with bovine serum albumin to improve their dispersibility and increase drug loading sites.

[0007] (3) There is currently a lack of theoretical basis for the application of naturally derived pine soot ink as a drug delivery system.

[0008] In contrast, carbon nanomaterials, due to their remarkable physical and chemical properties, have shown broad application potential in a variety of fields, including catalyst supports, electronic devices, drug delivery, and bioimaging. Naturally derived pine soot carbon particles (CNPs), a nanoscale material composed of carbon atoms, typically exhibit a spherical or near-spherical geometry and are a zero-dimensional carbon nanomaterial. The development of novel drug delivery systems using pine soot carbon particles not only aims to reintroduce medicinal ink into modern life and leverage its unique medicinal value, but also contribute to the innovative development of Traditional Chinese Medicine. Summary of the Invention

[0009] In view of this, the present invention aims to overcome the defects in the prior art and proposes bovine serum albumin-functionalized pine soot ink carbon nanoparticles and a preparation method and application thereof.

[0010] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0011] In a first aspect, the present invention provides a method for preparing bovine serum albumin-functionalized pine soot ink carbon nanoparticles, comprising the following steps:

[0012] Pine soot ink carbon nanoparticles are dispersed in water, and then a bovine serum albumin solution is added and stirred evenly to obtain a mixed solution; unbound bovine serum albumin in the mixed solution is removed by centrifugation to obtain bovine serum albumin-functionalized pine soot ink carbon nanoparticles.

[0013] In some specific embodiments of the present invention, the mass ratio of the pine soot carbon nanoparticles to bovine serum albumin is (0.5-10):2.

[0014] In some specific embodiments of the present invention, the mass ratio of the pine soot carbon nanoparticles to bovine serum albumin is 2.5:2.

[0015] In a second aspect, the present invention further provides bovine serum albumin-functionalized pine soot ink carbon nanoparticles prepared by the above preparation method.

[0016] In a third aspect, the present invention also provides a use of bovine serum albumin-functionalized pine soot ink carbon nanoparticles in the preparation of a nano-delivery system for delivering hydrophobic drugs.

[0017] In some embodiments of the present invention, the hydrophobic drugs include, but are not limited to, one or more of methotrexate, plumbagin, curcumin, doxorubicin, docetaxel, camptothecin, oridonin, and andrographolide.

[0018] In some embodiments of the present invention, the hydrophobic drug is resveratrol.

[0019] In a fourth aspect, the present invention further provides a nanocomposite CB-Res, wherein the nanocomposite CB-Res is bovine serum albumin functionalized pine soot carbon nanoparticles loaded with resveratrol.

[0020] In a fifth aspect, the present invention also provides a method for preparing the nanocomposite CB-Res, comprising the following steps: adding pine soot carbon nanoparticles functionalized with bovine serum albumin to a resveratrol solution, mixing in the dark, and then centrifuging and washing to remove unbound resveratrol to obtain the nanocomposite CB-Res.

[0021] In some specific embodiments of the present invention, the mass ratio of the resveratrol to the bovine serum albumin functionalized pine smoke ink carbon nanoparticles is (0.5-6):6.

[0022] In some specific embodiments of the present invention, the mass ratio of the resveratrol to the bovine serum albumin functionalized pine smoke ink carbon nanoparticles is 1:1.

[0023] This invention uses bovine serum albumin (BSA) to functionalize pine soot ink carbon nanoparticles, replacing the glue added during the preparation of traditional pine soot medicinal inks. This improves particle dispersibility and increases drug loading sites. The drug delivery system effectively delivers the hydrophobic drug resveratrol, enhancing its biological activity and therapeutic efficacy.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention significantly improves the water solubility, stability and in vitro drug release performance of the drug by loading the hydrophobic drug onto the naturally derived pine soot ink carbon nanoparticle carrier, thereby enhancing the activity and efficacy of the drug.

[0026] (2) The nanomaterial developed in the present invention has a simple preparation process, excellent biocompatibility, can adapt to the delivery needs of a variety of hydrophobic drugs, and has wide applicability and practical value.

[0027] (3) This invention innovatively utilizes naturally derived pine soot ink carbon nanoparticles, enriching the material options for drug delivery systems and providing new ideas and possibilities for further optimizing drug delivery technology. Furthermore, the development of this new drug delivery system not only reintroduces medicinal ink into modern life, leveraging its unique medicinal value, but also contributes to the innovative development of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the solubility of Res and CB-Res;

[0029] Figure 2 In vitro drug release curves of Res and CB-Res;

[0030] Figure 3 is the retention rate (%) of free Res and CB-Res during exposure to UV light for 180 min at room temperature;

[0031] Figure 4 (A) Observation of the effects of different concentrations of Res and CB-Res on the optical density (OD) of yeast cells at 600 nm at 24 h, 48 h, and 72 h. 600nm ); (B) shows the yeast cell CLS treated with different concentrations of Res and CB-Res (CB in the figure refers to pine smoke black carbon nanoparticles functionalized with BSA at a concentration of 2 μg / mL). DETAILED DESCRIPTION

[0032] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0033] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] Where values ​​are described herein as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values ​​falling within that range, regardless of whether a specific value or sub-range is explicitly stated.

[0035] In this document, "a plurality of" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0036] In this document, the terms “preferably” and “more preferably” are only used to describe implementation methods or examples with better effects. It should be understood that they do not limit the scope of protection of the present invention.

[0037] In this document, the word "further" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0038] In this article, the term "and / or" is used to describe the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0039] As used herein, the term "about" means + / - 10%, preferably + / - 5%, more preferably + / - 1% of the specified value.

[0040] In this document, the terms “include,” “including,” “have,” “contain,” etc. are open-ended terms, meaning including but not limited to.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0042] The following meanings in the embodiments are as follows:

[0043] CNP: pine smoke carbon nanoparticles (purchased from Tianjin Qiandi Biotechnology Co., Ltd.);

[0044] BSA: bovine serum albumin;

[0045] CB: bovine serum albumin-functionalized pine smoke ink carbon nanoparticles;

[0046] Res: resveratrol;

[0047] CB-Res: resveratrol-loaded bovine serum albumin-functionalized pine smoke ink carbon nanoparticles;

[0048] CLS: Chronological Lifespan;

[0049] The present invention will be described in detail below with reference to the embodiments.

[0050] Example 1

[0051] (1) Preparation of nanocomposite (CB-Res):

[0052] 400 mg of CNPs were dispersed in 40 mL of sterile distilled water and sonicated for 120 minutes to obtain a CNP suspension with a mass concentration of 10 mg / mL. 320 mg of BSA was then dispersed in 16 mL of PBS to obtain a 20 mg / mL bovine serum albumin (BSA, Sigma-Aldrich, America). The CNP suspension and BSA solution were then mixed at various volume ratios (0.5:1, 2.5:1, 6:1, and 10:1). The mixture was magnetically stirred at 4°C for 12 hours. During this time, unbound BSA was removed by centrifugation at 12,000 rpm for 10 minutes, yielding functionalized CNPs, designated CBs. The encapsulation efficiency (EE) and drug loading (LA) of CBs at various volume ratios are shown in Table 1.

[0053] Table 1 shows that the optimal volume ratio of CB is CNP:BSA = 2.5:1. 30 mg of CB at this optimal volume ratio was dissolved in 5 mL of PBS to obtain a 6 mg / mL CB solution. 16 mg of resveratrol (Res) was first completely dissolved in 0.25 mL of DMSO, and then 7.75 mL of PBS (DMSO <5%) was added to obtain a 2 mg / mL resveratrol (Res, Sigma-Aldrich, America) solution. The resveratrol (Res) and CB solutions were then mixed at various volume ratios (0.25:1, 0.5:1, 1.5:1, 2:1, and 3:1) for 4 hours. The pellets were centrifuged at 13,000 rpm for 10 minutes to remove unbound Res, and the supernatant was analyzed by UV-visible spectroscopy (Agilent Cary 8454). The particles were washed three times with double-distilled water to remove unbound Res. The encapsulation efficiency (EE) and drug loading (LA) of CB and CB-Res were evaluated according to the following formulas (1) and (2).

[0054] EE (%) = (total amount of drug added - amount of drug in supernatant) / total amount of drug added × 100

[0055] LA (%) = (total amount of drug added - amount of drug in supernatant) / (total amount of drug added + mass of carrier) × 100

[0056] The results are shown in Table 1.

[0057]

[0058] The results are shown in Table 2.

[0059]

[0060] As shown in Table 2, we selected CB-Res with a volume ratio of Res:CB = 3:1 for subsequent experiments.

[0061] (2) Determination of the solubility of Res and CB-Res: Solubility studies were performed by adding 1 mg of free Res and CB-Res (containing 1 mg of Res) to 1 mL of deionized water. The mixture was gently shaken at 37°C for 48 h, and the supernatant was centrifuged and analyzed using a UV-visible spectrophotometer at a wavelength of 305 nm.

[0062] The results are as follows Figure 1 As shown in Figure 3, the solubility of resveratrol in CB-Res increased by 69.68% (52.68 μg / mL) compared with free resveratrol (36.71 μg / mL).

[0063] (3) Determination of in vitro drug release properties of Res and CB-Res: The release of CB-Res was measured in Tween 80-phosphate buffered saline (PBS-Tween 80) (0.05% Tween 80, pH 7.2-7.4). Equal amounts (1 mg) of Res and CB-Res were encapsulated in separate dialysis bags (molecular weight cutoff 3500 Da) (Shanghai Yuanye Biotechnology Co., Ltd., China) and immersed in 25 mL of PBS-Tween 80 while shaking at 120 rpm at 37°C. During this period, 2 mL of Res and CB-Res samples were taken out at specific times. After sampling, 2 mL of fresh PBS-Tween 80 was added. The extracted samples were analyzed using a high-performance liquid chromatography method produced by Shandong Wukong Instrument Co., Ltd. The data shown are the average of three measurements.

[0064] The results are as follows Figure 2 As shown in Figure 3, the release rate of Res was less than 40% at 36 h. In contrast, the release percentage of Res in CB-Res reached approximately 55% at the same time point.

[0065] (4) Determination of UV stability of Res and CB-Res: The stability of Res samples placed in a 60 mm diameter culture dish was tested using a 4W UV lamp with a wavelength of 365 nm; the amount of retained Res was measured at 305 nm using a UV-Vis spectrophotometer at different time points. The results are shown in Figure 4. Figure 3 shown.

[0066] (5) Cell culture

[0067] Yeast cells were selected as a model of aging. The Saccharomyces cerevisiae BY4741 strain was provided by Dr. Yan Xiaohui of Tianjin University of Traditional Chinese Medicine. The cells were cultured at 28°C using SDC medium (Shanghai Ruichu Biotechnology Co., Ltd.). After 24 hours of culture, the cells were centrifuged to obtain cell pellets, which were then reconstituted into fresh SDC medium. Finally, cell suspensions with an optical density (OD) of 0.16–0.20 at 600 nm were selected for subsequent studies.

[0068] (6) Timing life analysis

[0069] A series of lifespan assessment experiments were conducted using 96-well plates (200 μL of yeast culture per well). In these experiments, yeast inocula were introduced into wells containing varying concentrations of Res and CB-Res. Incubation was controlled at 28°C, and growth was systematically assessed at 24, 48, and 72 hours.

[0070] Propidium iodide (PI) fluorescence (Sigma-Aldrich, USA) was used to assess the survival of yeast cells at different ages. During the CLS assay, 40 μL of yeast cells were added to separate 96-well plates at specified time intervals. The cells were then placed in 100 μL of 1× PBS containing 5 μg / mL PI and incubated in the dark for 15 minutes. In the same plate, cells treated with boiling water for 12 minutes were used to establish a positive control and stained with PI. Cells not stained with PI were used as negative controls. After incubation, the cells were washed with 200 μL PBS and resuspended. A full-wavelength microplate reader (Tecan Infinite F50, Tecan Trading AG, Switzerland) was used to measure the fluorescence intensity of each sample excited at 535 nm (emission wavelength was 617 nm) and the optical density at 600 nm. The cell survival rate was calculated using the following formula.

[0071]

[0072] Where I ij(535nm / 617nm) Refers to the specific fluorescent signal detected from a specific well when excited by 535 nm light and emits 617 nm light in the experiment. D(535nm / 617nm) Refers to the fluorescence intensity of the control sample consisting entirely of PI-stained dead cells. C(535nm / 617nm) is the fluorescence intensity of live cells not stained with PI. The optical density (OD) value at 600 nm provides a measure of light absorbance for different cell populations.

[0073] The results are as follows Figure 4 As shown in A and 4B, Figure 4 A shows that cell growth reached saturation at about 48 hours after incubation with different concentrations of Res and CB-Res. Starting from 72 hours, the overall cell growth showed a downward trend. Therefore, the 72-hour stationary period of cell culture was used as the first day for chronological life span (CLS) detection. The survival curves of yeast cells at different age time points showed that CLS had a concentration-dependent extension effect in the case of free Res and CB-Res. Figure 4 As shown in Figure B, on day 10, the viability of cells treated with 32 μg / mL Res was approximately 92%, while that of cells treated with 0 μg / mL Res was approximately 43% and that of cells treated with 32 μg / mL Res was approximately 48%. On day 20, the viability of cells treated with 32 μg / mL Res exceeded 65%, while that of cells treated with 0 μg / mL Res was less than 30%. Furthermore, after treatment of aged yeast cells with 4-32 μg / mL Res, cell viability dropped below 43%. These results clearly demonstrate that CB-Res significantly extends the lifespan of yeast cells compared to free Res.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing bovine serum albumin-functionalized pine soot ink carbon nanoparticles, characterized by: The steps include: Pine soot ink carbon nanoparticles are dispersed in water, and then a bovine serum albumin solution is added and stirred evenly to obtain a mixed solution; unbound bovine serum albumin in the mixed solution is removed by centrifugation to obtain bovine serum albumin-functionalized pine soot ink carbon nanoparticles.

2. The method for preparing bovine serum albumin-functionalized pine soot ink carbon nanoparticles according to claim 1, characterized in that: The mass ratio of the pine soot carbon nanoparticles to bovine serum albumin is (0.5-10):

2.

3. The method for preparing bovine serum albumin-functionalized pine soot ink carbon nanoparticles according to claim 1, characterized in that: The mass ratio of the pine soot carbon nanoparticles to the bovine serum albumin is 2.5:

2.

4. Bovine serum albumin-functionalized pine soot carbon nanoparticles prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the bovine serum albumin-functionalized pine soot carbon nanoparticles according to claim 4 in preparing a nano-delivery system for delivering hydrophobic drugs.

6. The use according to claim 5, characterized in that: The hydrophobic drugs include, but are not limited to, one or more of methotrexate, plumbagin, curcumin, doxorubicin, docetaxel, camptothecin, oridonin, and andrographolide.

7. The use according to claim 5, characterized in that: The hydrophobic drug is resveratrol.

8. A nanocomposite CB-Res, characterized by: The nanocomposite CB-Res is bovine serum albumin-functionalized pine smoke ink carbon nanoparticles loaded with resveratrol, and the bovine serum albumin-functionalized pine smoke ink carbon nanoparticles are the bovine serum albumin-functionalized pine smoke ink carbon nanoparticles according to claim 4.

9. A method for preparing the nanocomposite CB-Res according to claim 8, characterized in that: The method comprises the following steps: adding pine smoke ink carbon nanoparticles functionalized with bovine serum albumin into a resveratrol solution, mixing in the dark, and then centrifuging and washing to remove unbound resveratrol to obtain a nanocomposite CB-Res.

10. The method for preparing the nanocomposite CB-Res according to claim 9, characterized in that: The mass ratio of the resveratrol to the bovine serum albumin functionalized pine smoke ink carbon nanoparticles is (0.5-6):6, preferably 1:1.

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