CA2951729C - Albumin-indocyanine green-paclitaxel complex and preparation method and use thereof - Google Patents

Abstract

An albumin-indocyanine green-paclitaxel complex consists of indocyanine green, paclitaxel and human serum albumin. The preparation method comprises adsorbing indocyanine green and paclitaxel onto human serum albumin by hydrophobic interaction force. The compound is used to prepare drugs for treating cancer, and to conduct photo-thermal treatment and chemotherapy in fluorescence imaging mode.

Description

ALBUMIN-INDOCYANINE GREEN-PACLITAXEL COMPLEX AND
PREPARATION METHOD AND USE THEREOF
This application claims the priority of the China Patent Application No.
201410265653.2, filed with the Patent Office of China on June 13, 2014, titled "ALBUMIN-INDOCYANINE GREEN-PACLITAXEL COMPLEX AND PREPARATION
METHOD AND USE THEREOF".
FIELD OF THE INVENTION
The present invention relates to the field of biomedicine, specifically to an albumin-indocyanine green-paclitaxel complex and preparation method and use thereof.
BACKGROUND OF THE INVENTION
Cancer is one of the major malignant diseases threatening human health.
Although since the 1950s, a lot of human, material and financial resources have been devoted to the prevention and treatment of cancer for decades, but progress achieved by mankind is very .. limited in this regard.
Paclitaxel is a widely used anticancer drug following adriamycin and cisplatin, and is mainly used for ovarian and breast cancer, and displays relatively affirmative clinical therapeutic effects on lung cancer, large intestine cancer, melanoma, head and neck cancer, lymphoma, brain tumor as well. However, due to the fact that paclitaxel is highly insoluble in water, and it requires polyoxyethylene castor oil and anhydrous ethanol as hydrotropy agent, and that polyoxyethylene castor oil can lead to different degrees of allergic reactions, neurotoxicity, haematological toxicity in the body, thus the used dosage of paclitaxel is restricted. In addition, polyoxyethylene castor oil forms a large number of droplets in the blood circulation and encapsulates paclitaxel, reducing the amount of paclitaxel that leaves .. from blood circulation and enters into the tissue, thereby reducing the dose-effect relationship of paclitaxel. In order to reduce toxicity and improve efficacy, new dosage forms of paclitaxel have been developed clinically in recent years, in which albumin-bound paclitaxel is a new type of albumin-bound paclitaxel which is free from polyoxyethylene castor oil and uses human serum albumin as the drug carrier and stabilizer, and which has characteristics of exemption from anti-allergy pretreatment, better efficacy, lower toxicity etc.
However, albumin-bound paclitaxel can only be used as chemotherapeutic agent for tumor chemotherapy. Chemotherapy belongs to the therapeutic method for cancer having systemic effects, which can kill tumor cells at the cellular level and the molecular level. In theory, it 11623767.1 can kill all of the tumor cells, but it also inevitably has killing effect against normal cells.
Therefore, it has relatively large toxic and side effects in clinical use, and brings extreme pain to patients.
Photothermal therapy is a novel method for thermal ablation of tumor cells, and the main principle thereof is that tumor cells are directly killed via heat effect generated by photothermal conversion under the irradiation of excitation light. Whether a strong light absorption and a high efficiency of photothermal conversion can be produced on cancer cells are key factors influencing the success of photothermal therapy. Near-infrared light is a new non-invasive tool for photothermal therapy of cancer, can effectively penetrate normal tissues to reach the tumor site, and can reduce the damage to the normal tissues.
Indocyanine green (abbreviated as ICG) is a long wavelength tricarbocyanine dye, widely used in medical clinical diagnosis. ICG is basically not toxic and can be rapidly eliminated from the body, is at first mainly used to examine choroidal tumors, central serous chorioretinopathy, various choroidal inflammations, degenerative diseases, vascular streaks and vascular occlusive disease and the like. Since ICG has good fluorescent and optical absorption properties in the near infrared region and has potential applications in biomedicine, it is used for fluorescent imaging and photothermal therapy in recent years.
However, some physical characteristics such as aggregation at high concentration, poor stability, nonspecific adsorption and poor targeting capacity limit the development and application of ICG in biomedicine. Currently, some literatures report that adsorbing ICG onto albumin can enhance its fluorescence and stability. However, the binding stability of ICG and albumin is not very good, and the blood circulation time is not very good, and the enriched amount thereof at tumor sites is not high.
At present, for the treatment of tumor, the scholars universally believe that the combined application of various therapeutic methods is at present relatively effective and feasible. Thus, several therapeutic methods having different mechanisms, timing and routes of action are selected and used in combination to treat tumor.
SUMMARY OF THE INVENTION
In view of this, the object of the present invention is to provide an albumin-indocyanine green-paclitaxel complex. The complex has strong fluorescent and optical absorption properties in the near infrared region, and can kill tumor cells, and can implement combination therapy and imaging on both cellular and in vivo levels.
In order to achieve the object of the present invention, the present invention employs the following technical solutions:
11623767.1 2 An albumin-indocyanine green-paclitaxel complex consisting of indocyanine green, paclitaxel and human serum albumin, wherein the indocyanine green and the paclitaxel are adsorbed onto the human serum albumin by hydrophobic interaction force.
Among others, human serum albumin (Human Serum Albumin, abbreviated as HSA) is a protein in human plasma, the non-glycosylated single chain polypeptide of which comprises 585 amino acids, has a molecular weight of 66 kD. In plasma, its concentration is 42g/L, accounting for about 60% of total plasma protein. Human scrum albumin can transport fatty acids, bile pigments, amino acids, steroids, metal ions, and a number of therapeutic molecules in body fluid, while maintaining the normal blood osmotic pressure. Human serum albumin can be clinically used to treat shock and burns, to replenish blood loss due to surgery, accident or hemorrhea, and can also be used as plasma expanders.
Indocyanine green, commonly known as cardiogreen, green needles for diagnostic use, foxgreen, having a molecular weight of 774.9Da, is a long wavelength tricarbocyanine dye, and is widely used in medical clinical diagnosis. Meanwhile, indocyanine green has good fluorescent and optical absorption properties in the near infrared region, has potential applications in biomedicine, and can be used for fluorescent imaging and photothermal therapy.
Paclitaxel (Paclitaxel, PTX), also known as Taxol, Onxol, Nov-Onxol, has the chemical name 5 p,20-epoxy-1,2a,4,7 [3,1013,13n-hexahydroxytaxane- 11-en-9-one-4,10-diacetate-2-benzoate-13R2'R,3S)-N-benzoy1-3-phenylisoserine ester], with a molecular weight of 853.9 Da.
The albumin-indocyanine green-paclitaxel complex of the present invention consists of indocyanine green, paclitaxel and human serum albumin, wherein the indocyanine green and the paclitaxel are adsorbed onto the human serum albumin by hydrophobic interaction force, so as to obtain a nanoparticle of albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX). Since the indocyanine green in the albumin-indocyanine green-paclitaxel complex has good optical absorption in the near infrared region, and generates heat under laser irradiation and can facilitate more materials entering into the cell, and meanwhile, the paclitaxel in the albumin-indocyanine green-paclitaxel complex is an ideal chemotherapeutic agent that can kill tumor cells, the albumin-indocyanine green-paclitaxel complex of the present invention is an ideal agent for combination therapy. On the other hand, since indocyanine green in the albumin-indocyanine green-paclitaxel complex has strong fluorescence in the near infrared region, it is a good imaging agent and can be used for fluorescent imaging of cells in vivo. Thus, by in vivo fluorescent imaging technique, .. biodistribution can be studied, and the amount of the albumin-indocyanine green-paclitaxel 11623767.1 3 complex that reaches the tumor site can be monitored, so that combination therapy can be guided under fluorescence imaging mode.
In a particular embodiment of the present invention, the particle size of the albumin-indocyanine green-paclitaxel complex of the present invention is determined by transmission electron microscopy (TEM) and dynamic light scattering. TEM
photographs show that the particle size of HSA-ICG is about 7 to 8 nm, whereas the particle size of the albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX) of the present invention is about 50 to 60 nm. It shows that the addition of paclitaxel can induce HSA-ICG
to self-assemble into large nanoparticles, such that the indocyanine green and paclitaxel adsorbed by hydrophobic interaction are more stable. The laser particle size distribution chart shows that the hydration diameter of HSA-ICG is about 10 nm, and the hydration diameters of HSA-PTX and HSA-ICG-PTX are about 100 nm, which further demonstrates that the addition of paclitaxel can induce albumin or HSA-ICG to self-assemble into large nanoparticles. It is shown that paclitaxel not only acts as a chemotherapeutic drug, but also acts as a crosslinking agent that renders proteins to self-assemble into large nanoparticles.
Thus, preferably, the albumin-indocyanine green-paclitaxel complex has the particle size of 50 nm to 60 nm.
Further, preferably, the molar ratio of human serum albumin to indocyanine green is 1:2.
Further, preferably, the loading amount of the paclitaxel is 5% to 8%.
The present invention also provides a method for preparing the albumin-indocyanine green-paclitaxel complex, which comprising dissolving indocyanine green to obtain an indocyanine green solution, dissolving paclitaxel to obtain a paclitaxel solution, dissolving human serum albumin in phosphate buffer solution to obtain a phosphate buffer solution of human serum albumin; then simultaneously adding the indocyanine green solution and the paclitaxel solution to the phosphate buffer solution of human serum albumin, and stirring overnight in protection from light to obtain the complex.
Wherein, by controlling the amounts of the indocyanine green solution and the paclitaxel solution added to the phosphate buffer solution of human serum albumin, albumin-indocyanine green-paclitaxel complexes with different molar ratios of human serum albumin to indocyanine green or with different loading amounts of paclitaxel can be obtained.
Similarly, when preparing albumin-indocyanine green complex and albumin-paclitaxel complex, by controlling the amounts of the indocyanine green solution or the paclitaxel solution added to the phosphate buffer solution of human serum albumin, 11623767.1 4 albumin-indocyanine green complexes with different molar ratios of human serum albumin to indocyanine green or albumin-paclitaxel complex with different loading amounts of paclitaxel can be obtained.
ln some embodiments, the method for preparing the albumin-indocyanine green-paclitaxel complex of the present invention comprises dissolving indocyanine green to obtain a 10 mg/ml indocyanine green solution, dissolving paclitaxel to obtain a 20 mg/ml paclitaxel solution, dissolving human serum albumin in phosphate buffer solution to obtain a

2 mg/ml phosphate buffer solution of human serum albumin; then adding 10 111 of the indocyanine green solution and 411 of the paclitaxel solution into 1 ml of the phosphate buffer solution of human serum albumin, and stifling overnight in protection from light to obtain an albumin-indocyanine green-paclitaxel complex with a molar ratio of human serum albumin to indocyanine green of 1:2, and a loading amount of paclitaxel of 5%.
Wherein, preferably, the solvent for dissolving indocyanine green is dimethylsulfoxide.
Preferably, the solvent for dissolving paclitaxel is ethanol.
Further, in some embodiments, the preparation method of the present invention further comprises a step of purifying the albumin-indocyanine green-paclitaxel complex.
Preferably, the purification is specifically centrifugating the mixture obtained by stirring overnight to collect the supernatant, so as to remove indocyanine green and paclitaxel that are not adsorbed onto the protein.
Further, preferably, the centrifugation is a centrifugation at 14800 rpm for 5min.
The present invention also provides an albumin-indocyanine green-paclitaxel complex obtained by the above-described preparation method.
In one particular embodiment, the temperature variation curve of the albumin-indocyanine green-paclitaxel complex of the present invention is assayed by infrared thermal imager. The results show that within 5min, the temperature of the albumin-indocyanine green-paclitaxel complex of the present invention increases significantly, indicating that the albumin-indocyanine green-paclitaxel complex has a relatively strong optical absorption property and can be used as a material for photothermal therapy.
In one particular embodiment, the effect of combination therapy of HSA-ICG-PTX
was investigated at cellular level. The results show that, under the circumstances of laser irradiation, the gentle warming generated by the photothermal property of HSA-ICG-PTX
can increase the permeability of the cell membrane, thereby facilitating HSA-ICG-PTX' s U23767 .I 5 ability of entering into cells, and in turn killing cells by using PTX. It is shown that the effect of combination therapy of HSA-ICG-PTX at the cellular level is significant.
In one particular embodiment, 4T1 cells incubated with HSA-ICG-PTX having different paclitaxel concentrations are subjected to viability assay by standard MTT
reagents. The results show that, HSA-ICG-PTX has very significant chemotherapeutic effects on cells when the concentration of paclitaxel is very low.
In a particular embodiment, the HSA-ICG-PTX is injected into the bodies of mice bearing 4T1 tumor through the tail vein, a volume of blood is taken at different time points, and the behavior of ICG in the blood circulation system of mice is analyzed by measuring the changes of ICG fluorescent intensity in the blood. The results show that, as time increases, the amount of the material remaining in the blood of mice decays gradually, but the amount of HSA-ICG-PTX remaining in the blood of mice is significantly higher than the amount of HSA-ICG remaining in the blood of mice. It is shown that the blood circulation time of HSA-ICG-PTX is obviously higher than that of HSA-ICG.
In another particular embodiment, the HSA-ICG-PTX is injected into the body of mice bearing 4T1 tumor through the tail vein, the tumor site was exposed to 808 nm laser for 10 min irradiation, and the relationship between the temperature of the tumor site of mice and laser irradiation time, changes in tumor volume arid survival rate of mice were subjected to statistics. The results show that the temperature of the tumor site of mice injected with HSA-ICG-PTX can be rapidly raised to 48 C and maintained for 10 min, facilitating more materials entering into the cells, and killing tumors by using the toxicity of PTX. The tumors in mice are eliminated entirely after two days under the photothermal and chemotherapeutic combined therapeutic effects. All the mice injected with HSA-ICG-PTX are still alive after 50 days, and the tumor site does not regenerate. It is shown that the combined therapeutic effect of HSA-ICG-PTX at in vivo level is significant.
Accordingly, the present invention also provides use of the albumin-indocyanine green-paclitaxel complex in the manufacture of a medicament for treating cancer.
Seen from the above technical solutions, the present invention provides an albumin-indocyanine green-paclitaxel complex and preparation method and use thereof. The albumin-indocyanine green-paclitaxel complex of the present invention consists of indocyanine green, paclitaxel and human scrum albumin, wherein the indocyanine green and the paclitaxel are adsorbed onto the human serum albumin by hydrophobic interaction force.
In the albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX) of the present invention, the chemotherapeutic drug paclitaxel (PTX) acts as a crosslinking agent, causing a 11623767.1 6 plurality of HSA-ICG to combine into 50-60 nm nanoparticles, which not only improves the stability of HSA-ICG, but also greatly increases the blood circulation time of HSA-ICG, greatly improves the amount enriched in the tumor site. On the other hand, the photothermal effect of ICG can facilitate the HSA-ICG-PTX entering into the cells, thereby enhancing the effect of combination therapy.
The albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX) of the present invention has a very good water-solubility and biocompatibility, and has good dispersibility under both aqueous and physiological conditions, and does not precipitate significantly at 14800 rpm; not only has very good optical absorption in the near infrared region, but also generates heat under laser irradiation, facilitating more materials entering into the cell, and can kill tumor cells, and is an ideal combined therapeutic agent; also has a strong fluorescent property, can guide the combination therapy of photothermal therapy and chemotherapy in fluorescence imaging mode.
The albumin-indocyanine green-paclitaxel complex of the present invention was injected to the bodies of tumor-bearing mice via the tail vein, and was monitored by fluorescent imaging technique. It is found that the enrichment of HSA-ICG-PTX at the tumor site is greatly improved as compared to HSA-ICG. Then photothermal therapy was carried out using laser, which has a significant effect, and will not damage other sites, and will not relapse after cure.
The method for preparing the albumin-indocyanine green-paclitaxel complex of the present invention is simple to manipulate, uses readily available raw materials, and can be used for mass preparation of the albumin-indocyanine green-paclitaxel complex.
DESCRIPTION OF DRAWINGS
Figure 1 shows the TEM photographs of HSA-ICG and HSA-ICG-PTX in Example 4;
wherein a is the TEM photograph of HSA-ICG, and b is the TEM photograph of HSA-ICG-PTX.
Figure 2 shows the laser particle size distribution profiles of HSA-ICG, HSA-PTX and HSA-ICG-PTX in Example 4; wherein (black straight line) is HSA-ICG, = = = (black dotted line) is HSA -PTX, (gray straight) is HSA-ICG-PTX.
Figure 3 shows the loading curve of HSA-PTX obtained by high performance liquid chromatography in Example 5.
Figure 4 shows the UV absorption spectra of HSA-ICG-PTX with different ratios but the same HSA concentration in Example 5; wherein, ¨ (black straight line) is HSA-ICG-PTX
11623767.1 7 (1:1:10), ¨ (black dotted line) is HSA-ICG-PTX (1:2:10), ¨ (gray straight) is HSA-ICG-PTX (1:4:10).
Figure 5 shows the fluorescent spectra of HSA-ICG-PTX with different ratios but the same HSA concentration in Example 5; wherein, ¨ (black straight line) is HSA-ICG-PTX
(1:1:10), ¨ ( black dotted line) is HSA-ICG-PTX (1:2:10), (dotted line) is HSA-ICG-PTX
(1:4:10).
Fig. 6 shows the temperature-rising curves of different materials under 808 nm laser irradiation in Example 6; wherein, is water (aqueous solution); is HSA-PTX; is HSA-ICG; is HSA-ICG-PTX.
Figure 7 shows the cell survival curves after incubating 4T1 cells with varying concentrations of PTX and HSA-PTX and HSA-ICG-PTX in Example 7; wherein, ¨
(black straight line) is free PTX (paclitaxel solution), - - (black dotted line) is HSA-PTX, (dotted line) is HSA-ICG-PTX.
Figure 8 shows the effect diagram of combination therapy of HSA-ICG-PTX at cellular level in Example 8; wherein, is HSA-ICG-PTX, is HSA-ICG + laser; is HSA-ICG-PTX + laser.
Figure 9 shows the blood circulation diagram of HSA-ICG and HSA-ICG-PTX in the bodies of mice in Example 9, wherein, --40¨ is HSA-ICG-PTX, is HSA-ICG.
Figure 10 shows the diagram of enrichment conditions of HSA-ICG and HSA-ICG-PTX
.. at the tumor site of mice in Example 10; wherein Figure a is a fluorescent imaging diagram of mice in vivo, with the upper diagram in Figure a being HSA-ICG-PTX, and the lower diagram in Figure a being HSA-ICG, from left to right: 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h;
Figure b is the fluorescent signal analysis diagram; in Figure b, is HSA-ICG-PTX, is HSA-ICG.
Figure 11 shows the distribution profiles of HSA-ICG and HSA-ICG-PTX in vivo in Example 11; wherein Figure a is the fluorescent imaging diagrams of different organs and of tumor sites, with the upper diagram in Figure a being HSA-ICG-PTX, and the lower diagram in Figure a being HSA-ICG; Figure b is the fluorescent signal analysis diagrams of different organs and of tumor sites; in Figure b, is HSA-ICG-PTX, is HSA-ICG.
Figure 12 shows the relationship diagram of the temperature of tumor sites of mice and the time of laser irradiation in Example 12; wherein, is HSA-ICG-PTX; is PBS.
Figure 13 shows the diagram of changes in tumor volume of mice in Example 12;
wherein, is PBS +
laser; -0- is HSA-PTX; is HSA-ICG-PTX; is HSA-ICG-PTX +
11623767.1 8 laser.
Figure 14 shows the statistical graph of the survival rates of mice in Example 12;
wherein, is PBS + laser; ¨'¨is HSA-PTX; is HSA-ICG-PTX; is HSA-ICG-PTX +
laser.
DETAILED ENBODIMEN TS
The Examples of the present invention discloses an albumin-indocyanine green-paclitaxel complex and preparation method and use thereof. Those skilled in the art can use the content herein for reference and appropriately improve the process parameters to achieve it. It should be specifically noted that all the similar substitutions and alterations are .. obvious to those skilled in the art, and they are deemed to be included in the present invention.
Products and methods as well as uses of the present invention have been described by the preferred Examples, and relevant personnel obviously can alter or appropriately change and combine the products and methods as well as uses described herein so as to realize and apply the technology of the present invention without departing from the content, spirit and scope of the present invention.
For further understanding the present invention, the present invention is described in detail in conjunction with the following Examples. Among them, the meanings of abbreviations used in the specification and claims are set forth in the following table:
HSA human serum albumin ICG indocyanine green PTX paclitaxel HSA-ICG albumin-indocyanine green complex HSA-PTX albumin-paclitaxel complex HSA-ICG-PTX album in- indocyan ne green-paclitaxel complex PBS phosphate buffer Example 1. Preparation of albumin-indocyanine green-paclitaxel complex:
Albumin (HAS), as the substrate, adsorbs indocyanine green and paclitaxel by hydrophobic interaction, wherein paclitaxel can act as a crosslinking agent to cause albumin 11623767.1 9 to self-assemable into large nanoparticles, and form the albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX). The specific preparation method is as follows:
Indocyanine green (ICG) was dissolved in dimethyl sulfoxide, and formulated into 10 mg/mL indocyanine green solution. Meanwhile, paclitaxel (PTA) was dissolved in ethanol, and formulated into 10 mg/mL paclitaxel solution. 2mg of human serum albumin (HSA) was weighted, dissolved in 2 mL of phosphate buffer solution, and formulated into 1 mg/mL
phosphate buffer solution of human serum albumin.
The indocyanine green solution and the paclitaxel solution were added to the phosphate buffer solution of human serum albumin in different proportions to obtain albumin-indocyanine green-paclitaxel complexes with different molar ratios of human serum albumin to indocyanine green or different loading amounts of paclitaxel.
6 uL of indocyanine green solution and 25 jiL of paclitaxel solution were added to 1 ml of phosphate buffer solution of human serum albumin, stirred overnight (12h) in protection from light; finally, the mixture obtained by stirring overnight was subjected to centrifugation for 5 min at 14800 rpm, and the unreacted PTX and ICG were removed. The supernatant was albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX), with a molar ratio of human scrum albumin to indocyanine green of 1:2, and a loading amount of paclitaxel of 5%.
Example 2. Preparation of albumin-indocyanine green complex Indocyanine green (ICG) was dissolved in dimethyl sulfoxide, and formulated into 10 mg/mL indocyanine green solution; 2 mg of human serum albumin (HSA) was weighted, dissolved in 2 mL of phosphate buffer solution, then 6 pi, of indocyanine green solution was added, stirred overnight (12 h) in protection from light; finally, the mixture obtained by stirring overnight was subjected to centrifugation for 5 min at 14800 rpm, and the unreacted ICG was removed. The supernatant is albumin-indocyanine green complex (HSA-ICG).
Example 3. Preparation of albumin-paclitaxel complex Albumin-paclitaxel complex of different proportions can be obtained by adding paclitaxel solution to phosphate buffer solution of human serum albumin in different proportions.
Wherein, paclitaxel (PTX) was dissolved in ethanol, and formulated into 10 mg/mL
paclitaxel solution; 2 mg of human serum albumin (HSA) was weighted, dissolved in 2 mL of phosphate buffer solution, then 25 !IL of paclitaxel solution was added, stirred overnight (12 h); finally, the mixture obtained by stirring overnight was subjected to centrifugation for 5 min at 14800 rpm, and the unreacted PTX was removed. The supernatant is 0.2 mg/ml 11623767.1 10 albumin-paclitaxel complex (HSA-PTX).
Example 4. Characterization of albumin-indocyanine green-paclitaxel complex The particle size of each complex prepared by Examples 1-3 were observed by employing transmission electron microscope and dynamic light scattering, respectively. The results are shown in Figure 1 and Figure 2.
After albumin-indocyanine green complex (HSA-ICG) and albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX) were dropped onto copper net, they were stained by phosphotungstic acid to improve protein contrast, and were characterized by transmission electron microscope (FEI Tecnai F20).
Albumin-indocyanine green complex (HSA-ICG) and albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX) (wherein the HSA concentration is 1 mg/ml) were measured by dynamic light scattering instrument to determine the hydrated radius thereof.
Figure 1 is the TEM photographs of albumin-indocyanine green complex (HSA-ICG) and albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX). It can be seen from Figure 1 that the particle size of HSA-ICG is about 7 to 8 nm, whereas the particle size of HSA-ICG-PTX is about 50 to 60 nm. It is shown that the addition of paclitaxel can induce HSA-ICG to self-assemble into large nanoparticles, such that the indocyanine green and paclitaxel attached by hydrophobic interaction are more stable.
Figure 2 is the laser particle size distribution profiles of albumin-indocyanine green complex (HSA-ICG), albumin-paclitaxel complex (HSA-PTX) and albumin-indocyanine green-paclitaxel complex (HSA-ICG-PTX). It is shown in Figure 2 that the hydrated diameter of HSA-ICG is about 10 nm, and the hydrated diameters of HSA-PTX and HSA-ICG-PTX
are about 100 nm. It is further shown that the addition of paclitaxel can induce albumin or HSA-ICG to self-assemble into large nanoparticles. It is shown that paclitaxel not only acts as a chemotherapeutic drug, but also acts as a crosslinking agent to induce protein to self-assemble into large nanoparticles.
Example 5. The determination of the loading amounts of indocyanine green and paclitaxel The amount of PTX loaded on HSA was measured using high performance liquid chromatography (HPLC) to study the effects of loading amounts of paclitaxel.
HSA-PTXs of different proportions were synthesized in accordance with the procedure described in Example 3: HAS and PTX were respectively dissolved in chromatographic pure methanol and incubated at room temperature for 12 h, centrifuged at 14800 rpm for 10 minutes to remove precipitate, and passed through a needle for measurement.
The amount of 11623767.1 11 paclitaxel was detected with HPLC loaded with UV detector (detection wavelength 227nm), with a mixture of methanol and water at a volume ratio of 1:1 being used as the mobile phase, wherein the elution time of paclitaxel is 7.8 min. Then the UV - visible absorption spectroscopy and fluorescence spectroscopy were used to study the loading of ICG The results are shown in Figures 3-5.
Figure 3 is the loading curve of HSA-PTX, the amount of PTX loaded onto HAS is increased with the increase of the added amount of PTX. The concentration of PTX is selected to be 0.2 mg/ml through the loading curve, i.e. the loading amount 5%
is used for the following experiments.
Figure 4 is the ultraviolet absorption spectrum of HSA-ICG-PTX in different proportions (molar ratio of human serum albumin to indocyanine green) with the same HSA
concentration (0.2 mg/ml). From the figure, it can be clearly seen that HSA-ICG-PTX has very good optical absorption in the near infrared region of 700 nm to 850 nm and is a very good photothermal therapeutic agent. With the increase in the loading proportion of ICG, the absorption peak of ICG around 800 nm is increased significantly.
Figure 5 is the fluorescence spectrum of HSA-ICG-PTX in different proportions (molar ratio of human serum albumin to indocyanine green) with the same HSA
concentration (0.2 mg/ml). From the figure, it can be clearly seen that when ICG is adsorbed onto HSA, fluorescence in the near infrared region is significantly enhanced; when the ratio of HSA to ICG is 1:2, the fluorescence is the strongest; when the ICG amount is continually increased, there will be some fluorescence quenching. Therefore, the molar ratio of human serum albumin to indocyanine green is selected to be at 1:2 in the subsequent experiments.
Example 6. Test of temperature-rising curve of HSA-ICG-PTX under laser irradiation 2 ml of HSA-PTX, HSA-ICG and HSA-ICG-PTX prepared in Examples 1-3 with the same HSA concentration (2 mg/ml) and aqueous solutions thereof were placed in cuvettes, and 808 nm laser light (the power is 0.5 W/cm2) was irradiated onto the samples directly. An infrared thermal imager was used to test the temperature change curve thereof, and the results are shown in Figure 6.
From the results of Figure 6, it can be seen that within 5 min, the temperatures of HSA-ICG and HSA-ICG-PTX solutions are increased significantly, indicating that nanoparticles adsorbed with ICG have high photothermal conversion rate;
whereas the control group HSA-PTX and water are essentially free of change under the same laser irradiation. It is shown that HSA-ICG-PTX complex has a relatively strong optical absorption property, and can be used as a material for photothermal therapy.
11623767.1 12 Example 7. Toxicity study of HSA-ICG-PTX nanoparticles at cellular level PTX solution (PTX dissolved in a solution of ethanol and castor oil with a volume ratio of 1:1), HSA-PTX and HSA-ICG-PTX with different concentrations of paclitaxel were respectively incubated with 4T1 cells for 72h, and standard MTT reagents were used for cell viability assay. The results are shown in Figure 7.
From the results of Figure 7, it can be seen that, all of PTX solution (PTX
dissolved in a solution of ethanol and castor oil with a volume ratio of 1:1), HSA-PTX and HSA-ICG-PTX
have very obvious chemotherapeutic effect on cells when the concentration of paclitaxel is very low, and the chemotherapeutic effect of HSA-ICG-PTX is stronger than those of PTX
solution and HSA-PTX.
Example 8. Combination therapy of HSA-ICG-PTX at cellular level 25 .1_ of HSA-ICG prepared in Example 2 and HSA-ICG-PTX material prepared in Example 1 with different concentrations were respectively added to a 96-well plate containing 100 uL of 4T1 cells liquid, irradiated using 808 nm, 0.4 W/cm2 laser device for 30 min and the temperature was monitored to not exceed 45 'C. After culturing for 1 h continually, excess materials were washed away. After cultured for 24 h continually, cell viability was detected by Mrt method to investigate the combination therapeutic effect of HSA-ICG-PTX at the cellular level. The results are shown in Figure 8.
As can be seen from Fig. 8, HSA-ICG-PTX in absence of laser irradiation and HSA-ICG
in the case of laser irradiation have certain impacts on cell viability.
However, in the case of laser irradiation, the photothermal property of HSA-ICG-PTX produces gentle warming, which can increase the permeability of the cell membrane, thereby facilitating the ability of HSA-ICG-PTX entering into the cells, and killing the cells using PTX. It is shown that the combination therapeutic effect of HSA-ICG-PTX at the cellular level is significant.
Example 9. Analysis of in vivo blood circulation of HSA-ICG and HSA-ICG-PTX
200 !IL of the aqueous solutions of HSA-ICG prepared in Example 2 and HSA-ICG-PTX
prepared in Example 1 (CiisA = 5 mg/mL) were injected into the bodies of mice through the tail vein, a volume of blood was taken at different time points, and the behaviors of the above materials in the blood circulation system of mice were analyzed by measuring the changes of ICG fluorescent intensity in the blood. The particular assay method was: the sampled blood were weighted and then the withdrawn blood was dissolved by dissolving solution (1% SDS, 1% Triton-100, 40 mM Tris buffer solution), subjected to low revolution speed centrifugation to remove cell debris, and the supernatant was determined in terms of the fluorescent I 1623767.1 13 intensity of ICG; wherein the excitation wavelength of ICG was 730 nm, the emission peak was at about 810 nm, the receiving spectrum range was from 750 nm to 900 nm.
The statistical results are shown in Figure 9.
Figure 9 is the blood circulation diagram of HSA-ICG and HSA-ICG-PTX in the bodies of mice. As is shown in Figure 9, as time increases, the amount of the material remaining in the blood of mice decayed gradually, but the amount of HSA-ICG-PTX remaining in the blood of mice was significantly higher than the amount of HSA-ICG remaining in the blood of mice. It is shown that, since the crosslinking of PTX leads to the outcome that the particle size of nanoparticles is significantly increased, the blood circulation time of HSA-ICG-PTX
is obviously higher than that of HSA-ICG, which can gain time for the material to be enriched at the tumor site.
Example 10. In vivo imaging of HSA-ICG and HSA-ICG-PTX
HSA-ICG prepared in Example 2 and HSA-ICG-PTX prepared in Example 1 were injected into the bodies of mice through the tail vein, real-time image acquisition was performed on small animal imaging system (CRI) at different time points, and the enriched amount of materials at the tumor site was observed. The results are shown in Figure 10.
Among them, the selected excitation light source is 730 nm, the exposure time is 100 ms.
Figure 10 is the diagram of enrichment conditions of the materials at the tumor site of mice. As is shown in Figure 10(a), the enriched amount of HSA-ICG-PTX at the tumor site is .. significantly higher than the enriched amount of HSA-ICG in the tumor, which is in consistent with the blood circulation data. As the time passed, HSA-ICG was rapidly metabolized out due to the smaller particle size thereof. Figure 10 (b) is the curve of the fluorescence signal value at the tumor site changing over time, which shows that the enriched amount of HSA-ICG-PTX at the tumor site was significantly higher than that of HSA-ICG, and reached to peak at 8 h.
Example 11. Distribution of HSA-ICG and HSA-ICG-PTX in vivo HSA-ICG prepared in Example 2 and FISA-ICG-PTX prepared in Example I were injected into the bodies of mice whose back bears 4T1 tumor via the tail vein.
24 hours later, the mice were sacrificed and the tumor and important organs were removed and placed on .. watch glass. Fluorescent photographs were taken on small animal imaging system to observe the enriched amounts of the materials at various organs and tumor sites. The results are shown in Figure 11.
Figure 11 is the diagram of the enrichment condition of the materials in mice in vivo. As 11623767.1 14 is shown in Figure 11(a), at 24 h, the enriched amount of HSA-ICG-PTX at the tumor site is the highest, and the kidney site also has some enrichment, indicating that HSA-ICG-PTX has certain degree of depolymerization in the body, some materials are metabolized out from the kidney slowly; however, the enriched amounts of HSA-ICG at various parts are very low, indicating that HSA-ICG is easily metabolized out due to the small particle size thereof. This result is consistent with the results of in vivo imaging. Figure 11(b) is the fluorescent signal values of different organs and tumor site semiquantitatively obtained by small animal imaging system. The experimental data further illustrates that the blood circulation time of HSA-ICG-PTX is obviously higher than that of HSA-ICG.
Example 12. Combination therapy of HSA-ICG-PTX at in vivo level Five mice whose back bears 4T1 tumor were selected as the experimental group, and were injected with HSA-ICG-PTX prepared in Example 1 through the tail vein.
After 4 hours, the tumor sites were exposed to 808 nm laser at the power of 0.3 W/cm2 for 10 min, and the temperature of the tumor sites was controlled to be at 48 C. Additional 3 groups of mice (n =
5/group) whose back bears tumor were used as a control group test, and were treated as follows, respectively: (1) the mice were injected with phosphate buffered saline (PBS) and were irradiated for 10 min under the laser with the same power; (2) the mice were injected with the same dose of HSA-PTX prepared in Example 3; (3) the mice were injected with the same dose of HSA-ICG-PTX prepared in Example 1, but without the implementation of laser irradiation. After each group of mice was treated, the tumor volumes in mice's back were measured once every other day. The calculating method for the volume was:
length *
width2/2. When the tumor volume exceeds 1 cm3, it is regarded that the mouse is demised.
The relationship between the temperature of the tumor site of mice and laser irradiation time, changes in tumor volume and survival rate of mice in each group were subjected to statistics.
The results are shown in Figures 12 to 14.
Figure 12 is the relationship between the temperature of tumor sites of mice and the time of laser irradiation. As is shown in Figure 12, the temperature of the tumor sites of mice injected with HSA-ICG-PTX can rapidly rise to 48 C and maintain for 10 min, facilitating more materials entering into the cells to kill tumor by using the toxicity of PTX; while the temperature of the tumor in mice from the control group was almost unchanged, and would not affect the growth of tumor in mice.
Figure 13 is the diagram of changes in tumor volume. As is shown in Figure 13, the tumors in the experimental group of mice eliminated after two days under the combined therapeutic effects of photothermal therapy and chemotherapy, while the photothermal therapy and chemotherapy alone only had certain inhibitory effect on tumor growth in the 11623767.1 15 early stage.
Figure 14 is the statistical chart of the survival rates of mice in each group. As can be seen from Figure 14, on Days 18 to 22, all mice in the control group were died, while all the mice in the experimental group were still alive after 50 days, and the tumor site did not regenerate.
The illustrations of the above Examples are only used to help understanding the method of the present invention and the core concept thereof. It should be noted that, for an ordinary skilled in the art, a number of improvements and modifications can be made to the present invention without departing from the principle of the present invention. These improvements and modifications also fall within the protection scope of the claims of the present invention.
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Claims (10)

1. An albumin-indocyanine green-paclitaxel cornplex consisting of indocyanine green, paclitaxel and human serum albumin, wherein the indocyanine green and the paclitaxel are adsorbed onto the human serum albumin by hydrophobic interaction force.

2. The albumin-indocyanine green-paclitaxel complex according to claim 1, wherein the albumin-indocyanine green-paclitaxel complex has the particle size of 50 nm to 60 nm.

3. The albumin-indocyanine green-paclitaxel complex according to claim 1, wherein the molar ratio of human serum albumin to indocyanine green is 1:2.

4. The albumin-indocyanine green-paclitaxel complex according to claim 1, wherein the loading amount of the paclitaxel is 5% to 8% based on the total weight of the albumin-indocyanine green-paclitaxel complex.

5. A method for preparing the albumin-indocyanine green-paclitaxel complex according to claim 1, comprising dissolving indocyanine green to obtain an indocyanine green solution, dissolving paclitaxel to obtain a paclitaxel solution, dissolving human serum albumin in phosphate buffer solution to obtain a phosphate buffer solution of human serum albumin; then adding the indocyanine green solution and the paclitaxel solution to the phosphate buffer solution of human serum albumin, and stirring overnight in protection from light to obtain the complex.

6. The preparation method according to claim 5, wherein the solvent for dissolving indocyanine green is dimethylsulfoxide.

7. The preparation method according to claim 5, wherein the solvent for dissolving paclitaxel is ethanol.

8. The preparation method according to claim 5, wherein it further comprises a step of purifying the albumin-indocyanine green-paclitaxel complex.

9. An albumin-indocyanine green-paclitaxel complex obtained by the preparation method of any one of claims 4 to 8.

10. Use of the albumin-indocyanine green-paclitaxel complex of any one of claims 1, 2, 3, 4 and 9 in the manufacture of a medicament for treating cancer.