WO2020037752A1 - Modified indocyanine green and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a modified indocyanine green, a preparation method therefor and the use thereof. One of two sulfonic groups of the molecular structure of indocyanine green is substituted by a modified PEG group, thereby obtaining a new type of indocyanine green (ICG) modified by PEG. By modifying ICG, the plasma half-life of ICG can be prolonged, so that the retention time thereof in blood is notably prolonged, and thereby the performance of uses in multiple aspects, such as angiography, intraoperative navigation, and detection and treatment of tumors, is optimized. The modified ICG shows a similar good fluorescent property and spectral absorption capability to the original unmodified ICG; moreover, the modified ICG has a very good stability, and therefore possesses good application prospects in the field of vascular imaging.

Description

Modified indocyanine green, preparation method and application thereof Technical field

The invention relates to a modification method of vascular fluorescence contrast agent, in particular to indigocyanine green modified with PEG, and a preparation method and application thereof.

Background technique

Indocyanine Green (ICG) is a tricarbonyl cyanine dye with a molecular weight of 751.4 Da. It is a negatively charged ion and belongs to the family of cyanine dyes. The indocyanine green structural formula is shown in FIG. 1A of the accompanying drawings. It is a dye medicine commonly used to check liver function and liver effective blood flow. Immediately after being injected into the body, it is combined with plasma proteins, and is quickly distributed in the blood vessels of the whole body with blood circulation. It is efficiently and selectively taken up by hepatocytes, and then excreted from the hepatocytes into the bile in a free form, enters the intestine through the biliary tract, and is discharged with feces. in vitro. Due to fast excretion, about 97% of normal people are excluded from the blood after 20 minutes of intravenous injection, do not participate in chemical reactions in the body, no enterohepatic circulation (ICG entering the intestine is no longer absorbed into the blood), no lymphatic reflux, and no kidney Wait for other liver organs to be excreted.

ICG is widely used in surgery for angiography. Compared with other angiography methods (X-ray, CT, MRI and PET), ICG can be easily and economically injected for angiography, such as neurosurgery, bypass coronary surgery , Flap surgery, reconstruction surgery, trauma surgery and laparoscopic surgery, used to check blood circulation and intraoperative navigation. The ICG bolus enters the systemic blood circulation, and imaging is completed within minutes after the injection, and it can usually be re-injected after about 15 minutes as needed.

ICG is also used in tumor detection and treatment. ICG's fluorescence quantum yield is about 10%, and the fluorescence and photoacoustic signals increase and decrease with the fluorescence quantum yield, respectively. When an ICG molecule is excited, it can further transfer energy to other molecules. Therefore, ICG can be used as a contrast agent for fluorescence imaging and photoacoustic imaging. ICG can use the EPR effect to non-specifically aggregate in tumors, or target modification, specifically to aggregate in tumors, so as to perform tumor detection, localization, and photothermal therapy and photodynamic therapy.

However, the ICG has a very short half-life in plasma, which limits the application of ICG as a near-infrared fluorescent imaging material. At present, clinical practice is based on the use of multiple injections or slow injections to obtain accurate fluorescent detection results for the clinic. difficult. For example, when measuring liver blood flow, 25 mg of ICG is dissolved in as little sterilized injection water as possible, diluted with physiological saline to a concentration of 2.5 to 5.0 mg / ml, and the above-mentioned solution equivalent to 3 mg of ICG is injected intravenously. Next, the intravenous infusion was continued at a rate of 0.27 to 0.49 mg per minute for about 50 minutes at a certain rate until the blood sample was taken (both peripheral blood and hepatic vein blood were required).

It can be seen that the half-life of ICG in plasma is too short, which brings many difficulties and limitations to its practical application.

Therefore, the present invention is expected to modify or modify the existing ICG to extend the plasma half-life, eliminating the need to give patients multiple injections or slow and continuous injections in the blood fluoroscopy test; at the same time, the modified or The modified ICG has good fluorescence properties and spectral absorption capabilities close to or better than the original ICG, and does not affect its application characteristics in angiography.

Summary of the Invention

According to the first aspect disclosed herein, a modified indocyanine green is proposed, in which one of the two sulfonic groups in the molecular structure of the indocyanine green is substituted with a modified PEG group. The modified PEG is also called modified polyethylene glycol, which is a PEG modified by a chemical modification group or a biologically active group. The modified PEG group refers to an active group formed after the modified PEG loses an H atom.

In some embodiments of the disclosed method, the modified PEG includes, but is not limited to, mPEG (monomethoxy ether PEG), mPEG-NH ₂ (mPEG-amino), mPEG-SS (mPEG-succinimide) Succinate), mPEG-SC (mPEG-succinimide carbonate), mPEG2-NHS (mPEG2-N-hydroxysuccinimide ester), mPEG-SPA (mPEG-succinimide propionate) , MPEG-ALD (mPEG-propionaldehyde), mPEG-MAL (mPEG-maleimide), HO-PEG-COOH (a-hydroxy-w-carboxy polyethylene glycol), mPEG-b-PS (poly Block copolymer of ethylene glycol and polystyrene), mPEG-b-PI (block copolymer of polyethylene glycol and polyisoprene), mPEG-b-PAN (embedding of polyethylene glycol and polyacrylonitrile Segment copolymer), mPEG-b-PCL (block copolymer of polyethylene glycol and polyε-caprolactone), mPEG-b-PMMA (block copolymer of polyethylene glycol and polymethyl methacrylate) , Α-hydroxyl-PEG-ω-amide, dihydroxyPEG, HO-PEG-NH2 (α-hydroxy-ω-amino polyethylene glycol), mPEG-carboxyl (mPEG-carboxyl), mPEG-cyan (mPEG-nitrile Base) and so on.

In some embodiments of the disclosed method, the molecular weight of the modified PEG group ranges from 500 to 20,000, preferably 500, 2000, 5000, or 10,000, 20,000.

In some embodiments of the disclosed method, the molecular structural formula of the PEG-modified indocyanine green is represented as follows:

Here, n = 9 to 450.

According to the first aspect disclosed herein, a method for preparing a modified indocyanine green is proposed. The molecular structure formula of the modified indocyanine green is as follows:

Where n = 9 to 450; the method includes:

An ICG derivative modified with -CO-NHS ester group and mPEG-NH _{2 are used} as reactants, in which mPEG-NH ₂ undergoes an ester substitution reaction with -CO-NHS in the ICG derivative to remove a molecule of N-hydroxyl Succinimide, a new type of ICG modified by mPEG-NH- (see structural formula C); the reaction process is expressed as:

In some embodiments of the disclosed method, the reaction conditions in the above reaction process are: the -CO-NHS ester group-modified ICG derivative is mixed with mPEG-NH _{2 at} a molar ratio of 1: 5, at a pH of 7.0 to In 8.0 PBS, the reaction was stirred at room temperature for 5-12 hours, and purified by HPLC and other methods to obtain PEG-ICG (see structural formula C).

In short, the main invention of the present invention is to replace one of the two sulfonic acid groups of the indocyanine green molecular structure with a modified PEG group to extend the plasma half-life of ICG without affecting or even strengthening it. The application performance of ICG in angiography, vascular fluorescence imaging and EPR effect. However, during synthesis, the sulfonic acid group of ICG is difficult to directly replace with modified PEG. Therefore, the preparation method of the present invention uses the ICG derivative modified with -CO-NHS ester group as the reactant instead of ICG. [The CO-NHS Ester-modified ICG derivatives can be synthesized by themselves or can be purchased commercially], because the -CO-NHS ester group is highly active, it is easy to transesterify with mPEG-NH ₂ and remove one molecule of NHS to mPEG -NH- is connected to the carbonyl group -C = O-, thereby preparing a new type of ICG modified by PEG, and completing the process of ICG modified by PEG. Wherein, if mPEG-NH ₂ with different molecular weight is used as a reactant, after the reaction is completed, mPEG-NH- of various molecular weights are bonded to -C = O- to achieve the purpose of completing the modification process of PEG with different molecular weights. The reaction is highly controllable, and the reaction product can be designed according to needs, and the reaction conditions are mild (room temperature). In addition, the synthetic route has high selectivity and yield, and can be easily converted for industrial production.

In the present invention, one of the sulfonic acid groups -SO ₃ H of ICG is substituted with a modified PEG group, thereby achieving the effect of significantly extending the half-life of ICG in human plasma. PEG-ICGs and ICG were injected into the tail vein of mice, and near-infrared region II fluorescence imaging was performed on the femoral veins of the mice. From 0.06421h of unmodified ICG to 1.12731h to 6.2413h, compared to unmodified ICG, ICG modified with modified PEG groups with molecular weights of 500, 2000, 5000, 10,000, and 20,000 has a longer retention time in blood. Significantly extended, optimizing its applications in angiography, intraoperative navigation, tumor detection and treatment. At the same time, after examining the absorption spectrum intensity of the modified PEG-ICGs, it can be found that the modified and unmodified ICGs have basically the same spectral absorption capabilities, and are in the near infrared I region (wavelength 650nm-900nm) and II region (wavelength 900nm). -1200nm) have good fluorescence properties, and are consistent with the absorption and emission spectrum properties of unmodified ICG. This shows that PEG-ICGs have the same application value of near-infrared I and II region imaging as ICG. In addition, after testing, the modified PEG-ICGs showed good stability in both PBS (phosphate buffered saline) and FBS (fetal bovine serum) at pH 7.4, so the modified PEG-ICGs of the present invention have Prospects for commercial production and applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a molecular structural formula of indocyanine green ICG, and FIG. 1B is a molecular structural formula of modified PEG-ICGs according to the present invention.

FIG. 2A shows that the intermediate product ICG-NHS of modified ICG is purified by high-performance liquid chromatography analysis and an absorption peak is detected in a 780 nm channel.

Figure 2B, Figure 2C, Figure 2D, Figure 2E, and Figure 2F are PEG500-ICG, PEG2000-ICG, PEG5000-ICG, and PEG10000-ICG, respectively, obtained by mPEG-NH ₂ with molecular weights of 500, 2000, 5000, 10,000, and 20,000. ICG and PEG20000-ICG were purified by HPLC analysis and the absorption peaks were detected in the 780nm channel.

Figure 3 shows the relative absorption spectra of unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG at a concentration of 20 μg / ml at a wavelength of 600 nm to 950 nm.

Figure 4 shows the relative fluorescence intensities of unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG in the near-infrared I region (wavelength 650nm-900nm).

Figure 5 shows the relative fluorescence intensities of unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG in the near infrared II region (wavelength 900nm-1200nm).

Fig. 6 shows the decrease rate of fluorescence intensity of modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG in PBS (components Na ₂ HPO ₄ and NaH ₂ PO ₄ ) at pH 7.4. Time variation curve.

FIG. 7 is a graph showing the change rate of the fluorescence intensity decrease rate of FPEG (fetal bovine serum) with time for modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG.

Figure 8 shows unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG in the near-infrared region II after mice were injected with 2 mg · mL ^-1 tail vein. Group of fluorescence imaging photos.

Fig. 9 is a photo of fluorescence imaging of near-infrared region II in mice with tumors by injecting unmodified ICG and modified PEG5000-ICG into the tail vein, respectively, and testing the EPR of modified PEG5000-ICG. effect.

It should be noted that the drawings are not drawn to scale, and for the purpose of illustration, elements having similar structures or functions are generally indicated by the same reference signs in all drawings. It should also be noted that the drawings are only intended to facilitate the description of preferred embodiments of the invention. The drawings do not illustrate every aspect of the described embodiments and do not limit the scope of the invention.

detailed description

In the present invention, the vascular fluorescent imaging material ICG is mainly modified with a modified PEG group to extend its half-life in the blood. Modified PEG is also called modified polyethylene glycol. It is a PEG modified by a chemical modification group or a biologically active group, that is, the conventional PEG changes its terminal group, and -OH, -COOH, -NH ₂ , methoxy, nitrile and the like are substituted or linked. The modified PEG group refers to an active group formed after the modified PEG loses the H atom at the terminal. Modified PEGs include, but are not limited to, mPEG (monomethoxy ether PEG), mPEG-NH ₂ (mPEG-amine), mPEG-SS (mPEG-succinimide succinate), mPEG-SC (mPEG-amber Imide carbonate), mPEG2-NHS (mPEG2-N-hydroxysuccinimide ester), mPEG-SPA (mPEG-succinimide propionate), mPEG-ALD (mPEG-propionaldehyde), mPEG -MAL (mPEG-maleimide), HO-PEG-COOH (a-hydroxy-w-carboxy polyethylene glycol), mPEG-b-PS (block copolymer of polyethylene glycol and polystyrene) , MPEG-b-PI (block copolymer of polyethylene glycol and polyisoprene), mPEG-b-PAN (block copolymer of polyethylene glycol and polyacrylonitrile), mPEG-b-PCL (poly Block copolymer of ethylene glycol and polyε-caprolactone), mPEG-b-PMMA (block copolymer of polyethylene glycol and polymethyl methacrylate), α-hydroxyl-PEG-ω-amide, bis HydroxyPEG, HO-PEG-NH ₂ (α-hydroxy-ω-aminopolyethylene glycol), mPEG-carboxyl (mPEG-carboxyl), mPEG-cyan (mPEG-nitrile), and the like. The molecular weight of the modified PEG group ranges from 500 to 20,000, and is preferably 500, 2000, 5000, 10,000, or 20,000.

Preferably, the molecular structure formula of the modified indocyanine green of the present invention is represented as follows:

Here, n = 9 to 450.

Preferably, the method for preparing the above-mentioned PEG-modified indocyanine green: using the ICG derivative and mPEG-NH ₂ modified by the -CO-NHS ester group as reactants, wherein mPEG-NH ₂ and the ICG derivative are reacted -CO-NHS undergoes an ester substitution reaction to remove a molecule of N-hydroxysuccinimide to prepare a new type of ICG modified by mPEG-NH- (see structural formula C); the reaction process is expressed as:

Preferably, the reaction conditions in the above-mentioned reaction process are: the ratio of the molar ratio of -NHS-modified ICG derivative (Structural Formula B) to mPEG-NH ₂ (Structural Formula A) of different molecular weight is 1: 5, at pH = 7.0 ～ In 8.0 PBS, shake at room temperature for 5-12 hours and purify by HPLC to obtain PEG-ICG, which is the product represented by (see structural formula C).

The ICG (see structural formula C) modified by the modified PEG group of the present invention is significantly prolonged in the blood, which optimizes its applications in angiography, intraoperative navigation, tumor detection and treatment, and other aspects.

According to the above method, specific preparation examples and test examples are given below.

Production Example 1

Preparation method: The ratio of the ICG derivative (structural formula B, purchased from Intrace Medical SA ) modified with -CO-NHS ester group and mPEG-NH ₂ (structural formula A) with a molecular weight of 500 is 1: 5, at pH = In 7.4 PBS, shake for 12 h at room temperature and purify by HPLC to obtain PEG-ICG (see structural formula C), as shown in Figure 1B.

Production Example 2

This example is the same as Preparation Example 1, except that mPEG-NH ₂ with a molecular weight of about 2000 was used for the reaction. The product is designated as PEG2000-ICG.

Production Example 3

This example is the same as Preparation Example 1, except that mPEG-NH ₂ with a molecular weight of about 5000 was used for the reaction. The product is designated as PEG5000-ICG.

Production Example 4

This example is the same as Preparation Example 1, except that mPEG-NH ₂ with a molecular weight of about 10,000 was used for the reaction. The product is designated as PEG10000-ICG.

Production Example 5

This example is the same as Preparation Example 1, except that mPEG-NH ₂ with a molecular weight of about 20,000 is used for the reaction. The product is designated as PEG20000-ICG.

Product property test

1. Product synthesis characterization test

The PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG prepared in Preparation Examples 1 to 5 are purified by high performance liquid chromatography (HPLC), and a separation column (C4 Column, 300A, 3.5 μm, 4.6 mm × 250 mm), and the absorption peak was detected under the 780 nm channel. The procedure is shown in Table 1:

Table 1:

Time (min) Flow rate (mlmin- ¹ ) Mobile phase A (%) Mobile phase B (%) 0 1 95 5 3 1 95 5 twenty three 1 5 95 26 1 5 95 27 1 95 5 30 1 95 5

See FIG. 2A to FIG. 2F. Figure 2A is an HPLC chart of the reactant ICG-NHS (ICG modified with -CO-NHS ester group) of structural formula B. The retention time in the separation column is 22.4943min. ICG-NHS has low polarity and lipophilicity. Poor water solubility and other characteristics.

FIG. 2B is an HPLC chart of PEG500-ICG in Preparation Example 1. The peak time is 20.7236min earlier than ICG-NHS, which indicates that the polarity of ICG is relatively larger and the water solubility is better, which proves that mPEG-NH ₂ and The ICG connection was successful. Since mPEG-NH ₂ are not connected to the ICG is no specific absorption peak, it is not detected by HPLC mPEG-NH _2. The peak time of Figure 2B is earlier than ICG-NHS, which proves that mPEG-NH ₂ is successfully connected to ICG.

FIG. 2C is an HPLC chart of PEG2000-ICG in Preparation Example 2. The peak time is 19.6685min earlier than ICG-NHS and PEG500-ICG. It also shows that mPEG-NH _{2 has} a larger molecular weight, which further polarizes ICG and changes its water solubility. Good, so the peak time of PEG2000-ICG is earlier than that of PEG500-ICG, which proves that mPEG-NH ₂ is successfully connected to ICG.

FIG. 2D is an HPLC chart of the PEG5000-ICG chart of Preparation Example 3. The peak time was 19.2725min earlier than ICG-NHS, PEG500-ICG, and PEG2000-ICG. Similarly, mPEG-NH _{2 was} successfully connected to ICG.

FIG. 2E is an HPLC chart of PEG10000-ICG in Preparation Example 4. The peak time is 19.6829min earlier than ICG-NHS, but the peak time is later than PEG5000-ICG. It may be because the molecular weight of the connected mPEG-NH _{2 is} too large. Due to the position in the column, although the water solubility is better, the peak time is later than PEG5K-ICG. But again, mPEG-NH ₂ that has not been successfully connected to ICG has no specific absorption peak, so FIG. 2E can prove that mPEG-NH ₂ is successfully connected to ICG.

FIG. 2F is an HPLC chart of PEG20000-ICG in Preparation Example 5. The peak time is 19.4424min. The peak time is earlier than ICG-NHS, and the peak time is later than PEG5K-ICG. It may also be because the molecular weight of the attached PEG is too large, so although the water solubility is better, the peak time is later than PEG5K-ICG. But for the same reason, FIG. 2F can prove that mPEG-NH ₂ is successfully connected with ICG.

PEG-ICGs can be detected only after the connection is successful, and the peak time is earlier than that of ICG-NHS, indicating that the connection is successful, and the PEG-ICGs after the successful connection have better water solubility than ICG-HNS. .

2. Product spectral property test

PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG prepared in Preparation Example 1-5 were tested for their spectral properties (including absorption spectral properties and emission spectral properties), and compared with unmodified ICG Compare. The specific operation is as follows:

The UV-Vis-NIR spectrometer cary 5000 was used to measure the spectral absorption intensities of ICG, PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG in the spectral range of 500nm-1000nm, respectively. See Figure 3. The weighted continuous curve represents PEG20000-ICG, the gray continuous line represents ICG, the dark heavy dotted line represents PEG500-ICG, the thin dotted line represents PEG2000-ICG, the one-dot chain line represents PEG5000-ICG, and the two-dot chain line represents PEG10000-ICG. As can be seen from FIG. 3, the modified PEG-ICGs exhibited a spectral absorption ability substantially consistent with that of the unmodified ICG.

Using the spectrophotometer 200, set the excitation light at 808nm, and test the relative of ICG, PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG in the near infrared I region (650nm-900nm range). The fluorescence intensity. The test results are shown in Figure 4. The weighted continuous curve represents PEG20000-ICG, the gray continuous line represents ICG, the dark heavy dotted line represents PEG500-ICG, the thin dotted line represents PEG2000-ICG, the one-dot chain line represents PEG5000-ICG, and the two-dot chain line represents PEG10000-ICG. As can be seen from FIG. 4, in the near-infrared I region, the modified products PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG have good fluorescent properties comparable to unmodified ICG. It can be seen that the modification of ICG by modified PEG did not affect the fluorescent properties of the original ICG.

Using spectrometers NIRQuest512 and CVH100 / M, set the excitation light at 808nm, and test ICG, PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG in the near infrared II region (in the range of 900nm-1200nm) Relative fluorescence intensity. The test results are shown in Figure 5. The weighted continuous curve represents PEG20000-ICG, the gray continuous line represents ICG, the dark heavy dotted line represents PEG500-ICG, the thin dotted line represents PEG2000-ICG, the one-dot chain line represents PEG5000-ICG, and the two-dot chain line represents PEG10000-ICG. It can be seen from FIG. 5 that in the near-infrared region II, the modified products PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG have good fluorescent properties comparable to unmodified ICG.

The above spectral property tests show that ICG modified by mPEG-NH ₂ has good fluorescence properties in the near-infrared I and II regions, and is consistent with the absorption and emission spectral properties of ICG. Therefore, PEG-ICGs have the application value of near-infrared I and II region imaging equivalent to ICG.

3. Product stability test

(1) Stability test under neutral solution

Take the modified products PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG prepared in Preparation Example 1-5 and dissolve them in PBS solution at pH 7.4 to obtain 5 test samples with a concentration of 20 micrograms. / mL. The components of the PBS buffer solution are Na ₂ HPO ₄ and NaH ₂ PO ₄ . Let the above five test samples stand in a constant temperature box protected from light and test the fluorescence intensity of the five solution samples at 3h, 6h, 9h, 12h, 15h, 18h, 21h, and 24h, respectively, and calculate the decline rate. For the test, a spectrophotometer 200 was used, and the excitation light was set at 808 nm. The test results are shown in Figure 6. The results show that except for the decrease in the fluorescence intensity of PEG500-ICG, which is slightly faster within 1 to 12 hours, the fluorescence intensity of other PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG are stable over time . This shows that the modified products PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG have good stability in a near neutral environment.

(2) In vitro serum stability test

The modified products PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG prepared in Preparation Example 1-5 were respectively dissolved in FBS (fetal bovine serum, SR01C-500 purchased from SAILY BIO) to obtain 5 test samples (20 μg / mL).

Let the above five test samples stand in an incubator. At 3h, 6h, 9h, 12h, 15h, 18h, 21h, and 24h, test the fluorescence intensity of the five test samples and calculate the decline rate. For the test, a spectrophotometer 200 was used, and the excitation light was set at 808 nm. The test results are shown in Figure 7. The results show that the fluorescence intensity of the five test samples is in a very stable state over time. This shows that the modified products PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG have good stability in serum. The above results show that the modified product PEG-ICGs has a good practical application prospect and has commercial conditions.

4. Plasma half-life test

Eighteen healthy mice (about 20 g) were divided into 6 groups of 3 mice each. The 6 groups of mice were respectively injected with ICG and PEG500 in the tail vein according to the amount of ICG in the composition of 1 mg · kg ^-1 (the injection amount was 0.1-1 mg · kg ^-1 , and the concentration of ICG in the injection solution was 20 μg / mL). -ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG. After 3min, 8min, 30min, 120min, 240min, 270min, and 600min, the blood clearance rates of ICG and the above five mPEG-ICG were compared by near-infrared region II fluorescence imaging and vascular fluorescence imaging. The specific operation method and calculation are as follows:

Imaging and photographing conditions: Excited by a 808nm laser, and a 900nm long-pass filter FELH0900 separates the excitation light from the emitted light, and is photographed by a SWIR near-infrared camera.

Mice were anesthetized with the animal anesthesia system VIP3000, and five kinds of PEG-ICGs (PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG) and ICG were injected into the tail vein at the same time point for 3 min. At 4min, 5min, 6min, 7min, 8min, and 12min, near-infrared region II fluorescence imaging was performed on the femoral vein of the mouse, and pictures were taken, and see the attached photo group shown in FIG. 8.

The calculation of ICG or PEG-ICGs half-life in plasma is to compare the fluorescence intensity of the blood vessel site in the image photograph with the fluorescence intensity of the skin of the leg to find the ratio to reflect the blood metabolism time of PEG-ICGs and ICG. Among them, in the ICG group, the blood vessels in the medial leg of the leg were clearly visible 3 minutes after the injection, and the fluorescence signal of the blood vessel part disappeared around 8 minutes. The IC ₍ T _(1/2) = 0.06421h _{) was} calculated.

In the PEG500-ICG group, the blood vessels in the medial leg of the leg were also clearly visible 3 minutes after the injection, and the fluorescence signal disappeared around 12 minutes. The T _(1/2) = 0.16121h of the PEG500-ICG was calculated. Compared with the mice in the ICG group, the metabolism time of PEG500-ICG in the PEG500-ICG group was not significantly prolonged.

In the PEG2000-ICG group, the fluorescence signal disappeared in the blood vessels at about 135 minutes after injection, and T _(1/2) = 1.12731h was calculated. The blood retention time was longer than that of ICG and PEG500-ICG alone.

In the PEG5000-ICG group, the fluorescence signal disappeared in the blood vessels about 240 minutes after the injection, and T _(1/2) = 2.417 h was calculated. Compared with the injection of ICG, PEG500-ICG and PEG2000-ICG alone, the blood retention time was longer.

In the PEG10000-ICG group, the fluorescence signal was still weakly visible in the blood vessels around 270 minutes after injection, and T _(1/2) = 2.6667h was calculated. Compared with the injection of ICG, PEG500-ICG, PEG2000-ICG and PEG5000-ICG alone, the blood retention time was prolonged.

In the PEG20000-ICG group, the fluorescence signal could be detected about 5 hours after the injection, which lasted to 10 hours. Calculated T _(1/2) = 6.2413h. Compared with the single injection of ICG, PEG500-ICG, PEG2000-ICG, PEG5000-ICG and PEG10000-ICG, the blood retention time was longer.

This shows that the PEG2000-ICG, PEG5000-ICG, PEG10000-ICG, and PEG20000-ICG obtained by modifying the ICG by the method of the present invention have a significantly longer residence time in the plasma of the animal compared to the unmodified ICG. Therefore, through the modification of ICG, it has been optimized for many applications such as angiography, intraoperative navigation, tumor detection and treatment.

5. EPR effect test in animals

EPR effect, that is, the high permeability and retention effect of solid tumors. Specifically, it refers to the property that certain sizes of molecules or particles tend to aggregate in tumor tissue compared to normal tissue. The main reason is that the microvascular endothelial space in normal tissues is dense and structurally complete, and macromolecules and lipid particles cannot easily penetrate the vascular wall, while solid tumor tissue is rich in blood vessels, wide vascular wall space, poor structural integrity, and lack of lymphatic reflux, resulting in Macromolecular substances and lipid particles have selective high permeability and retention. This phenomenon is called the high permeability and retention effect of solid tumor tissues, which is referred to as the EPR effect. An important indicator for measuring the suitability of drugs or diagnostic materials for tumor detection is the EPR effect. Here, the EPR effect of the PEG-ICGs prepared by the present invention was tested. The test method is as follows:

Prepare skov-3 tumor cells (purchased from EK-Bioscience) and 2 mice weighing 300 g / mouse. Each tumor has a population of 2,000,000 cells and is injected subcutaneously. After 3-4 weeks, the tumor size reaches 5-10 mm. Two tumor-bearing mice were anesthetized by the animal anesthesia system VIP3000, based on the amount of ICG (due to the different molecular weights of ICG and PEG5000-ICG, here 1mg · kg ^-1 was calculated based on the amount of ICG contained; injection At that time, ICG and PEG5000-ICG were dissolved in PBS at pH 7.4), and ICG and PEG5000-ICG were injected into the tail vein at 1 mg · kg ^-1 each. ICG-injected mice were imaged by near-infrared region II at 3min, 4h, and 6h, and PEG5000-ICG-injected mice were imaged by near-infrared region II at 3min, 12h, and 48h. Imaging and photographing conditions: Excited by a 808nm laser, and a 900nm long-pass filter FELH0900 separates the excitation light from the emitted light, and is photographed by a SWIR near-infrared camera. The photos taken are shown in Figure 9.

Among them, in mice injected with PEG5000-ICG, the blood vessels at the tumor were very clear after 3 minutes, the fluorescence signal at the tumor was obvious at 12 hours, and there was a strong EPR effect, which was clearer than that of mice injected with IEG at 3 minutes. Imaging of PEG5000-ICG-injected mice after 48h was close to imaging of IEG-injected mice at 3min. It can be seen that the PEG-ICGs of the present invention have a strong EPR effect and can be applied to the detection of tumors in animals.

The foregoing content of the present invention has proved that the modification and modification of ICG with mPEG-NH ₂ can significantly extend the half-life of ICG in the blood, thereby significantly extending the residence time of ICG in the blood, and optimizing its navigation in angiography and surgery. , Tumor detection and treatment and other applications.

Similarly, compared to mPEG-NH ₂ , other modified PEGs have properties similar to mPEG-NH ₂ , such as mPEG, mPEG-SS, mPEG-SC, mPEG2-NHS, mPEG-SPA, mPEG-ALD, mPEG-MAL , HO-PEG-COOH, mPEG-b-PS, mPEG-b-PI, mPEG-b-PAN, mPEG-b-PCL, mPEG-b-PMMA, α-hydroxyl-PEG-ω-amide, HO-PEG -OH, HO-PEG-NH ₂ , mPEG-carboxyl, mPEG-cyan, etc. Therefore, in the present invention, these modified PEGs can also be used to modify or modify ICG. The process / conditions of the modification reaction may be all different due to the structure of the modified PEG molecule or the end groups, but it will be understandable to those skilled in the art based on the facts and product properties tested and confirmed by the present invention Some ICGs modified by other modified PEGs should also have half-life and blood metabolic properties similar to those of ICGs modified by mPEG-NH ₂ , can stay in the blood for a longer time, and also show the same as unmodified ICGs. Equivalent / even better fluorescence properties and spectral absorption capabilities can be used in angiography, intraoperative navigation, tumor detection, etc., to optimize the application performance of ICG.

The embodiments described in the present invention are susceptible to various modifications and alternative forms, and specific examples and methods thereof have been illustrated in the drawings and described in detail herein. It should be understood, however, that the invention is not limited to the particular forms or methods disclosed, but on the contrary, the disclosed embodiments are intended to cover all modifications, equivalents, and alternatives.

Claims (10)

A modified indocyanine green is characterized in that one of the two sulfonic groups in the molecular structure of the indocyanine green is substituted with a modified PEG group. The modified indocyanine green according to claim 1, wherein said modifying group is selected from PEG _{mPEG, mPEG-NH 2, mPEG} -SS, mPEG-SC, mPEG2-NHS, mPEG- SPA, mPEG-ALD, mPEG-MAL, HO-PEG-COOH, mPEG-b-PS, mPEG-b-PI, mPEG-b-PAN, mPEG-b-PCL, mPEG-b-PMMA, α-hydroxyl- Any of PEG-ω-amide, HO-PEG-OH, HO-PEG-NH ₂ , mPEG-carboxyl, and mPEG-cyan modified PEG is a group in which H is deleted. The modified indocyanine green according to claim 1, wherein the molecular weight of the modified PEG group ranges from 500 to 20,000. The modified indocyanine green according to claim 3, wherein the molecular weight of the modified PEG group is 500, 2000, 5000, 10,000, or 20,000. The modified indocyanine green according to claim 1, wherein: The molecular structural formula of the modified indocyanine green is as follows:

Here, n = 9 to 450. A method for preparing modified indocyanine green. The molecular structure formula of the modified indocyanine green is as follows:

Wherein n = 9-450; the method includes: using an ICG derivative modified with -CO-NHS ester group and mPEG-NH ₂ as reactants, and mPEG-NH ₂ and -CO-NHS in the ICG derivative occur The ester substitution reaction removes a molecule of N-hydroxysuccinimide to obtain a new type of ICG modified by mPEG-NH- (see structural formula C); the reaction process is expressed as:

The method according to claim 6, characterized in that the reaction conditions of the method are: the ICG derivative modified with -CO-NHS ester group and mPEG-NH ₂ are mixed in a molar ratio of 1: 5 A new ICG modified with mPEG-NH- was prepared in PBS at pH = 7.0-8.0 with reaction at room temperature for 5-12 hours. A novel ICG modified by modified PEG for angiography, intraoperative navigation and tumor detection. The application according to claim 8, wherein the modified PEG includes, but is not limited to, mPEG, mPEG-NH ₂ , mPEG-SS, mPEG-SC, mPEG2-NHS, mPEG-SPA, mPEG-ALD, mPEG -MAL, HO-PEG-COOH, mPEG-b-PS, mPEG-b-PI, mPEG-b-PAN, mPEG-b-PCL, mPEG-b-PMMA, α-hydroxyl-PEG-ω-amide, HO -PEG-OH, HO-PEG- NH 2, mPEG-carboxyl and any one of mPEG-cyan. The application according to claim 8, wherein the molecular weight of the modified PEG is 500 to 20,000.

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