# Patent

## Title
Kit for preparation of target radiopharmaceuticals and method of using it

## Abstract

The invention relates to a kit for preparation of target radiopharmaceuticals, a method of using the kit to prepare target radiopharmaceuticals and use of the target radiopharmaceuticals. The target radiopharmaceuticals comprise a radio-nuclear loading on liposome and inhibit the tumor growth and metastatic progression of head and neck cancer, lung cancer and brain cancer. The radiopharmaceuticals may be used for treating the mentioned cancers.

## Background
FIELD OF THE INVENTION
The invention relates to a kit for preparation of target radiopharmaceuticals, a method of using the kit to prepare target radiopharmaceuticals and use of the target radiopharmaceuticals. The target radiopharmaceuticals comprise a radio-nuclear loading on liposome and inhibit the tumor growth and metastatic progression of head and neck cancer, lung cancer and brain cancer.
BACKGROUND OF THE INVENTION
It is known that tumor cells grow fast if they are supplied with nutrients by angiogenesis. However, tumor cells growth during angiogenesis will result in vascular hypoplasia, which in turn results in a lot of leakages in micrometer order on blood vessels. Such a loose structure of blood vessels allows drug penetrate into tumor cells cavity easily. Moreover, since tumor tissue lacks of normal lymphatic network, if drugs enter into tumor cells, it is very difficult to leave through normal lymphoid tissue so that it could prolong residence time of drugs in tumor cells, which is so-called Enhanced Permeability and Retention (EPR) effect. Due to such a property, drugs in a size of from 100 nm ̃200 nm is considered be a potential for treating tumor.
Liposomes were first discovered by Alec Bangham, a British scientist, in 1960s. Liposomes are a spherical vesicle composed of a lamellar phase lipid bilayer, which structure is similar to the composition of cell membrane. Liposomes are biocompatible and biodegradable in human body. Liposomes comprise phospholipid, which phosphate end is hydrophilic and lipid end is hydrophobic so that liposomes can be used as a carrier both for hydrophobic and hydrophilic drug. Particle size of liposomes is from tens nanometers (nm) to tens micrometers (μm), they do not easily enter into the blood vessels in normal tissue, since the gaps between normal vascular endothelial cells are closed. However, since the gaps between tumor endothelial cells are enough large to yield the EPR effect, liposomes easily enter tumor endothelial cells and accumulate on tumor sites, thus improve the therapy efficiency of the drug carried on them. Thus, if therapeutic radionuclides are encapsulated in liposomes, it could improve tumor therapeutic effects of β-ray emitted by radionuclides. Methods for labeling radioisotopes onto liposomes are generally divided into two ways. One is a surface labeling method, which comprises directly labeling radionuclides on liposome on which surface bonded with chelators. Another is an embedding (after loading) method, which is the most common method. For example, Bao et al. published that N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (BMEDA) labeled with rhenium-188 (Re-188), rhenium-186 (Re-186) and technetium-99m were embedded in liposome and studied its effect on radiotherapy and imaging agents of radiodiagnosis on normal mice (Bao et al. J. Pharm. Sci (2003) 92, 1893-1904; J. Nucl. Med (2003), 44, 1992-1999; U.S. Pat. No. 7,718,160 B2). The common features in the after loading methods for preparing radiolabeled liposomes are: (i) Radioisotopes require the helps of chelators or ionophores to enter into liposomes. Therefore, a step of labeling of chelators with radioisotopes is necessary in this process. (ii) After entering into liposomes, radioisotopes need to react with other chelators or buffers solution to remain within liposomes stably. But there are disadvantages in using the after loading methods for preparing radiolabeled liposomes: (i) The used chelator, i.e. BMEDA is liquid state, it is easy oxidized so that the efficiency labeling with Re-186 or Tc-99m will be reduced. (ii) Additional purifying steps are required because of the low loading efficiency (generally the loading efficiency is about 60% to about 80%). (iii) The kit is un-stable for long time storage so that it should be prepared just before use. Furthermore, the whole labeling process is complex and time-consuming, it will reduce the radioactivity of radiopharmaceuticals to be prepared (for example, Re-188 radionuclides, which half-life is about 16.9 hours) so that the kinds of radioisotopes which can be used are limited.
There are many researches about therapy efficiency of Re-188 or Re-186-radiolabeled liposomes for treating colon cancer, head and neck cancer and breast cancer. In 2010, French et al. studied interventional therapy effect of head and neck cancer with 186Re-liposome (185 MBq (5 mCi)/cm3 tumor) (J Vasc Intery Radiol. 2010; 21(8): 1271-1279) and reported that average tumor volume of the 186Re-Liposome group on post-treatment day-14 was decreased to 87.7±20.1% (<0.001) and radiation absorbed dose on tumor was 526.3±93.3 Gy, which is far higher than 186Re-perrhenate and 186Re-BMEDA groups. And no systemic toxicity was observed. In 2012, Phillips et al. used 186Re-Liposome for treatment of glioblastoma at a dose of up to 1850 Gy by local injection and no overt clinical or microscopic evidence of toxicity was found (Neuro-Oncology 2012; 14(4):416-425). Animals treated with 186Re-Liposomes in a dose of 1850Gy had a median survival of 126 days (95% confidence interval [CI], 78.4-173 days), while control group is 49 days (95% CI, 44-53 days). And no systemic toxicity was observed. Although it showed that 186Re-Liposome is effective to treat the head and neck cancer and glioblastoma, the administration is limited to local injection, which will limit 186Re-Liposome to only use in the treatment of carcinoma in situ, and cannot be used in treatment of metastatic cancer.
Comparing with rhenium-186, rhenium-188 is a carry free and can be obtained in high radioactivity from tungsten-188 generator, thus it is more convenient for clinical use. Further, the half-life of rhenium-188 is 16.9 hr which is shorter than rhenium-186 (its half-life is 90 hr), thus rhenium-188 is safer and can use lower dose in clinical use.
SUMMARY OF THE INVENTION
In view of the above, the present invention relates to a system for preparation of target radiopharmaceuticals, a method of using the kit to prepare target radiopharmaceuticals and use of the target radiopharmaceuticals. By using 188Re as a radionuclide embedded in liposomes through BMEDA chelater to produce 188Re-Liposomes can avoid the reticuloendothelial system (RES) and pass through the vascular slit in lively angiogenesis for passive target drug accumulating in tissues by the mechanism of EPR (enhanced permeability and retention) effect.
The main object of the present invention is to provide a system for preparation of target pharmaceuticals, which comprises: (1) vial A which contains lyophilized mixture of N,N-bis(2-mercaptoethyl)-N′,N′-diethyl-ethylenediamine (BMEDA), sodium gluconate, and stannous chloride; (2) vial B which contains an aqueous solution of radionuclide including 188Re and/or 186Re; and (3) vial C which contains an aqueous liposome solution comprising phospholipid, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000) in a mole ratio of 30 ̃95:20 ̃45:3 ̃7.5.
In the present kit, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or hydrogenated soybean phosphatidylcholine.
In the present kit, the mole ratio of BMEDA, sodium gluconate, and stannous chloride in vial A is BMEDA:sodium gluconate:stannous chloride=10:2 ̃100:1 ̃40.
In the present kit, the specific activity of 188Re and/or 186Re is 1 mCi ̃100 mCi/mL.
In the present kit, the volume ratio of vial A and vial C is 1:1 ̃1:10.
In the present kit, the aqueous solution in vial B and C are both saline solution.
Another object of the present invention is to provide a method of using the present kit to prepare target radiopharmaceuticals, which comprises the steps of: (1) injecting the solution in the vial B into the vial A and reacting at an appropriate temperature for appropriate period; (2) adjusting the pH of the mixture obtained in step (1) in a range of 6 ̃7 by adding aqueous sodium hydroxide solution, and (3) injecting the liposome solution in the vial C into the vial A obtained in step (2) and reacting at an appropriate temperature for an appropriate period to obtain target pharmaceutical, i.e 188Re- and/or 186Re-Liposome.
In the present method, the step (1) is carried out in a temperature of from 4° C. ̃110° C. for 30 minutes to 75 minutes.
In the present method, the step (3) is carried out in a temperature of from 4° C. ̃80° C. for 15 minutes to 60 minutes.
Further object of the present invention is to provide a target pharmaceutical, which is prepared by the present method, which is liposome labeled with 188Re- and/or 186Re through chelator BMEDA (hereinafter, it is called as 188Re- and/or 186Re-Liposome).
Rhenium-188 (188Re) is a radionuclide having a short half-life of 16.9 hours and emitting 155 keV γ emission suitable for imaging and 2.12 MeV β emission suitable for treating tumor. Therefore, in clinical use, a patent can first be administered with a low dose, for example, 3 ̃14 mCi of “188Re- and/or 186Re-Liposome” to evaluate the size and position of tumor, then be administered with a high dose, for example more than 14 mCi for treatment, so that “188Re- and/or 186Re-Liposome” possess both functions of treatment and diagnostic of tumor.
The present target pharmaceutical, i.e. 188Re- and/or 186Re-Liposome is useful for treatment of head and neck cancer, lung cancer, brain cancer and their metastatic. Advantages of the present kit are follows: (1) easy use, (2) simple operation, (3) high purity without further purification; (4) suitable for clinical use; and (5) possess both functions of treatment and diagnostic of tumor.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows photographs of a mouse implanting with FaDu-GLT cells to establish head and neck cancer (FIG. 1(a)) and its bioluminescent image photograph (FIG. 1(b)).
FIG. 2 shows SPECT/CT imaging photographs of the mouse suffering head and neck cancer at 4, 24, and 48 hours after administration of present 188Re-Liposome.
FIG. 3 shows a graph of strength of bioluminescence in tumor administrating with 188Re-BMEDA and 188Re-Liposome, respectively.
FIG. 4 shows MRI imaging photographs of a patent suffering with head and neck metastases before administration of 188Re-Liposome (FIG. 4(a)) and SPECT/CT imaging photographs after administration of 188Re-Liposome (FIG. 4(b)).
FIG. 5 shows nasopharynx endoscopic photographs of a patent suffering with head and neck metastases before (FIG. 5(a)) and after (FIG. 5(b)) administration of 188Re-Liposome.
FIG. 6 shows tumor growth photographs for BALB/c nude mice implanting with H292-GLT cell to establish small cell lung cancer via microCT (FIG. 6(a)) and bioluminescent imaging (FIG. 6(b)).
FIG. 7 shows a Kaplan-Meier survival curve of 188Re-BMEDA treated group and 188Re-Liposome treated group.
FIG. 8 shows a Kapla-Meier survival curves of Fischer 344/F98luc orthotopic glioma bearing rat model after single intravenous injection of normal saline (●) and 333 MBq 188Re-Liposome (◯), respectively (n=7).



DETAILED DESCRIPTION OF THE INVENTION
The present invention will be illustrated by the followings, which are merely for example without limiting the scope of the present invention.
The following abbreviations are employed:
BMEDA: N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine
DSPC: Distearoyl phosphatidylcholine
PEG: Polyethylene glycol
EXAMPLE 1
The Preparation of Lyophilized Mixture of N,N-Bis(2-Mercaptoethyl)-N′,N′-Diethyl-Ethylenediamine (BMEDA), Sodium Gluconate, and Stannous Chloride
Vial A
148 mg sodium gluconate were dissolved in 1 mL 10% acetic acid solution to obtain an aqueous sodium gluconate solution and 20 mg stannous chloride were dissolved in 2 mL 0.1N HCl to obtain an aqueous stannous chloride solution. Then 35 mg BMEDA, 875 μL of the sodium gluconate solution and 840 μL of the stannous chloride solution were pipetted into a fresh vial, then flushing with nitrogen gas for 2 minute to avoid the oxygenation of stannous chloride to obtain a mixture containing BMEDA, sodium gluconate and stannous chloride. The resultant mixture solution was split charged into each vials in an amount of 152 μL/vial, which contains BMEDA/sodium gluconate/stannous chloride=3.08 mg/77.1 μL/74.1 μL (mole ratio of 1:4:0.3). Then these vials were subjected to a lyophilized process. The conditions of lyophilized process are described in Table 1.







TABLE 1







Condition of the lyophilized process











lyophilized process
Step
Time
Temperature
Pressure





Pre-frozen
1
 1 hr
−80° C.
<0.120 mBar


Primary lyophilized
2
36 hr
−80° C.
<0.120 mBar


Second lyophilized
3
12 hr
 18° C.
<0.120 mBar









Upon finishing the lyophilized process, each vials was flushed with N2 gas, sealed and stored in −20° C., which is corresponding to vial A.
EXAMPLE 2
Preparation of Vial C
DSPC (55.31 g, 70 μmole), cholesterol (18.14 g, 46.66 μmole), and DSPE-PEG2000 (20.59 g, 7 μmole) (mole ratio of 30:20:3) were weighted into a 200 mL round bottom and added with 8 mL choloform to dissolve them thoroughly. The resultant mixture was evaporated by using rotary evaporators at 60° C. to remove solvent and thus a lipid film was formed on the wall of the flask. Then 5 mL of 250 mM ammonium sulfate solution (250 mM (NH4)2SO4, pH 5.0, 530 mOs) were added into the flask and the flask was shaken in a water bath at 60° C. until the lipid film was dispersed in ammonium sulfate solution thoroughly to obtain multi-layer liposome (MLV) dispersion. The multi-layer liposome dispersion was frozen in liquid nitrogen and de-frozen in water bath at 60° C. with repeating six times. Then the multi-layer liposome dispersion was extruded by high pressure film extruder (Lipex Biomembrane, Vancouver, Canada) to obtain single two-layer liposome. The resultant two-layer liposome dispersion was loaded on Sephadex G50 gel permeation column and purified by using 0.9% NaCl solution as a eluent. The eluted fraction was collected and determined its particle size by using nano-ZX (Malvern, UK.) to be a normal distribution in a range of from 80 ̃120 nm. Using Bartlett's Method to determine the phospholipid concentration in the liposome is found to be 13 ̃14 μmole/mL.
EXAMPLE 3
Preparation of 188Re-Liposome
A 188ReO4− solution (vial B) added into vial A in a radioactivity of 4 mCi/mL, and the resultant mixtures reacted at 80° C. for 60 minutes. 55 uL 2N NaOH were added to the vial A to adjust the pH of the mixture to 6. The labeling reaction is as follows.

188ReO4−+Sn4+→Reduced 188Re+Sn4+

188Re-gluconate

Reduced 188Re+gluconate→

188Re-gluconate+2BMEDA→188Re(BMEDA)2+gluconate

Then an aqueous liposome solution in vial C into the above vial A (having added with the solution in vial B) in a volume ratio of vial A to vial C of 1:1). The resultant mixtures reacted in 60° C. for 30 minutes. Completing the reaction, the vial was cold down at room temperature to obtain a target radiopharmaceutical, i.e. 188Re-Liposome. The encapsulating efficiency of the 188Re-Liposome was determined by PD-10 column (GE Healthcare) with using normal saline as the eluent. The PD-10 column was first conditioned with 20 mL normal saline, then loaded with 100 μL 188Re-Liposome and eluted with saline. The eluted solution was collected in each tube in an amount of 0.5 ml/tube for total 20 tubes. The encapsulating efficiency of 188Re-Liposome was calculated according to the following standard formula:

The encapsulating efficiency (%)=[100%×(Rhenium-188 main peak activity/(Total radioactive)].

Wherein

total radioactive: the sum of total fraction radioactivity and column residue.

Rhenium-188 main peak activity: the sum of Radioactivity of fractions with 188Re-Liposome (fraction5-10).

A purification is not required if the encapsulating efficiency exceeds 90%.







TABLE 2







the labeling yield and encapsulating


efficiency in different radioactive.













Radioactive of 188Re
Labeling
Encapsulating



No.
(mCi/mL)
yield (%)
efficiency (%)
















1
4.01
100
93.32



2
5.02
100
93.54



3
6.59
100
90.83



4
9.03
100
92.51










EXAMPLE 4
Therapeutic Efficacy Studies of 188Re-Liposome Treatment in Human HNSCC Mice Model
The FaDu-GLT cells were implanted into BALB/c nude mice at a concentration of 106 cells/50 μl to to establish head and neck cancer. 16 days after implantation, the mice were obverted by naked eye and by bioluminescent imaging. FIG. 1 shows photographs of a mouse implanting with FaDu-GLT cells to establish head and neck cancer (FIG. 1(a)) and its bioluminescent image photograph (FIG. 1(b)).
The 188Re-BMEDA and 188Re-Liposome were separately injected intravenously into tumor-bearing mice with 640 μCi in a single dose, each group has 9 BALB/c nude mice. The mice of 188Re-Liposome group were imaged at 4, 24, and 48 hours after administration which SPECT/CT imaging photographs were shown in FIG. 2. The reconstructed 3D nanoSPECT/CT image showed a clear signal in the lower oral cavity. Moreover, the bioluminescent image signal revealed that the tumor growth was repressed within 15 days after 188Re-Liposome administration (FIG. 3).
EXAMPLE 5
Clinical Study of 188Re-Liposome in Patients with Metastatic Cancer
Each patient with metastatic cancer receives single dose 3 mCi 188Re-Liposome injection. After 188Re-Liposome administration, single-photon emission computed tomography (SPECT) imaging were carried out on 1, 4, 8, 24, 48 and 72 hours, and computed tomography (CT) were carried out on 24 hours. Image registration was conducted by Velocity AI imaging software for the SPECT images each time point with the CT image of 24 hour to evaluate biodistribution and radiation dosimetry on normal organs and tumor.
One of the patients suffering head and neck metastases received 188Re-Liposome and subjected to SPECT and CT imaging. The MRI image was acquired for tumor diagnosis about one month before this study, and a nasopharyngeal tumor in left nasal cavity was observed (FIG. 4(a)). The SPECT/CT fusion image on 24 hour revealed that 188Re-liposome distributes at the same location with MRI image (FIG. 4(b)). In addition, from nasopharynx endoscopic examination results, it shown that the lesion has engorged blood vessels and irregular surface over left nasopharynx one month before 188Re-Liposome administration (FIG. 5(a)), but it has been in regression with much crust and mucoid over nasopharynx two month after 188Re-Liposome injection (FIG. 5(b)).
EXAMPLE 6
Therapeutic Efficacy Studies of 188Re-Liposome Treatment in Human NSCLC Mice Model
A centimeter incision was made on the left chest wall of nude mice to precisely locate the insertion of needle, and 106 H292-GLT cells were injected through 29-gauge syringe in a mixture with Matrigel. 16 days after the intrapulmonary implantation of human NSCLC, the solid tumor was also visible in the coronal view of microCT image (FIG. 6(a)). Moreover, the bioluminescent imaging (BLI) which is a crucial tool for quantifying the growth of tumors was assessed by the In Vivo Imaging System (IVIS) with the administration of D-luciferin (FIG. 6(b)).
The 188Re-BMEDA and 188Re-Liposome were separately injected intravenously into tumor-bearing mice with 640 μCi in a single dose, each group has 9 BALB/c nude mice.
The survival was recorded since the first mice died from the treatment or the disease up to 60 days. Median survival time (Table 3) exhibited a significant difference between these two groups, the result of 188Re-BMEDA group is 26 days, and the 188Re-Liposome group is 40 days.
The Kaplan-Meier survival curves (FIG. 7) showed mice given 188Re-Liposome was observed after 60 days and still 10% survival, whereas 188Re-BMEDA mice are all died on 40 days.







TABLE 3







median survival time analysis of 188Re-


BMEDA group and 188Re-Liposome group










188Re-BMEDA
188Re-Liposome















Sample size(n)
9
9



Median survival (days)
26
40











significance
P = 0.0045




95% CI
0.1131 to 0.9615










EXAMPLE 7
Therapeutic Efficacy Studies of 188Re-Liposome Treatment in Human NSCLC Mice Model
The rats (male, 12-13 week old) were anesthetized with Isofluorane® and then administered with atropine sulfate (0.1 mg/kg) via subcutaneous injection; subsequently, the rats were deeply anesthetized by intraperitoneal injection of Zoletil® 50 and Balanzine 2% mixture at a 5:2 volume ratio (0.1 mL/100 g rat body weight). After anesthesia, the rat head's hair on operative field was removed. Then, the rats were immobilized by a stereotactic frame (Stoelting®, USA). A 2 cm linear incision was carefully operated and the field-open immobilized for the following surgery. After removing the periosteum, a 1 mm diameter of hole was created with a high-speed drill at the right brain (located at 3 mm lateral to midline and 5 mm anterior to lambda) and the dura carefully pricked with a sharp tweezers. For implantation, the F98luc cells were harvested and re-suspended in Hanks' balanced salt solution (HBSS) plating on the ice before use. The 1×105 cells in 10 μL medium were inoculated into the brain (a depth of 5 mm from the skull bone) using a 100 μL Hamilton® syringe and 271⁄2 gauge needle through nanoliter syringe pump (KDS 310 plus; Holliston, Mass., USA) with the injection rate at 3 μL/min. After seeding, the needle was retained for 2 min and then drawn out carefully and slowly. Finally, paraffin was used to fill the surgical hole and the incision was sutured. The rats were observed closely until completely awakening.
To evaluate therapeutic efficacy of 188Re-Liposome in Fischer344/F98luc orthotopic glioma bearing rat model, 35 rats in total were used herein. For survival evaluation study, fourteen tumor bearing rats were randomly divided into two groups (seven rats per groups). Subsequently, the rats of two groups were administered with normal saline (control group) and 188Re-Liposome (333 MBq/0.5 mL; 2.5 μmol phospholipid/0.5 mL) via single intravenous injection on Day 7 post-inoculation, respectively. Then the rats were monitored for survival and body weight every day until death. The survival curves for treating with 188Re-Liposome and normal saline in Fischer344/F98luc glioma-bearing rat model. The result revealed that the lifespan for 188Re-Liposome was significantly increased as 10.67% compared to control group (P=0.007). The statistical analysis for therapeutic efficacy evaluation is summarized in Table 4. The maximum survival time for 188Re-Liposome and control groups was 24 and 19 days, respectively. In addition, the median survival time was 20.75 and 18.75 days, respectively.







TABLE 4







Therapeutic efficacy evaluation of 333-MBq 188Re-Liposome


in orthotopic glioma-bearing rat model.
















Maximum
Median





No. of
Dose
survival time
survival time
Prolongation in


njection
rat/group
(MBq)
(d)
(d)
lifespan (%)
P-value
















188Re-Liposome
7
333
24
20.75
10.67
0.007


Normal saline
7
None
19
18.75
—
—









While the invention has been described in detail, modifications within the spirit and scope of the invention will be readily apparent to those of skill in the art. In addition, it should be understood that aspects of the invention and portions of various embodiments and various features recited below and/or in the appended claims may be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.

## Claims

1. A method for preparing target radiopharmaceuticals, which comprises the steps of:
(1) mixing a vial B and a vial A and reacting the same at an appropriate temperature for an appropriate period, wherein the vial A contains lyophilized mixture of N,N-bis(2-mercaptoethyl)-N′,N′-diethyl-ethylenediamine(BMEDA), sodium gluconate, and stannous chloride, and the vial B contains an aqueous solution of radionuclide including 188Re and/or 186Re lyophilized, and wherein the lyophilized mixture is prepared by the steps of:
a) mixing BMEDA, sodium gluconate, and stannous chloride to produce a mixture;
b) freezing the mixture at −80° C. and flushing the same with N2 gas under 0.120 mBar for 1 hour;
c) lyophilizing the mixture at −80° C. and flushing the same with N2 gas under 0.120 mBar for 36 hours; and
c) lyophilizing the mixture at 18° C. and flushing the same with N2 gas under the pressure of 0.120 mBar for 12 hours;

(2) adjusting the pH of the mixture obtained in step (1) in a range of 6 ̃7 by adding aqueous sodium hydroxide solution, and
(3) injecting the liposome solution in a vial C into the mixture obtained in step (2) and reacting at an appropriate temperature for an appropriate period to obtain a target pharmaceutical, i.e., 188Re-and/or 186Re-Liposome, wherein the vial C contains an aqueous liposome solution comprising phospholipid, cholesterol, and 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG 2000) in a mole ratio of 30 ̃95:20 ̃45:3 ̃7.5.

2. The method according to claim 1, wherein the step (1) is carried out in a temperature of from 4° C. ̃110° C. for 30 minutes to 75 minutes.

3. The method according to claim 1, the step (3) is carried out in a temperature of from 4° C. ̃80° C. for 15 minutes to 60 minutes.