Patent Application
20190002596
The present invention is in the field of anti-tumor agents, and relates to a sulfated heparin oligosaccharide and a preparation method and use thereof.
Tumors are a major threat to human health, more seriously, malignant tumors, such as liver cancer and lung cancer etc. are prone to metastasis. Currently, there are no drugs for inhibiting tumor metastasis, making treatment for tumor metastasis difficult, and thus making tumor metastasis the most significant cause of death in cancer patients. Two processes are very critical for achieving invasion and migration of tumor cells: one is to break through a barrier formed by the extracellular matrix (ECM) and the basement membrane (BM), and the other is to form new blood vessels. ECM and BM are barriers against invasion and metastasis of tumor cells, and malignant tumor cells must enter into the circulation across the ECM and BM, so as to spread and metastasize. In this process, the degradation of various components of the ECM and heparin sulfate proteoglycans (HSPGs) is essential. Heparanase (HPA) is the only endo-β-D-glucuronidase currently found in mammals that can cleave heparan sulfate (HS). HPA identifies the specific structure of HS, and it does not completely cleaves the HS side chain, but only cleaves the glycosidic bonds at certain sites in the HS side chain, which is degraded as short saccharide chains of 10 to 15 saccharide units. In addition, HS side chains can bind a variety of biologically active molecules, such as growth factors, cytokines, chemokines, morphogens and blood coagulating proteins. HPA degrades HS to release active growth factors which can promote tumor angiogenesis, and tumor growth, invasion and metastasis. Therefore, HPA plays an important role in the invasion and metastasis of tumor cells, and the research and screening of HPA inhibitors have become a new direction for humans to seek potential drugs for the treatment of cancers.
For a long time, clinical evidence shows that heparin has an anti-tumor effect, and in recent years, clinical trials for low molecular weight heparin-assisted anti-tumor therapy are undergoing. After long-term research, the anti-tumor effect of heparin has been widely recognized and appreciated in the industry, and it might be realized mainly by inhibiting tumor metastasis, and further be realized by inhibiting the heparanase activity in vivo.
Heparin, as a traditional anticoagulant, is mainly used for its anticoagulant activity. However, it has a broad range of biological activity due to the structural diversity thereof. In non-anticoagulant applications of heparin, its anticoagulant activity is a common and important side effect. As such, the anticoagulant activity of heparin is a major disadvantageous factor in the application of anti-tumor metastasis, and it is prone to cause side effects such as bleeding. An important aspect in the research of the application of the non-anticoagulant activity of heparin is to destroy the anticoagulant activity while maintaining the basic structure of heparin. With respect to anti-tumor metastasis, heparin has high inhibitory activity on heparanase, but has no significant effect on the inhibition of tumor growth and on the inhibitory activity on tumor cell invasion and adhesion at the cellular level, and achieves poor results in an anti-tumor metastasis experiment in mice and thus cannot significantly inhibit the occurrence of tumor metastasis. The main reason therefor might be that the specificity of heparin is not high, and thus heparin can interact with a variety of endogenous substances, reducing its ability to bind to heparanase.
CN101824100A discloses a heparin oligosaccharide dodecamer having the following structural formula:
which has the use of preventing proliferation of vascular smooth muscle cells. However, the oligosaccharide of that invention has no significant ability of inhibiting tumor cell adhesion and migration.
CN 104764847A discloses a method of preparing a heparin oligosaccharide comprising a N-acetylated structure, and the following 4 hexasaccharide fragments and 3 octasaccharide fragments are disclosed in this reference:
That invention merely address the difficulties in the preparation and structural verification of a heparin oligosaccharide comprising a N-acetylated structure, and achieves no significant improvement in the heparanase inhibitory activity in vivo.
As such, in the art, it is desired to obtain a heparin oligosaccharide having heparanase inhibitory activity in vivo and reduced anticoagulant activity.
To address the disadvantages of the prior art, the object of the present invention is to provide a sulfated heparin oligosaccharide and a preparation method and use thereof.
To accomplish the object of the present invention, the following technical solution is employed in the present invention.
In the first aspect, the present invention provides a sulfated heparin oligosaccharide, containing an unsaturated double bond resulting from enzymolysis by heparinase at the non-reducing end thereof, comprising an uronic acid derivative and a glycosylamine derivative, and having a structure represented by Formula I:
wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, Ra, Rb, Rc and Rd are independently SO3− or H; Rx′, Ry′ and Rz′ are independently COCH3 or SO3−, and n is 1 to 3.
In the present invention, n is 1 to 3, i.e., 1, 2, or 3. When n=1, the sulfated heparin oligosaccharide is sulfated heparin hexasaccharide; when n=2, the sulfated heparin oligosaccharide is sulfated heparin octasaccharide; and when n=3, the sulfated heparin oligosaccharide is sulfated heparin decasaccharide.
There is a double bond in the structure of the sulfated heparin oligosaccharide of the present invention, which thus has a strong absorption peak in the ultraviolet region at about 232 nm, which can be very conveniently taken advantage of in the qualitative and quantitative determination of the heparin oligosaccharide.
The sulfated heparin oligosaccharide of the present invention has good inhibitory activity on heparanase and tumor metastasis. It has a short saccharide chain and low molecular weight, and has no anticoagulant activity at all. Meanwhile, the sulfated heparin oligosaccharide has high specificity, and thus can specifically inhibit heparanase and inhibit tumor metastasis.
In the sulfated heparin oligosaccharide of the present invention, in Formula I, the number of sulfonic groups in each disaccharide unit is no less than 2 on average, for example, the number of sulfonic groups in each disaccharide unit can be 2, 3, 4, or 5 on average; the number of acetyl groups in each disaccharide unit is no more than 0.5 on average, for example, it can be 0.5 or 0.4; and the numbers of sulfonic groups at position 6 and position 3 in glucosamine in each disaccharide unit are both no less than 0.5 on average, for example, it can be 0.5, 0.8, 1, 1.5, or 2.
The number of groups (for example, sulfonic groups, acetyl groups, or sulfonic groups at position 6 and position 3 in glucosamine) in each disaccharide unit refers to the number that is obtained by averaging the number of sulfonic groups in the whole heparin oligosaccharide to each disaccharide unit, for example, if there are a total of 2 acetyl groups in a sulfated heparin octasaccharide, the number of acetyl groups in each disaccharide unit is 0.5 on average.
Preferably, the uronic acid in Formula I is glucuronic acid or iduronic acid.
Preferably, the cation forming a salt with the carboxyl and/or sulfonic group contained in Formula I is selected from the group consisting of Na+, K+ or Ca2+.
In the above structural formula, the carboxyl or sulfonic group in glucuronic acid is negatively charged, and thus generally forms a salt with certain cations, typically Na+, K+ and Ca2+.
Preferably, the sulfated heparin oligosaccharide is any one of, or a combination of at least two of, the compounds having the following structures:
DP6, a heparin hexasaccharide derivative:
ΔU3S-ANS3S-I-ANS3S-G3S-ANS3S6S
ΔU3S-ANS-I3S-ANS3S-G3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S6S-G3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S6S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-I2S3S-ANS3S6S-G2S3S-ANS3S6S
DP8, a heparin octasaccharide derivative:
ΔU3S-ANS3S-I-ANS3S-I3S-ANS6S-G3S-ANS3S6S
ΔU3S-ANS-I3S-ANS3S-I3S-ANS6S-G3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S-I3S-ANS6S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S6S-I3S-ANS6S-G3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S6S-I3S-ANS6S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-T2S3S-ANS3S6S-I2S3S-ANS6S-G2S3S-ANS3S6S
DP10, a heparin decasaccharide derivative:
ΔU3S-ANS3S-I-ANS3S-I-ANS6S-I-ANS6S-G3S-ANS3S6S
ΔU3S-ANS-I3S-ANS3S-I3S-ANS6S-I3S-ANS6S-G3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S-I3S-ANS6S-I3S-ANS6S-G3S-ANS3S6S
ΔU2S3S-ANS3S6S-I3S-ANS3S6S-I3S-ANS6S-I3S-ANS6S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-I2S3S-ANS3S6S-I2S3S-ANS6S-I2S3S-ANS6S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-I2S3S-ANS3S6S-I2S3S-ANS3S6S-I2S3S-ANS3S6S-G2S3S-ANS3S6S
ΔU2S3S-ANS3S6S-I2S3S-ANS3S6S-I2S3S-ANS6S-G2S3S-ANS3S6S
In the above structures, ΔU represents
I represents α-L-iduronic acid, G represents β-D-glucuronic acid, and A represents α-D-glucosamine. NS represents a sulfonic group on the amino group, and 2S, 3S, 6S, etc. represent sulfonic groups at the 2-O, 3-O, and 6-O positions on the saccharide ring.
In the second aspect, the present invention provides a method of preparing the sulfated heparin oligosaccharide as described in the first aspect, comprising the following steps:
(1) degrading heparin with heparinase, separating and purifying to obtain a heparin oligosaccharide; and
(2) sulfating the heparin oligosaccharide obtained in step (1) with a sulfating reagent to obtain the sulfated heparin oligosaccharide.
In the method of preparing the sulfated heparin oligosaccharide of the present invention, the heparinase in step (1) is heparinase I.
In the method of preparing the sulfated heparin oligosaccharide of the present invention, a buffer, preferably a Tris-HCl buffer, pH 7.0, needs to be added when heparin is degraded with heparinase in step (1).
Preferably, the amount of heparinase added in step (1) is 15 to 25 IU/g heparin, e.g., 16 IU/g heparin, 16.5 IU/g heparin, 17 IU/g heparin, 17.5 IU/g heparin, 18 IU/g heparin, 18.5 IU/g heparin, 19 IU/g heparin, 19.5 IU/g heparin, 20 IU/g heparin, 21.5 IU/g heparin, 22 IU/g heparin, 23 IU/g heparin or 24 IU/g heparin, and preferably 18 to 23 IU/g heparin.
Preferably, the temperature for the degradation of heparin with heparinase in step (1) is 4° C. to 37° C., e.g., 5° C., 8° C., 10° C., 12° C., 15° C., 18° C., 20° C., 22° C., 25° C., 28° C., 30° C., 32° C., 35° C. or 37° C., and preferably 8° C. to 25° C.
Preferably, the time for the degradation in step (1) is 8 to 24 hours, e.g., 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours, and preferably 10 to 20 hours.
Preferably, the degradation of heparin with heparinase in step (1) comprises inactivation at 95° C. for 5 to 10 min, e.g., 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min or 9.8 min, preferably 5 to 8 min, and more preferably 6 min, after the degradation.
Preferably, the separation in step (1) comprises ultrafiltration, preferably ultrafiltration with a 10 KDa ultrafiltration centrifuge tube.
Preferably, the purification in step (1) is separation and purification through column chromatography.
After separation through column chromatography, heparin oligosaccharide is obtained through processes such as concentration, desalting and lyophilization.
In the method of preparing the sulfated heparin oligosaccharide of the present invention, before the sulfation in step (2), the heparin oligosaccharide obtained in step (1) is subjected to swelling treatment.
Preferably, the solvent used for the swelling treatment is DMF.
Preferably, the sulfating reagent in step (2) is (CH3)3N.SO3.
Preferably, relative to 1 g of the heparin oligosaccharide, the amount of the sulfating reagent is 1 to 10 g, e.g., 1.2 g, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 9.8 g.
Preferably, the temperature for the sulfation in step (2) is 60° C. to 120° C., e.g., 63° C., 65° C., 70° C., 73° C., 75° C., 78° C., 80° C., 83° C., 85° C., 88° C., 90° C., 93° C., 95° C., 98° C., 100° C., 115° C., 118° C. or 120° C.
Preferably, the time for the sulfation in step (2) is 1 to 12 hours, e.g., 1.5 hours, 2 hours, 2.3 hours, 2.5 hours, 2.8 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 10.5 hours, 11 hours or 11.5 hours.
In the present invention, the sulfation degree of the heparin oligosaccharide can be controlled by controlling the ratio of the heparin oligosaccharide to the sulfating reagent, the reaction temperature, and the reaction time, so as to obtain heparin oligosaccharides with various degrees of sulfate group substitution.
In the present invention, after the sulfation of the heparin oligosaccharide obtained in step (1) with the sulfating reagent, the reaction mixture needs to be worked up and purified, that is, the reaction mixture is added with 10-fold volume of purified water to dissolve the precipitate, transferred to a dialysis bag with a molecular weight cut-off of 100 to 500 Da, desalted with a P10 column after dialysis for three days, concentrated before cationic impurities are removed with a cation exchange column, neutralized with high purity NaOH, KOH or Ca(OH)2, etc., and concentrated before lyophilization or precipitation with ethanol, so as to obtain the corresponding sulfated heparin oligosaccharide.
As a preferred technical solution of the present invention, the method of preparing the sulfated heparin oligosaccharide of the present invention comprises the following steps:
(1) degrading heparin with heparinase I added at an amount of 15 to 25 IU/g heparin at 4° C. to 37° C. for 8 h to 24 h, in a buffer being a Tris-HCl buffer, pH 7.0, and after the degradation, inactivating at 95° C. for 5 to 10 min, ultrafiltering with a 10 KDa ultrafiltration centrifuge tube, and then separating and purifying through column chromatography to obtain the heparin oligosaccharide; and
(2) sulfating the heparin oligosaccharide obtained in step (1) at 60° C. to 120° C. for 1 to 12 hours with a sulfating reagent to obtain the sulfated heparin oligosaccharide.
The preparation method of the present invention can be employed to obtain sulfated oligosaccharides with various sulfation degrees. Each saccharide chain of the sulfated heparin oligosaccharide contains a double bond resulting from an elimination reaction of enzymolysis by heparinase at the non-reducing end thereof. Due to the presence of a double bond in the structure, the oligosaccharide has a strong absorption peak in the ultraviolet region at about 232 nm, which can be very conveniently taken advantage of in the qualitative and quantitative determination of the heparin oligosaccharide. The double bond does not substantially change during the sulfation of the heparin oligosaccharide. Heparin per se has no strong characteristic absorption peaks, and thus heparin substances can be detected only with DMB staining etc., which, however, has low sensitivity and cannot be accommodated to complex systems. As such, the presence of the double bond greatly facilitates the detection of the heparin oligosaccharide and derivatives thereof, and can also play an important role in the detection of biological metabolic processes.
In a third aspect, the present invention provides use of the sulfated heparin oligosaccharide as described in the first aspect in the manufacture of a medicament for anti-tumor metastasis.
The sulfated heparin oligosaccharide of the present invention has good inhibitory activity on heparanase and tumor metastasis, and can be used as an anti-tumor drug or as an anti-tumor active ingredient for the manufacture of an anti-tumor medicament for the treatment of a tumor, and for the prevention and inhibition of tumor metastasis.
In the sulfated heparin oligosaccharide of the present invention, heparin hexasaccharide, heparin octasaccharide and heparin decasaccharide with various sulfonation degrees all have significant inhibitory activity. At a same sulfonation degree, different chain lengths will lead to different inhibitory activity on heparanase, the inhibitory activity levels are specifically: sulfonated heparin hexasaccharide<sulfonated heparin decasaccharide<sulfonated heparin octasaccharide. At a same chain length, different sulfation degrees of the heparin oligosaccharides lead to different inhibitory activity on heparanase, specifically: low sulfated (10 to 40%) heparin oligosaccharide<highly sulfated (60 to 90%) heparin oligosaccharide<moderately sulfated (40 to 60%) heparin oligosaccharide. In the structure of the sulfated heparin oligosaccharide, sulfonation at position 6 in glycosylamine and sulfonation at position 3 in glycosylamine contribute more to the heparanase inhibitory activity.
Compared with the prior art, the present invention achieves the following advantageous effects.
The sulfated heparin oligosaccharide of the present invention has good inhibitory activity on heparanase and tumor metastasis, comprises short saccharide chains and a low molecular weight, and has no anticoagulant activity at all with no anticoagulant activity and bleeding risks. The preparation method of the present invention can prepare a sulfated oligosaccharide with a controllable sulfation degree, which has high inhibitory activity on heparanase in vitro, inhibitory activity on cell adhesion and migration that is 4 to 5 times higher than that of heparin, and in vivo activity of anti-tumor metastasis in mice that is 2 to 3 times higher than that of the heparin, thereby having a good anti-tumor metastasis effect and high specificity.
The technical solutions of the present invention will now be further illustrated by the following specific embodiments. As can be appreciated by a person skilled in the art, the Examples are given merely to aid understanding of the invention, and should not be considered limitative on the invention.
24 g of heparin was taken and added with 240 mL of a Tris-HCl buffer solution, which was stirred to dissolve, added with 480 IU of heparinase I, stirred until homogeneous, and underwent an enzymatic reaction at 10° C. for 16 h. After the reaction was complete, the reaction mixture was heated to 95° C. for inactivation for 6 min, and ultrafiltered with a 10 KDa ultrafiltration centrifuge tube. The filtrate was separated with a Bio-Gel P-10 (2.5×100 cm) chromatographic column with 0.2 M NH4HCO3 as the eluent, 1.75 to 2.15-fold column volume of fractions was collected to obtain a heparin tetrasaccharide mixture, 1.35 to 1.75-fold column volume of fractions was collected to obtain a heparin hexasaccharide mixture, 1.05 to 1.35-fold column volume of fractions was collected to obtain a heparin octasaccharide mixture, 0.85 to 1.05-fold column volume of fractions was collected to obtain a heparin decasaccharide mixture, and 0.75 to 0.85-fold column volume of fractions was collected to obtain a heparin dodecasaccharide mixture. After removal of NH4HCO3 through rotary evaporation, the corresponding heparin oligosaccharides were obtained by lyophilization.
As shown in
The structures of the heparin oligosaccharides were characterized and verified by means of UPLC-MS etc. For example,
0.54 g of the starting material of heparin octasaccharide was weighed and transferred to a reaction flask, added with 25 mL of anhydrous DMF, and stirred to dissolve. With stirring, 0.86 g of (CH3)3N.SO3 was weighed and gradually added to the above solution, and stirred for 10 min. The reaction flask was capped appropriately, placed in an oil bath at 80° C.n and stirred for 4 h. The reaction was stopped before left to cool to room temperature. The solid was dissolved in 90 mL of purified water, and the pH was adjusted to near neutral with 2 M NaOH. The solution was transfer to a dialysis bag with a molecular weight cut-off of 100 to 500 Da, and after three days of dialysis, the dialysate was detected with BaCl2 to determine whether a large amount of sulfate radicals are present therein. If not, the pH was first adjusted to neutral with 2 M NaOH, and concentrated to about 10 mL on a rotary evaporator. The mixture was loaded on a P2 column, eluted with purified water, and the eluent was collected with a fraction collector. The absorbance at 232 nm was detected to determine the location of the heparin octasaccharide derivative, the collected solution was detected with BaCl2 to determine whether a large amount of sulfate radicals are present therein, and the salt-free fraction of the heparin octasaccharide sulfated derivative was collected. The material was concentrated to about 10 mL, loaded on a Dowex cation exchange column, eluted with purified water, the eluent was collected with a fraction collector, and the absorbance at 232 nm was detected to determine the location of the heparin octasaccharide derivative. The fraction comprising the heparin octasaccharide derivative was collected, carefully adjusted to pH 7.0 with 0.1 M high-purity NaOH solution, and concentrated and lyophilized to obtain 20% sulfated heparin octasaccharide.
For 0.54 g of the starting material of heparin octasaccharide, heparin oligosaccharides with the indicated sulfation degrees can be obtained in a controllable manner by adjusting the feed amount of (CH3)3N.SO3 in the starting materials of the reaction and adjusting the reaction temperature and the reaction time as shown in Table 1.
For example, heparin oligosaccharides with corresponding sulfation degrees can be obtained through the adjustment of the reaction conditions and the feed amount as shown below.
The amount of (CH3)3N.SO3 was adjusted to 1.67 g, the reaction temperature was adjusted to 90° C., the reaction time was adjusted to 4 h, and the remaining procedures were the same, so as to obtain 40% sulfated heparin octasaccharide.
The amount of (CH3)3N.SO3 was adjusted to 3.34 g, the reaction temperature was adjusted to 90° C., the reaction time was adjusted to 6 h, and the remaining procedures were the same, so as to obtain 60% sulfated heparin octasaccharide.
The amount of (CH3)3N.SO3 was adjusted to 5.01 g, the reaction temperature was adjusted to 100° C., the reaction time was adjusted to 8 h, and the remaining procedures were the same, so as to obtain 80% sulfated heparin octasaccharide.
The heparanase inhibitory activity of sulfated heparin oligosaccharides was determined with the method as described in the reference [Hammond E, Li C P, Ferro V. Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening. Anal. Biochem. 2011; 396:112-116]. Specifically, the experimental procedure were as follows.
The reaction solution of the test group comprised 40 mM sodium acetate buffer (pH 5.0), 100 mM fondaparinux, and a specific concentration of the sulfated heparin octasaccharide, and in the reaction solution of the control group, the sulfated heparin octasaccharide was replaced with SST0001 control at a same concentration as that of the sulfated heparin octasaccharide. 100 μL of the reaction solution of the test group or the control group was added to each well in a 96-well plate, and heparanase was respectively added to a final concentration of 140 pM, to start the reaction. The 96-well plate was sealed with a tape, and incubated at 37° C. for 2 to 24 h. After the reaction was complete, 100 μL of a solution containing 1.69 mM WST-1 in 0.1 M NaOH was added to stop the reaction. The 96-well plate was resealed and incubated at 60° C. for 60 min. After cooling to room temperature, the absorbance value at 584 nm was measured. The inhibitory rate of the agent was calculated according to the following method:
Inhibitory rate=(1−the absorbance value of a sample/the absorbance value of a control)×100%
The heparanase inhibitory activity of the sulfated heparin oligosaccharides measured according to the above method is shown in
In this example, the following method was employed to determine the cell adhesion of 20% and 60% sulfated heparin octasaccharides.
(1) coating the basement membrane: 2 solutions were prepared with sterile double distilled water respectively: 10 g/L of BSA (1%), and 50 mg/L of Matrigel, 1:8 dilution; Matrigel was added to a 96-well culture plate at 50 μL/well respectively, and incubated at 4° C. overnight;
(2) hydrating the basement membrane: the residual liquid was aspirated from the culture plate, 50 μL of serum-free medium containing 10 g/L of BSA was add to each well, and allowed to stand at 37° C. for 30 min.
(3) preparing cells: normally cultured HeLa cells were taken and washed with PBS for three times, and the cells were divided into a control group (no drug but only a corresponding volume of PBS was added) and test groups (heparin and sulfated heparin octasaccharide were added, both at a concentration of 62.5 μg/mL), and cultured for 24 h.
(4) inoculating cells: tumor cells normally cultured or treated with heparin/HS oligosaccharide derivatives in step (3) for 24 h were taken, and washed with PBS for three times before digested with 0.5 mL of pancreatin. The digestion was closely monitored, and after the digestion, 5 mL of the culture medium was added, and the cells were dispersed by thoroughly pipetting to form a single cell suspension. The cells were counted, and the concentration of the cell suspension was adjusted according to the counting result to achieve a cell density of 105 cells/mL. The cell suspension was inoculated at 100 μL per well to a 96-well culture plate coated with Matrigel, and samples were processed in triplicate;
(5) culturing cells: the cells were cultured in a carbon dioxide incubator at 37° C. for 1 h, and washed with PBS once after the culture liquid in each well was discarded. 200 μL per well of fresh culture medium was then added, and the plated was observed and photographed.
(6) detecting: 10 μL of a CCK-8 dye solution was added to each well, and the plate was incubated in a carbon dioxide incubator at 37° C. for 3 hours, and read with a multifunctional microplate reader. The inhibitory rate was calculated according to the following formula, with the heparin control group as a reference.
Inhibitory rate=(1−the absorbance value of the treatment group/the absorbance value of the control group)×100%
According to the above method, the inhibitory rate of heparin on HeLa cell adhesion was 8.3%, the inhibitory rate of 20% sulfated heparin octasaccharide (Hep8-20%) on HeLa cell adhesion was 45.3%, and the inhibitory rate of 60% sulfated heparin octasaccharide (Hep8-60%) on HeLa cell adhesion was 37.9%.
In this example, cell migration with 40% and 60% sulfated heparin octasaccharides was determined by the following method.
(1) before the test, a Transwell chamber was placed in a 24-well plate, 600 μL of a DMEM liquid medium was added to the lower chamber, 100 μL of a DMEM liquid medium was added to the upper chamber, and the chamber was left in an incubator overnight for further use;
(2) preparation of cells: HeLa cells in logarithmic growth phase were taken and digested with pancreatin, the cell concentration was counted, and the cells were divided into a control group (no heparin or heparin oligosaccharides but only a corresponding volume of PBS was added) and test groups (heparin and sulfated heparin octasaccharide were added, both at a concentration of 62.5 μg/mL). The cells were diluted with a serum-free DMEM medium containing 2.5% BSA and serum-free, 2.5% BSA DMEM medium containing a corresponding heparin oligosaccharide to adjust the cell concentration to 2.5×105 cells/mL.
(3) 800 μL of a culture medium containing 5% FBS was added to the lower chamber of the Transwell chamber.
(4) 400 μL of the cell suspension in step (2) was added to the upper chamber of the Transwell chamber.
(5) The chamber was incubated in an incubator at 37° C. under 5% CO2 for 8 hours.
(6) The liquid in the upper chamber was carefully removed, and the inner surface of the chamber membrane was gently wiped with a cotton swab to remove non-migrating cells. It should be noted that the operation should be gentle so as to avoid damaging the chamber membrane.
(7) The chamber was transferred to another 24-well plate containing 400 μL of cell dye liquor, and the cells were stained at room temperature for 10 minutes.
(8) The Transwell chamber was gently washed with distilled water for 3 to 5 times, and left at room temperature for natural-air drying.
(9) The cells were counted under a microscope and photographed.
(10) The chamber was transferred to another clean 24-well plate, 200 μL of a cell lysis solution was added to each well, and the chamber was placed on a shaker.
(11) After incubated at room temperature for 10 minutes, 100 μL of the cell lysis solution was added to a 96-well plate, and the absorbance value was read at 560 nm. With the heparin control group as a reference, the transfer rate and inhibitory rate were calculated according to the following formulae:
Transfer rate=the absorbance value of the treatment group/the absorbance value of the control group×100%
Inhibitory rate=[1−(the adhesion rate of the treatment group/the adhesion rate of the control group)]×100%
According to the above method, the results of cell adhesion are shown in
In this example, the anti-tumor metastasis activity of 40% and 60% sulfated heparin octasaccharides was determined according to the following method.
B16-BL6 mouse melanoma cells (2×105) were injected into C57BL/6 mice via the tail vein, and the mice were divided into a control group (no heparin oligosaccharide was added, and PBS was employed as control) and test groups (40% sulfated heparin octasaccharide was added, 200 μg for each mouse), 10 mice per group. After 3 weeks, the mice were dissected, the lungs of the mice were fixed in the Bouin's solution, and the numbers of tumors in the lungs of the mice were calculated. We detected the formation of metastatic tumor weekly through an IVIS-200 fluorescence imaging system by employing luciferase-labeled B16-BL6 mouse melanoma cells. Pictures were taken 10 min after intraperitoneal injection of 2.5 mg of fluorescein.
According to the above method, the results of anti-tumor metastasis activity are shown in
The applicant hereby states that the above-mentioned examples in the present invention are provided by way of illustration of the sulfated heparin oligosaccharide of the present invention, the preparation method and use thereof, but the present invention is not limited to the above-mentioned examples, i.e. it is not intended that the present invention has to rely on the above-mentioned examples for implementation. It should be appreciated by a person skilled in the art that any modification to the present invention, equivalent replacement of materials employed and addition of an adjunct ingredient in the present invention, as well as selection of specific embodiments etc. would fall within the protection and disclosure scope of the present invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/099901 | 12/30/2015 | WO | 00 |
