| Endostatin(ES)is an endogenous anti-angiogenic factor with a relative molecular mass of 20 kD isolated from the supernatant of hemangioendothelioma in mice.ES can inhibit the neonatal angiogenesis of tumor sites and cut off the role of tumor nutrient supply and waste metabolism.It is currently used clinically for the treatment of various tumors.ES2(IVRRADRAAVP)is a fragment of the ES structure that has anti-angiogenic activity and is three times more active than the complete sequence of ES.However,ES2 has shortcomings such as poor stability and short half-life,and has not yet achieved clinical application.Chemical modification of proteins and peptides is an effective way to improve their shortcomings.Currently,the most widely used modifier is polyethylene glycol(PEG).PEG has many advantages,but its non-degradability will lead to other reactions in the long-term,large-scale use of drugs.Chitosan is a polysaccharide with a well-defined structure,good water solubility and certain anti-tumor activity.In the early stage,our group used quatemized chitosan oligosaccharides to modify ES2 in vitro.The modified product(HTCOSC-ES2),while retaining anti-angiogenic activity,has improved water solubility,stability,and increased affinity for endothelial cells.This paper will further discuss how HTCOSC-ES2 acts on endothelial cells and how to exert anti-tumor effects in vivo;in addition,the in vivo pharmacokinetic characteristics of HTCOSC-ES2 have also been studied.The research content and main achievements of this topic are as follows:1.Preparation of HTCOSC-ES2(1)After the quaternized group side was linked to the hydroxyl group at the C6 position of the chitooligosaccharide,quaternized chitosan oligosaccharide(HTCOSC)was obtained by dialysis and freeze-drying,and the yield was 92.64%.The analysis was verified by nuclear magnetic resonance spectroscopy('H NMR)and infrared spectroscopy(IR).The HTCOSC structure is in line with expectations(2)Using NHS/EDC as a cross-linking agent,HTCOSC-ES2 was prepared by forming an amide bond between a carboxyl group in the ES2 molecule and an amino group in the HTCOSC molecule,and was separated and purified by Sephadex G-25 gel chromatography.(3)Particle size and transmission electron microscopy experiments show that ES2 can form a nano-sized spherical structure with a hydrophilic amino acid on the outside and a hydrophobic amino acid on the inside in the aqueous phase.In the aqueous phase,HTCOSC-ES2 can form a nano-sized spherical structure with a hydrophilic chitosan oligosaccharide and a hydrophobic amino acid inside.2.Effects of HTCOSC-ES2 on endothelial cell proliferation in vitroBoth ES2 and HTCOSC-ES2 significantly inhibited endothelial cell proliferation in a concentration-dependent manner,with HTCOSC-ES2 activity being slightly stronger than ES2.When administered at concentrations of 5?g/mL,25?g/mL,50?g/mL,75?g/mL,100 ?g/mL,and 125 ?g/mL,the inhibition rate of ES2 on endothelial cell proliferation was(3.50±1.85)%,(13.866±2.01)%,(25.89±5.83)%,(34.83±1.88)%,(33.24±3.38)%and(39.84±1.71)%;HTCOSC-ES2 inhibits endothelial cell proliferation were(14.86±0.76)%,(25.22±3.40)%,(37.50±3.65)%,(42.93±1.96)%,(43.60±1.53)%and(54.04±5.28)%,respectively.3.Endothelial cells uptake and intracellular distribution of HTCOSC-ES2The endothelial cell uptake pathway of FITC-ES2 and FITC-HTCOSC-ES2 was studied by flow cytometry combined with the clathrin inhibitor chlorpromazine and the lipid raft inhibitor methyl-?-cyclodextrin.The results showed that endothelial cells can take up ES2 and HTCOSC through clathrin and lipid raft pathway,and the clathrin pathway is dominant.The distribution of FITC-ES2 and FITC-HTCOSC-ES2 in endothelial cells was studied by laser confocal microscopy.The results showed that both HTCOSC-ES2 and ES2 experienced the process of first entering the lysosome and then distributing it to the nucleus.HTCOSC-ES2 entered the lysosome at a slightly later time than ES2.At 0.5 h,some ES2 entered the lysosome,and HTCOSC-ES2 began to enter the lysosome for 1 h.HTCOSC-ES2 and ES2 enter the nucleus at the same time,starting at 2 h and completely entering the nucleus at 3 h.4.Effects of HTCOSC-ES2 on endothelial cell apoptosis and cell cycle(1)The effects of ES2,HTCOSC-ES2 and HTCOSC+ES2 mixtures on endothelial cell apoptosis were studied by flow cytometry.The results showed that endothelial cells had a certain degree of apoptosis when incubated with medium containing different doses of ES2,HTCOSC+ES2 mixture and HTCOSC-ES2.However,compared with the blank control group and the bFGF group,the apoptotic rate of the ES2 group and the HTCOSC+ES2 mixture group(except the high dose group of HTCOSC+ES2 mixture)showed no significant difference(p>0.05).The apoptotic rate of the HTCOSC-ES2 group was significantly different(p<0.05)compared with the blank control group,bFGF group and ES2 group,and was dose-dependent.When the doses were 5?g,10 ?g,25?g and 50?g,the apoptosis rates of endothelial cells in the HTCOSC-ES2 conjugate group were(14.13±0.45)%,(34.40±7.24)%,(51.77±5.23)%and(59.10±4.73)%,respectively.(2)The effects of ES2,HTCOSC-ES2 and HTCOSC+ES2 mixture on endothelial cell cycle were studied by flow cytometry.The results showed that when the endothelial cells were incubated with medium containing different doses of ES2,HTCOSC+ES2 and HTCOSC-ES2,,The cell cycle was affected to some extent.With the increase of the dose,the proportion of G0/G1 phase in each group increased to different extents,and the proportion of S phase and G2/M phase also decreased to varying degrees.Among them,HTCOSC-ES2 had the most obvious effect on endothelial cell cycle.When the dose was 5 ?g,the ratios of G0/G1 phase,S phase and G2/M phase were(54.87±0.60)%,(44.77±0.51)%and(0.35±0.23)%,respectively;when administered at doses of 10 ?g,G0/G1 phase,S phase and G2/M phase was(59.39±0.91)%,(39.24±1.91)%and(0±0)%,respectively.When the dose was 25 gg,the ratios of G0/G1 phase,S phase and G2/M phase were(61.92±1.38)%,(37.52±2.10)%and(0.1±0.17)%,respectively.When the dose was 50?g,the ratios of G0/G1 phase,S phase and G2/M phase were(63.79±10.08)%,(36.12±0.66)%and(0±0)%,respectively.Compared with the control group or the ES2 group,HTCOSC-ES2 can significantly block most of the endothelial cells in the G0/G1 phase,a small part into the S phase,and a very small part into the G2/M phase.5.In vivo anti-tumor effect and mechanism of HTCOSC-ES2(1)The inhibitory activity of ES2 and HTCOSC-ES2 on B16 melanoma was studied in vivo.The results showed that both ES2 and HTCOSC-ES2 significantly inhibited the growth of B16 melanoma in vivo.With the increase of the number of days,the tumor volume of the saline group increased from(52.30±14.25)mm3 to(3994.10±1501.34)mm3,and the tumor volume of the ES2 group increased from(29.66±5.13)mm3 to(2167.27±394.79)mm3.The tumor volume of the HTCOSC-ES2 group increased from(28.62±4.78)mm3 to(904.33±74.36)mm3 Although the tumor volume of each group increased continuously,the tumor volume growth rate of the HTCOSC-ES2 group was much lower than that of the control group and the ES2 group.After the mice were sacrificed on the 15th day,the tumor weight of the HTCOSC-ES2 group was(2.58±0.77)g,which was significantly lower than that of the control group(8.10±2.30)g and the ES2 group(5.24±0.41)g.The ES2 and HTCOSC-ES2 mice were stable in weight and showed no significant increase or decrease,suggesting that both may have less toxic side effects.In conclusion,ES2 and HTCOSC-ES2 significantly inhibited the growth of B16 melanoma in vivo(p<0.05),with HTCOSC-ES2 having a significantly higher tumor inhibition rate than ES2(p<0.05).(2)Immunohistochemistry The effects of HTCOSC-ES2 and ES2 on the expression of tumor growth-related factors were studied.The results showed that VEGF,CD31 and caspase-3 were expressed in the saline group,ES2 group and HTCOSC-ES2 group.The IOD values of VEGF in the saline group,ES2 group and HTCOSC-ES2 group were(25.55±2.59),(18.38±4.60)and(3.18±0.34);the IOD values of CD31 in the saline group,ES2 group and HTCOSC-ES2 group were(30.00±5.69),(17.86±3.34)and(10.43±2.88),respectively.The IOD expression of caspase-3 in the saline group,ES2 group and HTCOSC-ES2 group was(10.56±2.06),(53.48±11.00)and(347.14±155.33),respectively.Immunohistochemical analysis showed that both ES2 and HTCOSC-ES2 significantly reduced the expression of angiogenic factor VEGF and the density of microvessels near the tumor(p<0.05),and increased the expression of apoptosis-related protein caspase-3 in tumor cells.The expression of caspase-3 in the HTCOSC-ES2 group was statistically significant(p<0.05).ES2 and HTCOSC-ES2 inhibit the microvessel density near the tumor by down-regulating VEGF and up-regulating the expression of caspase-3,thereby inhibiting the formation of new blood vessels near the tumor and inhibiting tumor growth.The HTCOSC modification of ES2 preserves and enhances the anti-angiogenic and anti-tumor effects of ES2,and its efficacy is in line with the expected results.6.HTCOSC-ES2 pharmacokinetics and tissue distribution studies(1)C57BL/6 mice were selected as the in vivo pharmacokinetic model.The plasma and tissue were treated by TCA precipitation protein to study the pharmacokinetic parameters and tissue distribution such as half-life.By comparing the pharmacokinetic parameters of ES2 and HTCOSC-ES2,it was found that the half-life t1/2,of the conjugate HTCOSC-ES2 was extended from(18.04±2.38)h to(29.17±3.91)h after chemical modification of ES2.The area under the AUC increased from(226.67±95.20)?g/L*h to(11650.98±129.41)?g/L*h,and the maximum plasma concentration Co increased from(277.83±121.86)?g/L to(1931.81±75.35)?g/L,the plasma clearance rate CL also decreased from(90.26±39.94)L/h/kg to(10.32±0.89)L/h/kg,indicating that the conjugate HTCOSC-ES2 achieved the half-life extension(2)The results of tissue distribution studies show that ES2 and HTCOSC-ES2 are mainly distributed in the liver and kidney.The distribution of ES2 and HTCOSC-ES2 in the liver can reach the maximum at 2 h,and the aggregation of HTCOSC-ES2 in the liver is more obvious than that of ES2.In addition,ES2 and HTCOSC-ES2 are also distributed in other tissues at lower concentrations.In summary,ES2 and HTCOSC-ES2 are mainly taken up by endothelial cells through clathrin and lipid raft pathway,transported to the nucleus by lysosomal degradation,promote endothelial cell apoptosis and inhibit the process of G0/G1 phase of cells,thereby inhibiting angiogenesis.HTCOSC-ES2 can promote the increase of tumor tissue apoptosis protein caspase-3 and the decrease of microvessel density and VEGF expression,leading to the reduction of the number of microvessels in tumors and thus effectively inhibiting tumor growth.In addition,compared with ES2,HTCOSC-ES2 achieved longer endothelial cell uptake time and half-life in vivo,and showed a certain liver aggregation effect.The development of this subject lays the foundation for the development of HTCOSC-ES2 as a therapeutic drug for neovascularization-related diseases. |
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