Construction And Preliminary Study Of Carboxymethyl Panax Notoginseng Polysaccharide-chitosan Nanoparticles Loaded With Salvianolic Acid B/Doxorubicin

The combination of drugs and excipients as a carrier for drug delivery is an emerging direction of nano-preparation research in recent years.There is also the concept of"medicine and supplement in one"in traditional Chinese medicine preparations,but there are few related basic researches.Panax notoginseng polysaccharide is a natural polymer compound with anti-oxidation,anti-tumor and other pharmacological activities.Therefore,a new type of nano-medicine carrier was prepared by combining panax notoginseng polysaccharide after carboxylation with chitosan in this project.The combination of chemical drugs and natural products can reduce toxic and side effects while ensuring the therapeutic effect,and successfully prepared polyelectrolyte complex nanoparticles loaded with chemotherapeutic drug doxorubicin and salvianolic acid B,an active ingredient of traditional Chinese medicine with myocardial protective effect.The optimization of drug loading process,characterization of physical and chemical properties,preliminary evaluation of biological safety and investigation of drug release behavior under different p H conditions were carried out.It enriches the concept of"medicine and supplement in one"and provides support for related research on the combination of chemical drugs（doxorubicin）and natural products（salvianolic acid B）.In this study,the concept of"medicine and supplement in one"was applied to the construction of new polyelectrolyte complex preparations.Panax notoginseng polysaccharide is a natural polymer compound with anti-oxidation,anti-tumor and other pharmacological activities.It is an ideal substance for"medicine and supplement in one".Panax notoginseng polysaccharide is carboxymethylated as a polyanion and can form a polyelectrolyte complex carrier through electrostatic interaction with polycations,such as chitosan.And carried charged drugs（salvianolic acid B and doxorubicin）through electrostatic interaction.In this way,the concept of"medicine and supplement in one"that uses carboxymethyl panax notoginseng polysaccharide as medicine and excipients to construct a polyelectrolyte complex carrier and enhance the activity.Carboxymethyl panax notoginseng polysaccharide was prepared by using sodium hydroxide as alkalization reagent and monochloroacetic acid as etherification reagent.The success of carboxymethyl modification was illustrated by carboxymethyl substitution degree,scanning electron microscope,infrared spectroscopy,differential scanning calorimetry,nuclear magnetic resonance spectroscopy analysis,molecular weight and viscosity measurement.At the same time,the scavenging rate of-OH·was used to evaluate the antioxidant activity of carboxymethyl panax notoginseng polysaccharide.The results showed that the carboxymethylated panax notoginseng polysaccharide significantly increased the scavenging capacity of-OH·(IC₅₀value decreased from 46.32 mg/ml to 2.03 mg/ml).Carboxymethyl panax notoginseng polysaccharide and chitosan polyelectrolyte complex was successfully prepared by polyelectrolyte titration.The morphology and peak of chitosan,carboxymethyl panax notoginseng polysaccharide,their physical mixture and polyelectrolyte complex were compared by scanning electron microscopy,infrared spectroscopy,differential scanning calorimetry and X-ray diffraction.The results showed that carboxymethyl panax notoginseng polysaccharide and chitosan polyelectrolyte complex was successfully prepared.Mouse breast cancer 4T1 cells and mouse cardiomyocyte H9C2 cells were used as cell models in vitro.Cell Counting Kit-8 was used to detect the toxic effects of salvianolic acid B and doxorubicin on the two kinds of cells in different ratios.The optimal drug mass ratio of the drug loading system was determined to be 20:1-25:1.The HPLC method for the content determination of the two components was established and the methodology was verified.Salvianolic acid B and doxorubicin were encapsulated in carboxymethyl pananx notoginseng polysaccharide and chitosan polyelectrolyte complex by micro jet method in this study.We successfully constructed salvianolic acid B/doxorubicin loaded carboxymethyl panax notoginseng polysaccharide and chitosan polyelectrolyte complex nanoparticles.We investigated the different mass ratio of chitosan and carboxymethyl panax notoginseng polysaccharide and different reaction p H with the entrapment efficiency and drug loading as evaluation indexes.And investigated the reconstitution conditions with the particle size and PDI as evaluation indexes.The optimal preparation conditions of drug loaded polyelectrolyte complex nanoparticles were determined.The mass ratio of chitosan and carboxymethyl pananx notoginseng polysaccharide was 3:1,p H of the reaction was 4.8,reconstituted solvent was 0.9%Na Cl injectionre dissolving,ultrasonic breaking time was 2 min and reconstitution stability was 27 h.The particle size,PDI and Zeta potential of carboxymethyl panax notoginseng polysaccharide and chitosan polyelectrolyte complex nanoparticles loaded with salvianolic acid B and doxorubicin were measured by Malvern laser particle size analyzer.The results showed that the nanoparticles loaded with salvianolic acid B/doxorubicin had a uniform particle size about 350 nm and a positive charge on the surface.Transmission electron microscope was used to detect the appearance of the nano preparation.The results showed that the nano preparation was clumpy,well dispersed and uniform in size distribution.Infrared spectroscopy,differential scanning calorimetry and X-ray diffraction analysis confirmed that salvianolic acid B and doxorubicin was successfully encapsulated in carboxymethyl panax notoginseng polysaccharide and chitosan polyelectrolyte complex nanoparticles.The hemolysis test was used to investigate the blood safety of the nanocarrier.The results showed that the nanocarrier had good biocompatibility and blood safety.Cytotoxicity experiments showed that panax notoginseng polysaccharide enhanced its anti-tumor activity after carboxymethylation.The dissolution characteristics of salvianolic acid B/doxorubicin loaded carboxymethyl pananx notoginseng polysaccharide and chitosan polyelectrolyte complex nanoparticles were investigated at p H 5.5 and p H 7.4 in vitro.The HPLC method for the determination of the two components in vitro dissolution experiment was established and validated.The results showed that compared with the solution group,the carboxymethyl panax notoginseng polysaccharide and chitosan polyelectrolyte nanoparticles loaded with salvianolic acid B/doxorubicin had obvious sustained-release effect,and the cumulative dissolution of salvianolic acid B/doxorubicin at p H 5.5 was higher than that at p H 7.4.The prepared nanoparticles were p H responsive.