Preparation, Characterization And Performance Research Of Poly Aspartic Acid Macromolecular Nano-carrier Drugs

Although the drugs with small molecular weight have high cu- rative effect, many of them lead to severe toxic effects and are poor cell selectivity. One way to improve the weakness is coupling drugs and targeting groups onto macromolecule to synthesize polymeric carrier drugs. This alters the drug disposition in vivo, limits drug uptake in the pinocytotic route, affords a mechanism for the concentration of the drug in the targeting cells, shows sustained-release and targeting function and decreases toxicity to normal organs and tissues, which may form drug delivery system （DDS） and become an crossing research subject in Macromolecu- lar Science, Pharmacy and Medical field.Synthetic polymers ofα-amino acid are safe and biodegrada- ble and are used for excellent drug carriers. In this paper we synthesized aspartic acid homo- and co-polymer through simply synthesis and modification by using aspartic acid and glutamic acid as initiators, which is as drug carriers to couple metronidazole （MTZ） to form a new polymeric drug, then evaluated their physico- chemical properties. Detailed results are as below:1. Synthesis of poly aspartic acid-metronidazole（PASP-MTZ）. Poly aspartic acid（PASP） was synthesized by polymerization of L-aspartic acid using strong phosphoric acid as catalyst, then PASP-MTZ was obtained by the reaction of MTZ with PASP by N-（3-dimethylaminopropyl）-N-ethylcarbodiimide hydrochloride （EDC）. The conjugated compound was characterized by infrared spectrum（IR）, hydrogen nuclear magnetic resonance（¹H-NMR）, gel permeation chromatography（GPC）, ultraviolet spectrum （UV） and transmission electron microscope（TEM）, and the results showed that MTZ was linked to PASP by covalent bonds, average molecu- lar weight of PASP-MTZ was 44163g·mol^-1, particle size was 404.8nm, drug loadings were about 30%, water absorption rate of PASP was 17% and condensed at 80℃.2. Synthesis of polyα,β-hydroxyethyl-aspartic acid-co-glutamic acid-metronidazole（PAG-MTZ）. Poly aspartic acid-co-glutamic acid （PAG） was prepared by acid-catalysed polycondensation the mix- ture of aspartic acid and glutamic acid in mixture solvents of trime- thylbenzene and sulfolane. The effect of monomer ratio of copoly- mers and reaction temperature was discussed. The metronidazole was coupled to PAG by using dicyclohexylcarbodiimide（DCC） and N,N-dimethylaminopyridine（DMAP） to form polymeric nano-carrier drug. The results showed the PAG-MTZ was random copolymers and elevated thermal stability, average molecular weight was 14281g·mol^-1, the diameter of the particle was 198.9nm, the drug loadings were about 12%, and the stability of the drug in laboratory was higher, water absorption rate was exceeded 20%.Respecting performances of copolymer were better than homopolymer, furthermore the cost of preparation was lower and easy to produce by industrialization, so select the PAG-MTZ as the drug for further study. 3. Metronidazole release behavior of PAG-MTZ in vitro and in vivo. Release of MTZ from PAG-MTZ polymeric nano-carrier drug were investigated under simulated in vivo conditions. The result showed metronidazole release of PAG-MTZ was sustained obviously. The accumulation percentage of metronidazole was 12.19% at 1 hour after released, and 47.51% at 24 hour while the MTZ to release about 100%. Metabolism in vivo of PAG-MTZ were determined with high performance liquid chromatography（HPLC）, the resules showed 2.18 times t_1/2 and 3.87 times MRT longer than MTZ active compound, which indicated that the sustained release of the drug also been found in vivo.4. Inhibitive activity of PAG-MTZ against Trichomonas vaginalis in vitro. The trichomonad was cultured in vitro and divided into groups of PAG-MTZ, free metronidazole, polymeric carrier and the control. The trichomonacidal activity was observed by MTT assay and the incorporation of ³H-TdR. The result demonstrated that the PAG-MTZ have trichomonacidal activity in vitro, there were significant difference between the group of PAG-MTZ and the control （P<0.05）. PAG-MTZ and MTZ have the equivalency anti-trichomonad activity when the concentrate of metronidazole was 40μg·ml^-1. The mechanism may be partial related to induction of apoptotic-like cell death in Trichomonas vaginalis by metronidazole released from PAG-MTZ.5. Therapeutic efficacy on BALB/c mice infected with trichomonads in vagina. BALB/c mice were infected with trichomonads in vagina to construct trichomonas genital tract infection animal model. The infected BALB/c mice were randomly divided into PAG-MTZ high-dose group, PAG-MTZ middle-dose group, PAG-MTZ low-dose group, free MTZ group, PAG carrier group and model group. Additionally, 10 uninfected mice were as normal control. Trichomonas genital tract infection animal model were confirmed by pathohistological, ultrastructural examination and culture of vaginal secretions. In animal experiment, the states of infected mouse group treated with PAG-MTZ were better than model group. The inflammatory cell infiltration of treated group decreased obviously, and tissue congestion and edema also lessened in cervix and vagina epithelial. Bridge corpuscle between cell and basement membrane was gradually recovered. Inflammation accumulated score of PAG-MTZ high-dose group was lowest among all groups except the normal control.6. Metabolism and disposition of PAG-MTZ and PAG in vivo. Because of amidated peptide bond and carboxyl group in PAG-MTZ, it is difficult to label with fluorescence, PAG-MTZ and PAG were directly labeled with ^99mTc. Combined with HPLC, drug releasing, distribution and excretion of PAG-MTZ and PAG after vagina administration were investigated. It was shown that PAG-MTZ can be hydrolyzed, released MTZ from PAG-MTZ, absorbed into blood through vaginal wall and distributed to the entire organism of rat rapidly, and 120 minutes after dosing, the concentration of drug almost cannot be detected. The drug seems not accumulate in rat. According to the test, the PAG carrier cannot enter into blood through vaginal wall, which illustrates that in vivo polymeric carrier drug must be released active compound to create drug effect. 7. Toxicological experiment of PAG-MTZ and PAG. The results revealed that PAG-MTZ and PAG may induce very slight mucosa irritation after vagina administration, especially for PAG carrier material. There was no any acute toxicity reaction in maximal tolerance dose test of PAG-MTZ and PAG.In this paper, the synthesized poly aspartic acid copolymer carrier has good water-soluble, low-toxic and biodegradable property. The prepared new macromolecular nano-carrier drugs can be sustained-release, have some trichomonacidal effect. These studies provided a reference for the synthesis of new polymeric drugs for mucosa administration.