Preparation Of Ursolic Acid Loaded Nanoparticles And Evaluation Of Its Antitumor Effect In Vitro And In Vivo

Background: Ursolic acid(UA), a pentacyclic triterpenoid derived from Catharanthus trichophyllus roots, Chamobates pusillus, has been reported for its anti-inflammatory, antioxidant and antitumor properties. It has also been demonstrated that UA could effectively induce apoptosis and inhibit angiogenesis. However, the clinical application of UA is limited due to several problems. For instance, the limited water solubility of UA leads to the low bioavailability and poor pharmacokinetics in vivo, which subsequently restricts its effectiveness. Thus, it is desirable to explore novel formulations of UA that overcome the limitations mentioned above. Polymeric nanoparticles as drug carriers demonstrate great advantages in delivering chemotherapeutics. For example, polymeric nanoparticles can enhance the solubility of hydrophobic drugs, make vulnerable drugs stable and release the drug in a sustained manner. Therefore, nanoparticle-based drug delivery system could prolong the circulation time of drugs in vivo and increase the targeted delivery to tumor, which makes it a novel way in cancer treatment.Objectives: UA-loaded nanoparticles were prepared by a nano-precipitation method using the synthesized methoxy poly(ethylene glycol)–polycaprolactone(m PEG–PCL) and poly(N-vinylpyrrolidone)-block-poly(?-caprolactone)(PVP-PCL) as drug carrier. The physical characteristics(size, zeta potential, stability and release pattern) and in vitro and in vivo antitumr efficacy were evaluated to demonstrate the advantage of drug loaded nanoparticles and provide fundamental evidences for the development of this novel nano-drug delivery system.Methods: m PEG–PCL and PVP-PCL were synthesized by ring-opening copolymerization. Drug loaded nanoparticles were prepared by a nano-precipitation method. Morphological examination of the nanoparticles was conducted with scanning electron microscope(SEM) and transmission electron microscope(TEM). Mean diameter and zeta potential were measured by photon correlation spectroscopy(DLS). Drug loading content(DLC) and encapsulation efficiency(EE) of different drug-loaded nanoparticles were determined and calculated. In vitro release pattern of drug-loaded nanoparticles were measured with the dialysis method. We explored the anticancer effect of UA-loaded nanoparticles and the potential mechanism in vitro and in vivo.Results: First, we prepared an efficient UA delivery system with amphilic copolymers(m PEG–PCL) as the drug carrier for the first time. SEM and TEM images clearly show the nearly smoothly spherical shape of UA-loaded nanoparticles with minor deformation. The size of UA-loaded nanoparticles is 144.0±4.0 nm. The drug loading content is 4.75 ± 0.45% with the encapsulation efficiency reaching 87 ± 5.3%. In vitro release study indicates that UA can be released from the core-shell structure of polymeric nanoparticles in a sustained manner. Second, UA was effectively transported into SGC7901 cells by nanoparticles and localized around the nuclei in the cytoplasms. The in vitro cytotoxicity and apoptosis test indicated that UA-NPs significantly elicited more cell death at almost equivalent dose and corresponding incubation time. Blank nanoparticles nearly did not suppress cells proliferation when its concentration reached up to 200?g/m L during different incubation periods, indicating the copolymers were nontoxic to tissues and cells. The DAPI staining showed the number of apoptotic cells treated with UA-NPs dramatically increased in a dose-dependent manner when compared with those treated with free UA. Moreover, UA-NPs led to more cell apoptosis through stronger inhibition of COX-2 and activation of caspase-3. The most powerful evidence from this report is that the significant differences between the cytotoxicity of free UA and UA-NPs are closely related to the expression levels of COX-2 and caspase-3, which demonstrates the superiority of UA-NPs over free UA in penetrating cell membrane. Third, we loaded UA into amphilic PVP-b-PCL nanoparticles and performed the physiochemical characterization as well as the releasing capacity. The drug loading content is above 12% with the encapsulation efficiency reaching 85%. In vitro release study indicates that UA can be released from the core-shell structure of polymeric nanoparticles in a sustained manner. Fourth, In vitro experiments indicate that UA-NPs inhibit the growth of liver cancer cells and induce cellular apoptosis more efficiently than free UA. Moreover, UA-NPs significantly delay tumor growth and localize to the tumor site when compared to the equivalent dose of UA. In addition, both western blotting and immunohistochemistry suggest the possible mechanism of the superior efficiency of UA-NPs is mediated by the regulation of apoptosis related proteins.Conclusion: In vitro and in vivo evaluation of UA-loaded nanoparticles reported in the current study demonstrates enhanced antitumor effects compared with free drugs. The current results provide a possible explanation for the superiority of UA-NPs that the efficient uptake of UA-NPs leads to an enhanced inhibition of COX-2 expression and the subsequent regulation of apoptotic proteins, thereby inducing apoptosis more effectively. Therefore, development of nanosized drug delivery systems emerges as anovel field in the research of anticancer drugs, which merits more intensive study and reveals potential application.