| Background:Cordycepin (Cor), also known as 3’-deoxyadenosine, is a nucleoside antibiotics, which was first isolated from Cordyceps militaris Link original liquid by Canadian scientists Cunningham et in 1950. It has been shown that cordycepin is a multifunctional compound, including antitumor, antibacteria, antivirus, antiinflammatory, antidiabetics, anti-hyperlipidemia, hyperglycemic, anti-leukemia, anti-aging, and so on. But cordycepin is converted to inactive metabolites 3’-deoxyinosine, because of rapid deamination caused by adenosine deaminase (ADA) in vivo, limiting its clinical applications. Ferthermore, side effects are serious when cordycepin is used in combination with ADA inhibitors.Objective:To solve the problem that cordycepin is rapidly metabolized under ADA in vivo, carbox-ymethyl chitosan (CMC) was chosed as the carrier to prepare cordycepin loaded carboxymethyl chitosan nanoparticles (Cor-CMC-Nps). And its release properties in vivo and in vitro and its action on the growth of human hepatoma cell HepG-2 were studied to investigate whether the new drug delivery system could alleviate the metabolism of cordycepin and prolong its action time.1. Preparation of cordycepin loaded carboxymethyl chitosan nanoparticles (Cor-CMC-Nps)Methods:1. Taking liquid paraffin/span-80 for oil phase and CMC saline solution for water phase, and sodium tripolyphosphate (TPP) as crosslinking agent, Cor-CMC-Nps was prepared by using emulsion ionic crosslinking method. The preparation technology and formulation were optimized by using single-factor test and orthogonal experiment.2. Ultracentrifugation-UV method was applied in the determination of entrapement ratio and drug loading of Cor-CMC-Nps. Diameter and surface potential of nanoparticles were determined by zetasizer meter. The surface morphology of sample was observed by transmission electron microscope (TEM). With differential scanning calorimetry (DSC) and X-ray diffraction (XRD), the physical state of cordycepin in nanoparticles was analyzed.Results:1. The results of single-factor test showed that the ultrasonic power, the dosage of span-80 and the concentration of CMC solution had a great influence on diameter and entrapement ratio of Cor-CMC-Nps, the quantity of Cor had less effect and the concentration of TPP had little influence.2. The results of orthogonal test showed that the dosage of span-80 and concentration of CMC had a significant effect on entrapement ratio, the former was greater than the latter, but the effect of ultrasonic power was not obvious. The optimum prescription and preparation technology were:span-80:2mg, CMC:6mg/ml, ultrasonic power:200 W.3. The prepared nanoparticles showed similar spherical by TEM. The mean diameter of the particle was 147.6 nm and zeta potention was-24.1 mv. Its entrapement ratio reached 69.6% with the drug loading of 9.02%. The results of DSC and XRD showed that the drug was dispersed in carrier in the amorphous form.2. The release of cordycepin loaded carboxymethyl chitosan nanoparticles in vitro and in vivoMethods:1. The release in vitro:The release of Cor-CMC-Nps in three kinds of medium was studied by dynamic dislysis. (1) Phosphate buffer (PBS, pH7.4);(2) PBS with serum; (3) PBS with ADA (8.8U/L). The concentration of Cor in medium was determinated by UV method and the cumulative amount of release was calculated. The curve equation of release was fitted respectively by the zero-order release, the first-order release, Higuchi equation and Weibull distribution model.2. The release in vivo:In order to find out the release of Cor-CMC-Nps in vivo, Blood was sampled from mouse eyes at different time after the intraperitoneal injection of Cor solution or Cor-CMC-Nps at dosage of 100mg/kg, respectively. The concentration of Cor in serum was determined by reverse-phase HPLC-PDA and AUC was calculated. Chromatographic conditions of HPLC:Column:ZOXBAX SB-C18 (4.6mmx250mm,5μm); Detector:PDA; mobile phase:water:methanol=88:12; flow rate:1ml/min; detection wavelength λ:260nm; column temperature:30℃; injection volume:20μl.Results:1. The release in PBS medium:The cumulative release rates of Cor solution were 80.8%,90.0% and 93.7% at 2h,6h,24h, respectively. The cumulative release rates of Cor-CMC-Nps were 27.2%,38.2%,57.1% and 38.2% at 2h,6h,24h,48h, respectively.2. The release in PBS medium with serum:The cumulative release rates of Cor solution were 62.2%, and 69.9% at 2h and 10h, respectively, then the drug concentration could not be detected later. The cumulative release rates of Cor-CMC-Nps were 35.3%,62.0%,70.9%, 80.4% at 2h, 10h,24h,48h, respectively.3. The release in PBS medium with ADA:The cumulative release rates of Cor solution were 17.5% and 20.1% at 2h and 8h, respectively, then the drug concentration could not be detected later. The cumulative release rates of Cor-CMC-Nps were 19.2%,25.8%,31.8%, 35.5% at 2h,8h,24h,48h, respectively. These results showed that Cor-CMC-Nps had a good sustained release effect in the presence of buffer, simulated blood environment and degradation enzymes.4. The rules of release:The release process of Cor-CMC-Nps in three kinds of medium accords with Weibull distribution model, and the correlation coefficient was in range of 0.9792-0.9945.5. After mice were given a intraperitoneal injection of Cor solution (100mg/kg), the peak concentration of Cor was about 8.85±0.43μg/ml at 0.5h, then the drug concentration decreased rapidly in 1h. The blood concentration of Cor reached to the maximum of 10.02 ±0.40μg/ml at 2.5h after giving a intraperitoneal injection of Cor-CMC-Nps solution (100mg/kg), then the drug concentration decreased quickly in 4h after injection, and did slowly after 4h. AUCNps/AUCCor was 1.11. When the drug concentration was above 6μg/ml, AUCNps/AUCCor was 1.76. These results showed that Cor-CMC-Nps had a good sustained release effect in vivo. The time maintained a high level of concentration after Cor-CMC-Nps was longer than that after Cor solution and AUC of Cor-CMC-Nps was also higher compared with that of Cor solution.3. The inhibition of cordycepin loaded carboxymethyl chitosan nanoparticleson HepG-2 cellsMethods:1. Experiment groups:(1) Blank control group:DMEM containing 10% newborn bovine serum; (2) Positive control group:5,10,20μg/ml DDP;(3) Negative control group:CMC-Nps; (4) Cor solution group:30,60,90,120,150μg/ml Cor; (5) Cor-CMC-Nps group:Cor-CMC-Nps solution containing 30,60,90,120,150μg/ml Cor.2. In vitro HepG-2 cells were cultured and drug was added according to the groups described above respectively, whose final concentration reached the concentration as above described. At 24h,48h,72h after incubation, the optical density value was determined by MTT method, and the cell inhibitory rate of each group was calculated. Cell morphology was observed under inverted microscope.3. In vitro HepG-2 cells were cultured and grouped and dosed as described above, then 0.3 μl ADA (2.0×10-3U/μl) was added in the each hole, the cell inhibitory rate of each group was calculated under the condition of ADA.Results:1. The cell inhibitory rate without ADA:Cor group:the inhibitory rate of 5 dosage groups of Cor on HepG-2 cells were in range of 21.6%～55.4%(P<0.001),27.2%-58.5%(P<0.001), 13.8%～47.8%(P<0.001) at different incubation times (24,48,72h). The cell inhibitory rate was enhanced with the increase of drug concentration at the same time. Cor-CMC-Nps group:the inhibitory rate of 5 dosage groups of Cor-CMC-Nps on HepG-2 cells were in range of 9.52%-48.2%(P<0.01,0.001),31.4%-68.5%(P0.001),35.9%～75.1% (P<0.001) at different incubation times (24,48,72h). The cell inhibitory rate was enhanced with the increase of drug concentration and extension of action time. The results showed that Cor-CMC-Nps and Cor could inhibit the growth of the HepG-2 cells.2. The comparation of cell inhibition between Cor-CMC-Nps and Cor:Compared with Cor group, the inhibition effects of Cor-CMC-Nps were less (P<0.01,0.001) at 24h. By contrast, those of Cor-CMC-Nps were significantly stronger than those of Cor group (P<0.001) at 48h and 72h. The cell inhibition rate of Cor-CMC-Nps reached the maximum of 75.1% at 72h, that of Cor peaked about 58.5% at 48h, the former is 1.28 times of the latter (increasing 28.5%). The results show that Cor-CMC-Nps lasted longer and had greater inhibition effects than that of Cor solution.3. The cell inhibitory rate with ADA:Cor group:the inhibitory rate of 5 dosage groups of Cor on HepG-2 cells were in range of 5.46%-35.6%,4.89%-26.9%,3.34%-17.8% at different incubation times (24h,48h,72h), its cell inhibition reached a peak at 24h. Cor-CMC-Nps group:the inhibitory rate of 5 dosage groups of Cor-CMC-Nps on HepG-2 cells were in range of 5.28%-29.1%,19.1%-43.6%,25.4%-46.0% at different incubation times (24h, 48h,72h), its cell inhibition also peaked at 72h. Compared with Cor group, the action time of Cor-CMC-Nps prolong significantly. Compared with the cell inhibitory rate without ADA, that of Cor-CMC-Nps group or Cor group in the condition of ADA decreased significantly at different incubation times (24h,48h,72h). Furthermore, the Cor group lasted shorter, while nanoparticles group remained unchanged. The results showed that nanocarriers had a protective effect on cordycepin, and could alleviate the metabolism of cordycepin and prolong its action time.4. Morphology of cells:Cells of the control group and the CMC-Nps group were spindle-shaped, after the intervention of Cor and Cor-CMC-Nps, the cells became spherical or round instead of spindle-shaped, and the adhesion of cells was decreased and some floated in the nutrient solution. The number of spherical or round cells increased with the extension of action time. Compared with Cor group, the inhibition of Cor-CMC-Nps on HepG-2 cells was more significant at 48h and 72h.Conclusion:1. Taking liquid paraffin/for oil phase and CMC saline solution for water phase, and TPP as crosslinking agent, Cor-CMC-Nps could be prepared by using emulsion ionic crosslinking method successfully. The mean diameter of the particle was 147.6 nm and zeta potention was-24.1 mv. Its entrapement ratio reached 69.6% with the drug loading of 9.02%.2. Cor-CMC-Nps had a good sustained release effect in PBS medium, PBS medium with serum and ADA.3. Cor-CMC-Nps had a good sustained release effect in vivo.4. Cor-CMC-Nps and Cor could inhibit the growth of the HepG-2 cells. Cor-CMC-Nps lasted longer and was greater inhibition than Cor solution under the same concentration.5. Cor-CMC-Nps had a protective effect on Cor, and could delay the degradation metabolism of Cor and prolong its action time. |
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