| Riccardin D is a macrocyclic bisbibenzyl compound extracted from liverwort Marchantia polymorpha L. It is a new substance and the study on pharmacology is ongoing. RD has shown its various biological activities and pharmacological actions, such as effectively reversing the multidrug resistance, its antifungal activity and inducing apoptosis. Furthermore, a systemic pharmacology research on lung cancer reported that riccardin D could inhibit angiogenesis in human lung carcinoma. Riccardin D has been regarded as a novel DNA topo Ⅱ inhibitor and a potential chemotherapeutic agent for treatment of lung cancers. However, its application in the clinic has been limited due to its poor solubility and low bioavailability. The study on RD was separated into two part. The 1st part was studied on RD nanosuspensions. And the 2nd part was studied on RD loaded multifunctional micelles.In the 1st part, the EPAS process as the bottom-up process and the microfluidisation as the top-down process are chosen. The physicochemical characterizations including the particle morphology, size distribution, DSC and XRPD were assessed. In the EPAS process, stabilizers were screened. RD was completely dissolved in ethanol as an organic phase. The organic phase was added slowly in iced water containing different concentrations of F68, PVP K30, and HPMC with a magnetic stirrer. After the mixing procedure the suspension was stirred to decrease the ethanol content and the nanosuspension in the bottom-up method was obtained. In the microfluidization process, the pressures and cycles were screened to obtain stable nanocrystal suspension. The nanocrystals made in EPAS process were proved to be smaller, more uniform and had a narrower distribution than the microfluidisation nanocrystals. Then the freeze-dried powder was analyzed. Due to its poor solubility, LC-MS and HPLC were both used to examine the solubility, dissolution rate and storage stability of the freeze-dried nanocrystals. Differential scanning calorimetry (DSC) and X-ray diffraction confirmed the crystalline states that were both reserved. At last, HPLC was used to analyze the pharmacokinetics and tissue distribution of the two kinds of nanocrystals. And we hope nanosuspensions with a passively lung targeting world be obtained with the help of reticuloendothelial system.After screening test, F68, HPMC, and PVPK30 were chosen as stabilizers and RD:F68:HPMC:PVP K30=2:1:2:1 was the best combination. RD nanocrystal suspension was prepared by an antisolvent precipitation technique. RD was completely dissolved in heated ethanol as an organic phase. The organic phase was added slowly in to iced water containing F68, PVP K30, and HPMC with a magnetic stirrer at 800 rpm. After the mixing procedure the suspension was stirred at 300 rpm for 2 h at room temperature to decrease the ethanol content. The nanosuspension in the bottom-up method was obtained. The same content of RD, F68, PVP K30, and HPMC as the optimal prescription were dispersed in water and treated with an ultrasound machine for 10 min. Then the suspension was in comminution process using an IKA homogenizer machine at 15,000 rpm for 1 min. The resultant suspension was performed on the microfluidizer model M-110P at 2000 bar and 8 cycles to obtain stable nanocrystal suspension. A cold water bath system was used during the microfluidisation process. The two methods were both easily performed and reproducible. The morphologies of the two nanosuspensions prepared by two methods were examined by TEM. The particles made in both methods were shaped regularly and homogeneous, and had a narrow size distribution. The size of the two kinds of nanocrystals was 184.1±3.15 nm and 815.37±9.65 nm. The Zeta potential of them was -27.89±0.84 mV and -17.43±0.76 mV.To sustain a long-term stability of the RD nanocrystals, lyophilization process was performed. The nanosuspensions were added with 5% mannitol as freezed dried protectant.3 mL nanosuspension in each Cillin-glass bottle (15 mL) was pre-frozen at-80℃ Ultra-low Temperature Freezer for 48 h. The frozen nanocrystals were freeze dried at -50℃ for 48 h with freeze drier The nanocrystal powder made in the EPAS process was called RD-NA (riccardin D nanocrystal A) and that made in the microfluidization process was called RD-NB (riccardin D nanocrystal B) for short. Storage stability was studied by storing RD-NA and RD-NB powders at 4℃ for one month. The changes in appearance, particle size and Zeta potential were measured after one month. During this storage period, the dried powder appearance barely changed after 1 month. The sizes of RD-NA and RD-NB increased to 208.37 ± 7.45 nm and 908.24 ± 16.23 nm and the Zeta potentials decreased to-23.24 ± 1.36 mV and-15.43 ± 1.28 mV respectively. According to the calibration curve, the solubility of RD in pure water is 0.619±0.245 μg·mL-1 while the physical mixture powder is 3.179 ±0.116μg·mL-1. The surprising news is that the solubility of RD-NA is 30.5± 2.1μg·mL-1 and the solubility of RD-NB is 222.084 ±0.5μg·mL-1. So the two methods can greatly increase the solubility of RD. And the dissolution rate was significantly improved after preparing nanocrystal powders.With the HPLC method, tissue distribution was analyzed after tail i.v. administration of RD-Sol, RD-NA and RD-NB. The results demonstrated that the nanosuspension delivery system had changed the tissue distribution of RD in mice. It was indicated that RD-NB had more significant lung passively targeting characteristics compared with RD-Sol and RD-NA, which made RD-NB a promising candidate for the treatment of lung diseases. On the other hand, the hydrophilic polymer PVPK30 acting as a stabilizer, had reduced opsonization, leading to a prolonged drug resistance in circulation.In the pharmacokinetics study, RD-NA and RD-Sol showed similar parameters, while RD-NB was quite different. For example, after administration of RD-Sol, the CLz was 4.2 L·h-1·Kg-1, MRT was 2.75 h and AUC was 16.7 h·μg·mL-1. Similarly, after administration of RD-NA, the CLz was 4.245 L·h-1·Kg-1, MRT was 2.183 h and AUC was 17.35 h·μg·mL-1. Compared with RD-Sol and RD-NA groups, the rats treated with RD-NB showed a longer MRT and lower CL. The CLz was 1.717 L·h-1·Kg-1, MRT was 4.277 h and AUC was 45.95 h·μgm·L-1, which was approximately 3-fold of other AUC.This part of study was designed for a practical problem in developing a new drug And we hope to solve a series of problems about drugs with poor solubility in a systematic way. In our study, it is indicated that RD-NB had more significant lung passively targeting characteristics compared with RD-Sol and RD-NA, which made RD-NB a promising candidate for the treatment of lung cancer.The 2nd part was studied on micelles. Self-assembled polymeric micelles are formed from an amphiphilic polymer, which could self-assemble into core-shell micelles with a hydrophilic outer shell and hydrophobic inner core in water via the molecular forces such as hydrophobic interaction, electrostatic interaction, hydrogen bonding and metal complexation. Amphiphilic polymeric self-assembled micelles have high thermodynamic stability and the hydrophobic inner core can be utilized as a cargo space for poorly water-soluble drugs, genes, peptides and proteins, while the hydrophilic outer shell keeps a long circulation of drugs in vivo. Amphiphilic polymeric self-assembled micelles have high potentials in drug delivery and receive more and more attentions. After chemical modification, they can achieve special pharmacological characteristics such as active targeting, micro-environment response (e.g. pH sensitivity, temperature sensitivity and magnetic targeting), escaping the engulfment of mononuclear phagocytic cells, and improving transportation through biomembrane.O-carboxymethylated chitosan (OCMC) was firstly hydrophobically modified with various deoxycholic acid (DOCA) to obtain a novel kind of polymer amphiphiles, and then covalently bounded with folic acid (FA) and PAE-PEG to develop a new cancer-targeted potential drug delivery system. The characterizations of the polymers were analyzed by FTIR and NMR. The physicochemical properties of self-aggregates in aqueous media were investigated by size distribution, Zeta potential, and transmission electron microscopy (TEM). The mean diameter of DOMC-FA and PEG-PAE-DOMC-FA self-aggregates in PBS solution (pH 7.4) was under 150 nm with a narrow size distribution. While in PBS solution (pH 5.0), the mean diameter of DOMC-FA and PEG-PAE-DOMC-FA self-aggregates was significantly larger with a wider size distribution. The TEM images of self-aggregates showed a spherical shape at pH 7.4. The two self-aggregates covered with negatively charged OCMC shells, exhibiting Zeta potentials near -20 mV in PBS solution (pH 7.4). While in PBS solution (pH 5.0), the Zeta potential was about -4 mV as the PEG-PAE was positively charged. As the results of size distribution and Zeta potential, the critical aggregation concentrations of DOMC-FA and PEG-PAE-DOMC-FA conjugates were changed with the pH value. Hemolytic experiment was completed to ensure the safety of the two kinds of micelles.The self-assemble method was screened for the incorporation of RD into DOMC-FA and PAE-PEG-DOMC-FA micelles. The average loading of RD in DOMC-FA and PAE-PEG-DOMC-FA micelles was 22.78% and 32.27% (45.57% and 64.53% encapsulation efficiency), respectively. The size and size distribution of drug-loaded micelles were characterized by Zetasizer and exhibited a mean particle diameter of about 153.6nm and 210.5 nm for the two formulations at pH 7.4 with narrow size distribution. All the drug-loaded micelles had relatively high negative Zeta potentials of around -20 mV. The negative Zeta potential indicated that the micelle surface was negatively charged. The appearance and drug-loading efficiencies hardly changed. While at pH 5.0, EE and DL of the two micelles were both decreased and particle size were increased, which indicated the change of the core-shell structure. In our study, the drug-load micelles were stable with good fluidity when sealed and stored under conditions such as refrigeration or at room temperature for three months. In vitro drug release study, at pH 7.4, there was a pronounced time prolongation of drugs release from micelles. Interestingly, the drugs cumulative release rates showed obvious pH dependence, which indicated that the micelles could release more drugs in tumor tissue. These results confirm that the RD loaded PEG-PAE-DOMC-FA micelles are useful controlled delivery system for cancer treatment.There was an increased level of uptake of folate-conjugated micelles compared with coumarin-6 solution in folate receptor overexpressed human breast cancer cells, MCF-7 cells, and the uptake mainly on account of the effective process of folate receptor-mediated endocytosis. The MTT assay, morphological changes and apoptosis test implied that the folate-conjugated micelles enhanced the cell-killing effect by folate-mediated active internalization, and the cytotoxicity of the pH sensitive micelles (PEG-PAE-DOMC-FA/RD) to cancer cells was much higher than DOMC-FA/RD or the RD solution. The results of this research demonstrated the PEG-PAE and folate-conjugated micelles could be beneficial in treatment of solid tumors by targeting delivery of micellar RD into the tumor cells and further reducing side effects and toxicities of the drugs.The tissue distribution of DOMC-FA/RD and PEG-PAE-DOMC-FA/RD micelles and RD injection was investigated after i.v. administrations into mouse. Compared with RD solution, PEG-PAE-DOMC-FA/RD micelles distinctly changed the distribution of RD in vivo and prolonged the MRT of RD in plasma greatly. The results of targeting-evaluation experiments showed PEG-PAE-DOMC-FA/RD micelles had a marked difference comparing with RD solution, i.e.3.79 for plasma, 1.33 for lung,0.80 for liver,0.65 for kidney,0.42 for spleen and 0.38 for heart. PEG-PAE-DOMC-FA/RD micelles show a high plasma targeting efficiency in vivo and can keep high drug levels for relatively long time. The levels of PEG-PAE-DOMC-FA/RD micelles in the heart and kidney tissues are significantly reduced which might decrease the side effects.The in vivo pharmacokinetics of DOMC-FA/RD and PEG-PAE-DOMC-FA/RD micelles, and RD solution were studied with DAS 2.0. The t1/2β value of DOMC-FA/RD micelles was similar with that of RD solution. While the t1/2β value of PEG-PAE-DOMC-FA/RD were 10-fold larger than the others. For PEG-PAE-DOMC-FA/RD, the values of AUC, CL, and MRT were biggest, slowest and longer of all. The results indicated that PEG-PAE-DOMC-FA/RD micelles could significantly lengthen the retention time of drugs in vivo and had a well-sustained release efficacy, which could be beneficial in treatment of solid tumors.MCF-7 cells were transplanted subcutaneously in mice to evaluate the PEG-PAE-DOMC-FA/RD, DOMC-FA/RD and RD solution on tumor cells in vivo. Tumor volume inhibition was measured and the results indicated that PEG-PAE-DOMC-FA/RD showed a better anticancer effect than DOMC-FA/RD and RD solution. During the treatment period, the body weight of the tumor-bearing mice in all the groups were not decreased, which confirmed the safety of the RD-loaded micelles.This is the first report on the preparation of FA-decorated pH-sensitive micelles as the carrier of RD. PEG-PAE-DOMC-FA micelles, which were implemented with FA-mediated cancer cell targeting and pH-triggered drug releasing properties, world be promising carriers for the specific delivery into cancer sites. Our studies contribute to the development of tumor-targeted delivery of the insoluble anticancer drugs, and play a very important role in clinical application of the NCE-riccardin D. |
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