Study On Hydroxycamptothecin Long-circulating Nanoparticles And Its Amphiphilic Carriers

Background & objectiveSince 1970s, Birrenbach introduced the concept and preparative method of nanoparticle firstly, the study of the pharmaceutical preparation has entered into a new micro realm——nanometric praeparatum. Compared to routine drugs, nanometric praeparatums have many good characters such as large specific surface area, high surface reaction activity, plenty of active centers, strong adsorbability and so on. They can also change the intracorporal distribution of the medicine to increase the dose distribution in target organs and raise therapeutic effects. However, after the intravenous injection of general nano-medicament, a variety of components (such as apolipoprotein, complement protein C, etc) in plasma will adsorb on the surface of nanoparticles to make it be easily identified by the phagocytic cells. This is just the process of body opsonization, the nanoparticles can be phagocytosed by mononuclear phagocyte system and rapidly cleared from the blood. Therefore, how to avoid MPS identifying and phagocytosing the nanoparticles is the key for transmiting them to other organs or tissues. It is found that nanoparticles'surface properties (such as lipophilic strength, surface potential value, etc.) are closely related to the adsorption of opsonin proteins on their surface. Generally speaking, the stronger the surface lipophilicity is, the lower the value of surface potential is, the easier that opsonin proteins absorb nanoparticles. This shows that the surface characteristics of nanoparticles are extremely important for preparing long-circulating nanoparticles. Currently, the study of nanoparticles for surface modification mainly focuses on two aspects:(1)To coat the surface of nanoparticles with hydrophilic polymers or surfactants; (2)To prepare amphiphilic polymer carrier materials containing hydrophilic fragments which contain PEG, PEO, Poloxamer, Poloxamine, Polysorbate-80 and Briji-35,etc. They can all form hydration protective clouds on the surface of nanoparticles' lipophilic cores and have certain three-dimensional steric effects, which can inhibit opsonin protein absorbing on the nanoparticles surface. Therefore, the nanoparticles have "stealths" feature for being not easily identified by MPS to achieve the purpose of long-circulating.Although the nanoparticles coated with surfactants (such as poloxamer, etc.) have long-circulating feature, in vivo micro-environment, some scholars believe that the vander wale force between blood materials and coating materials may easily lead the coated layer to fall off in the vivo circulation. As a foreign antigen, the deciduous exogenous substances may have the potential toxic effects on the human body and it is still in disputes. In the synthesis of amphiphilic polymer carrier, the copolymers reported are almost only the AB-type or ABA-type currently, which contain hydrophilic segments (such as PEG, PEO, etc.) and carrier materials (such as PEG-PLGA, PEG-PLA, PEG-PHDCA, etc.). The copolymers are mostly made through the polymerization reaction between PEG (or Monomethyl-substituted PEG molecule, PEO) and lactic acid monomers (or cyano-alkyl ester monomer, etc.). The reaction conditions are strict, such as requiring inert gas protection, sealing reaction tube, adding initiators, etc.This study chose poly (ethylene glycerol) (PEG) and poly(s-caprolactone) (PCL) of appropriate molecular weight as raw materials, succinic anhydride for the bridging agent, synthesized the amphiphilic copolymer carrier materials with different numbers of repeating units of PEG and PCL. PCL is semi-crystalline polymer material, as the lipophilic part of amphiphilic copolymer carriers, it can not only store the lipophilic drug, but also be beneficial to the stability of nanoparticles' morphology by its crystalline structure. The applications of PCL has a long history, such as the carrier material of long-acting contraceptive implant in vivo, tissue engineering materials, etc.; PEG is safe and non-toxic, and has been approved by FDA for clinical application. The synthetic reaction in this study is mild, simple and the reaction yield is high.10-hydroxycamptothecin (HCPT) is a kind of micro amount alkaloids extracted from Camptotheca acuminate Decaisne, and is one of the specific DNA topoisomerase I inhibitors. It is widely used to cure liver cancer, stomach cancer, leukemia and many malignant tumors. However, its clinical applications are confined by its special physico-chemical properties (for example, it is insolubilize in water and difficultly dissolves in lipids, the lactone band is also instable, etc.), so its curative effects decrease and the intracorporal half life is too short. This study choose it as model drug, and the amphiphilic PEG-PCL multiblock copolymers as the carrier material, to prepare long-circulating nanoparticles. Moreover, HCPT-SSLN (HCPT semisolid lipid nanoparticles) coated with solid and liquid lipids acted as the control group, their long-circulating abilities were compared by in vitro drug release experiment and in vivo pharmacokinetics tests. The goal of this study is to find a novel carrier for long-circulation and prepare long-circulating HCPT-NPs formulation. Materials and methods1 Preparation and characterization of HCPT-SSLNHCPT-SSLN was prepared by the method of "emulsion evaporation at a high temperature and solidification at a low temperature", using solid & liquid lipids as carrier materials, soya lecithin as surfactant. According to the morphology, particle size and entrapment efficiency, the affected factors in the preparing technology, including "types of surfactants", "molar ratio of solid/liquid lipids", "ratio of HCPT/lipids", "concentration of surfactants", "stirring time", "stirring velocity", etc. were investigated. On this basis, "ratio of HCPT/lipids", "molar ratio of solid/ liquid lipids" and "concentration of surfactants" were selected as major factors to make L9 (34) orthogonal design for optimization of formulation and technology. The zeta potential, particle size and size distribution (polydispersion) of HCPT-SSLN were determined on a Malvern Zetasizer HSA 3000; the morphology was observed by transmission electron microscopy. In order to control the quality of HCPT-SSLN, the methods for determination of open-loop form of HCPT quantity by UV spectrophotometry at 414nm and for determination of the entrapment efficiency and drug loading of HCPT-SSLN by sephadex gel filtration chromatography-UV were established. Different cryoprotectants were investigated to prepare the lyophilized HCPT-SSLN powder, and then the stability of the NPs powder was studied. By the comparison of HCPT sodium injection, the animal acute toxicity of HCPT-SSLN was conducted using kunming mice by tail intravenous administration.2 Synthesis of multiblock PEG-PCL copolymerThe multiblock polyethylene-polycaprolactone copolymer was synthesized, which PEG and PCL were used as starting meterials, succinic anhydride acted as cross-linking agent, DCC and DMAP as condensation agent and catalyst, respectively. Then FT-IR,1H-NMR,GPC,DSC,XRD analyses were used to characterize the copolymers. The cytotoxicity of the copolymers was evaluated with L929 cell line through MTT and LDH methods.3 Preparation and characterization of HCPT-PEG-PCL-NPsHCPT-PEG-PCL-NPs were prepared by co-solvent evaporation method, which copolymer was the carrier material, Span60 and Poloxamer 188 was the emulsifier and stabilizer. According to the morphology, particle size, entrapment efficiency and drug loading, the affected factors in the preparing technology, including "molar ratio of Span/PEG-PCL", "dosage of HCPT", "molar ratio of Span/Poloxamer", "stirring velocity", "stirring temperature", "volume of water phase", "hydration time", etc. were investigated. On this basis, "types of PEG-PCL", "dosage of HCPT", "molar ratio of Span/Poloxamer" and "molar ratio of Span/PEG-PCL" were selected as major factors to make L18(34) orthogonal design for optimization of formulation and technology. The zeta potential, particle size and size distribution (polydispersion) of HCPT-PEG-PCL-NPs were determined on a Malvern Zetasizer HSA 3000; the morphology was observed by transmission electron microscopy. In order to control the quality of HCPT-PEG-PCL-NPs, the methods for determination of lactone form of HCPT quantity by HPLC analysis in vitro at 384nm and for determination of the entrapment efficiency and drug loading of HCPT-PEG-PCL-NPs by centrifugal ultrafiltration—HPLC were established. Different cryoprotectants were investigated to prepare the lyophilized HCPT-PEG-PCL-NPs powder, and then the stability and XRD analysis of the NPs powder were studied.4 In vitro drug release study of HCPT-NPsIn vitro drug release studies were carried out as follows. Drug-loaded nanoparticles suspensions and aqueous solution of HCPT (the control) were placed in a dialysis membrane bag with molecular weight cut-off 3500g/mol, tied, and dropped into 250mL of a phosphate buffer solution media (pH=7.4). The entire system was kept dark and at 37℃with magnetic stirring at 100r/min. At predetermined time intervals,3 mL of aqueous solution was withdrawn from the release media. Meanwhile, an equal volume release medium was added. Then the drug content of the sample was assayed by HPLC. Furthermore, the drug release mechanisms of HCPT-NPs were studied with methematics modles including Weibull distribution model, Ritger-Peppas modle, etc.5 In vivo pharmacokinetic study of HCPT-NPs in ratsSix experiment groups including HCPT sodium injection, HCPT-SSLN and 4 kinds of HCPT-PEG-PCL-NPs formulations with the dosage of 5mg/kg were administrated to 54 Wistar rats (n=9) via the tail vein injection of a single dose. At predetermined time intervals, blood was taken and HCPT in plasma was extracted with ethyl acetate, and then measured by HPLC. The method for determination of lactone form of HCPT quantity by HPLC analysis in plasma was established, the plasma-drug concentration-time curve was drawn and the pharmacokinetic parameters were calculated by PKSolver 2.0 software. The long-circulating effects of different HCPT-NPs were evaluated and compared on order to choose the best long-circulating formulation and carrier material for HCPT.Results1 Preparation and characterization of HCPT-SSLNIt was showed that all the factors in single-factor experiment could influence the particle size and entrapment efficiency of HCPT-SSLN. Among them, only "concentration of surfactants" had significant effect on the entrapment efficiency of HCPT-SSLN. The optimal preparation parameters were as follows:ratio of HCPT/ lipids was 1:20, molar ratio of solid/liquid lipids was 2:1, concentration of surfactants was 5%, the velocity of oil phase into aqueous phase was 2.0mL/min, stirring temperature, time and velocity of emulsion evaporation was 75℃,2 h, 800 r/min, respectively. The average particle size, polydispersity, zeta potential, pH, entrapment efficiency and drug loading of the NPs was 130.5nm,0.19, 33.1mV,6.29,79.19%,2.51%, respectively. From the observation of transmission electron microscopy, the NPs exhibited a regular spherical shape. The regression equation of UV assay was A=0.0681C—0.004 (r=1.000) and the linear range was 0.2～10.0μg/mL. Furthermore, the lyophilized powder of HCPT-SSLN embodied good appearance and redissolving ability, in which 2.5% lactose added as cryoprotectant. After redissolving, the average particle size, polydispersity, zeta potential, pH and entrapment efficiency of the NPs was 138.9nm,0.25,—36.2mV, 6.29,75.47%, respectively. The stability study results showed that HCPT-SSLN lyophilized powder should be sealed and kept under dark at 4℃. In animal acute toxicity test, at the maximum tolerated dose of 214.6mg/kg, no poisonous effect and side effects were found..2 Synthesis of multiblock PEG-PCL copolymerThe results of FT-IR,1H-NMR,GPC,DSC and XRD analyses showed that the multiblock PEG-PCL copolymer synthesized contained fragment PEG and PCL, in accordance with the target substance. The GPC polydispersities of the copolymers was less than 1.1, as there was a narrow molecular weight distribution. The result of cytotoxicity showed that the multiblock PEG-PCL copolymers had good biocompatibility and was in line with biological evaluation criteria. 3 Preparation and characterization of HCPT-PEG-PCL-NPsIt was showed that all the factors in single-factor experiment could influence the particle size, entrapment efficiency and drug loading of HCPT-PEG-PCL-NPs. Among them, "molar ratio of Span/PEG-PCL" and "dosage of HCPT" were major factors and had significant effect. The optimal preparation parameters were as follows:dosage of HCPT was 5mg, molar ratio of Span/PEG-PCL was 20:1, molar ratio of Span/Poloxamer was 3:1,40mL water, stirring velocity was 800r/min, the velocity of organic phase into aqueous phase was 2.0mL/min, hydration time was 2 h, the best carrier material was PEG4000-PCL2000. When using PEG4000-PCL2000, PEG4000-PCL1250, PEG2000-PCL2000, PEG2000-PCL1250 as the carrier material to prepare NPs, the average particle size of NPs in turn were 116.1,110.0,119.9, 99.1nm; the zeta potential were—22.4,—16.9,—33.5,—28.8 mV; the entrapment efficiency were 88.29%,.83.10%,80.67%,77.46%; and the drug loading were 2.96%,2.56%,2.31%,2.14%, respectively. From the observation of transmission electron microscopy,4 kinds of NPs exhibited a regular spherical shape. The regression equation of HPLC assay was A=78.215C—0.498 (r=1.000) and the linear range was 0.05～5.0μg/mL. With different molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the HCPT-PEG-PCL-NPs showed different zeta potential, the higher Mw of PEG, the higher zeta potential was; but the effect of PCL on the zeta potential was on the contrary. Furthermore, the lyophilized powder of HCPT-PEG-PCL-NPs embodied good appearance and redissolving ability, in which 4% trehalose and 4% sucrose used as cryoprotectants. After redissolving, the average particle size, polydispersity, zeta potential and entrapment efficiency of HCPT-PEG4000-PCL2000-NPs was 124.5nm,0.15,—25.2mV,85.24%, respectively. The stability study results showed that HCPT-PEG-PCL-NPs lyophilized powder should be sealed and kept under dark at room temperature. The XRD analysis results indicated that the existence of HCPT in HCPT-PEG-PCL-NPs was nano-amorphous state.4 In vitro drug release study of HCPT-NPsIt was showed that the aqueous solution of HCPT (the control) reached the maxi-mum releaseas the cumulative release percentage was about 80% only in 24h. Compared with it, HCPT-NPs all showed a certain degree of sustained-release characteristics. Moreover, the most significant sustained-release characteristics was from HCPT-SSLN, but its cumulative release percentage tended to be saturation and less than 60% in 96h. With different molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the HCPT-PEG-PCL-NPs showed different release rate and cumulative release percentage, the higher Mw of PEG, the higher the release rate and cumulative release percentage were; but the effect of PCL on the release rate and cumulative release percentage was on the contrary. The release mechanisms of HCPT-NPs were fitted to Weibull modle, and it showed that the drug release process included passive diffusion and matrix-eroded procedure.5 In vivo pharmacokinetic study of HCPT-NPs in ratsIt was showed that there were significant difference among in vivo pharmacokinetic charac-teristics in rats of different HCPT-NPs (P<0.01). The in vivo metabolism of HCPT sodium injection was so fast that its in vivo half-life (t1/2) was only 0.1418h. However, the t,/2 of HCPT-PEG4000-PCL1250-NPs, HCPT-PEG2000-PCL1250-NPs, HCPT-PEG4000-PCL2000-NPs, HCPT-PEG2000-PCL2000-NPs and HCPT-SSLN were 18.07,9.08,5.25,5.14,4.28 times in turn as great as that of the control, which showed all HCPT-NPs prepared had a certain degree of long-circulating effect. The turn of all HCPT-NPs prepared which had stronger long-circulating effect was HCPT-SSLN≌HCPT-PEG2000-PCL2000-NPs≌HCPT-PEG2000-PCL1250-NPs (P>0.05)