| 1.PurposeIn this project,we prepared the magnetic ultrasound-sensitive liposomes of chlorin e6?Ce6?in accordance with the expected requirements,and established a subcutaneous tumor model of human lung adenocarcinoma with SPCA-1 in BALB/c nude mice.The microdialysis technique was used as the sampling method.Hydroquinone e6 magnetic-acoustic-sensitized liposome preparations were used for local pharmacokinetic studies of tumors to evaluate their localization and acoustic susceptibility to tumors,and to compensate for the inability of traditional blood drug concentration methods to scientifically evaluate tumor-targeting agents.At the same time,the overall pharmacokinetics of the preparations in rats was studied based on the blood drug concentration method and blood microdialysis method to fully describe the in vivo process and target distribution characteristics of the drugs,to further study the tumors related to the sonodynamic therapy technology.Targeted drug release system laid the foundation.2 Methods2.1 The establishment of Ce6 analysis methodUltraviolet spectrophotometry and high-phase liquid chromatography were used to determine the content of Ce6 and the methodological investigations were conducted.2.2 Preparation and quality evaluation of Ce6 magnetic ultrasound-sensitive Liposomes2.3 The overall pharmacokinetics of Ce6 magnetic sound-sensitive liposomes in rats.The stability of microdialysis recovery of blood probe Ce6 in vitro and in vivo was investigated within 12 hours.The blood concentration and blood microdialysis were used to compare the whole body of Ce6 magnetic ultrasound-sensitive Liposomes and Ce6 solution in rats.The pharmacokinetics process was studied and the relevant drug-time curves were plotted.The pharmacokinetic parameters were calculated and compared using DAS 2.1.1software.2.4 Local Pharmacokinetics of Tumor-bearing Nude Mice with Ce6 magnetic ultrasound-sensitive LiposomesA subcutaneous tumor model of human lung adenocarcinoma of SPCA-1 in BALB/c nude mice was established;a linear probe was implanted into the subcutaneous tumor tissue of the tumor-bearing nude mice to perform local pharmacokinetics of the tumor.The pharmacokinetics test was divided into four groups?A.Ce6solution External magnetic field group,B.Ce6 magnetic ultrasound-sensitive Liposomeslipidwithoutmagneticfieldgroup,C.Ce6magnetic ultrasound-sensitive Liposomes without magnetic field group;D.Ce6 magnetic ultrasound-sensitive Liposomes plus magnetic field ultrasound group?.Intraperitoneal injections were administered at a dose of 10 mg.kg-1?calculated as Ce6?.Modified Ringer's solution was used as the perfusate.The flow rate was 2?L·min-1.The in vivo recovery was measured by reverse dialysis.HPLC measurements were performed.The drug concentration was calculated and the drug curve was plotted.The pharmacokinetic parameters were calculated using software DAS 2.1.1.3 Results3.1 The establishment of Ce6 detection methodThe Ce6 magnetic ultrasound-sensitive liposomes were prepared by film dispersion method.The entrapment efficiency,particle size,zeta potential and drug release properties sensitive to ultrasound in vitro of prepared Ce6magnetic ultrasound-sensitive liposomes before and after lyophilization were determined to evaluate the quality of the preparation.HPLC conditions were:Agilent 1260 HPLC system,DAD detector;Detectionwavelength was 403 nm;Column temperature was 25°C;Column was WondaSil C18-WR column?4.6 mm×150 mm,5?m?;The phase was acetonitrile-0.2%phosphoric acid water?50:50?;the flow rate was 1 mL·min-1.Under this chromatographic condition,peaks of Ce6 modified Ringer's and Ce6-containing plasma samples were sharp and symmetrical with peak times of 5.78 and 6.66 min,respectively,and good separation of impurities,linear range,detection limit,and quantitation limit.All meet the experimental requirements.3.2 Preparation and Quality Evaluation of Ce6 magnetic ultrasound-sensitive LiposomesThe entrapment efficiencies of Ce6 magnetic ultrasound-sensitive Liposomes prepared by film dispersion method before and after freeze-drying were 92.21±1.13%and 91.89±0.99%.respectively,and the particle sizes before and after lyophilization were 157.90±3.66 nm and 177.63 nm,respectively.The zeta potentials were-20.70±0.92 mV and-7.77±0.85 mV,respectively.At the ultrasonic frequency of 1.0 MHz,the ultrasound intensity was 2.0 W/cm2,and the ultrasound time was within 2.5,5,8 and 12 minutes.From 2.5 to 8minutes,the cumulative release in vitro of Ce6 magnetic ultrasound-sensitive liposomes gradually increased with the increase of the ultrasound time,and the cumulative release in vitro from 8 to 12 minutes did not increase significantly.3.3.Pharmacokinetics of Dihydroquinone-e6 Magnetic Acoustic Liposomes in RatsWhether it is blood-microdialysis or plasma-drug concentration method,after intraperitoneal or caudal vein injection,the pharmacokinetic chamber models of Ce6 magnetic ultrasound-sensitive liposomes and Ce6 solution in rats were all fitted to the two-compartment model.When the dosage form administration method are the same,the AUC and Cmax of the group of blood-microdialysis method are slightly larger than those of the blood-drug concentration method group,and it is considered that the blood drug concentration method requires the continuous blood collection of rats which can lead to the blood volume reduce;the CL,MRT of group of Blood-drug concentration method is greater,it may be caused by anesthesia(the study blood-microdialysis method is carried out in anesthetized rats,their body metabolism is usually weakened;the other parameters are not very obvious differences,indicating microdialysis technique has little effect on the physiological state of the animal itself,and its operation is slightly more difficult than the implantation of the blood probe.The sample can be directly processed into the the high performance liquid chromatography or the liquid chromatography and mass spectrometry without further processing,and the sample collection process even can be automatically collected by an automatic sampler,which further verified the feasibility and superiority of blood-microdialysis in pharmacokinetic study in rats.Given the same dosage form?Ce6 solution or liposome formulation?,there is a relatively short period of absorption during intraperitoneal injection,and the maximum blood concentration can be reached quickly after tail vein administration?without direct absorption into the blood?.Relative to intraperitoneal injection,its Cmax is larger,its bioavailability is higher,its distribution phase half-life t1/2?and the elimination phase half-life t1/2?are shorter,its in vivo retention time MRT0-?is shorter,and it can be metabolized more rapidly.In the same administration method?tail vein injection or intraperitoneal injection?,the half-life t1/2?of the elimination phase of Ce6 liposomes by blood-drug method?t1/2?of Ce6 liposome is 1.24 times that of the Ce6 solution1.48 times?is longer,the retention time in the body?MRT0-?of Ce6 liposomes is 1.50 times that of the solution agent?is also longer,indicating that the prepared Ce6 liposome can prolong the time of the drug in vivo and has a good sustained release effect.3.4 Local Pharmacokinetics of Dihydrohomphine e6 Magnetic Acoustic Liposomal TumorsThe distribution of A,B,C,and D drugs in the local pharmacokinetics of the tumors all met the two-compartment open model.The Tmax of each group was 2.6±0.279,1.667±0.236,1.467±0.183,1.734±0.149 hours,respectively.Compared with the other three groups,the peak time was slower,indicating that the distribution of Ce6 magnetic sound-sensitive liposomes is faster.The peak time of C group was faster than that of B group,which indicated that magnetic sound-sensitive liposomes could accumulate to tumor tissue more quickly under the action of magnetic field.The lipid half-life t1/2?in the liposome in vitro magnetic solution group was longer than the liposome group,and the elimination half-life t1/2?was much shorter than other groups,and the in vivo retention time MRT0-?and AUC0-?were the minimum.This shows that liposome preparations allow faster drug distribution,slower elimination,increased retention time in vivo?sustained release?,and higherbioavailability.The liposome-enhanced magnetic field group was smaller than the liposome-free magnetic field group,t1/2?was smaller,t1/2?,MRT0-?value,and AUC0-?were greater,indicating that Ce6 magnetic sound-sensitive liposomes have good magnetic targeting Sex,plus a magnetic field makes the distribution faster,slower elimination,increased residence time in the body,and increasedbioavailability.Since the Tmax of the applied magnetic field group was 1.467 hours,the local ultrasound treatment of the liposome plus magnetic field ultrasound group was selected after 1 hour of administration,and the obtained pharmacokinetic parameter Cmax was larger than that of the lipid extracorporeal magnetic field group,and thereafter Within 4 hours,the drug concentration was greater in the lipid plus in vitro magnetic field group,and its t1/2?and MRT0-?values were slightly smaller,indicating that ultrasound promotes drug release?burst release?in liposomes.From the results of local drug concentration-time curves of nude mice tumors,the tmax?1.467 hours?of Ce6 magnetic ultrasound-sensitive can be used as the ultrasound irradiation time point for tumor-bearing nude mice.The results of local pharmacokinetic parameters of tumors in nude mice showed that Ce6magnetic ultrasound-sensitive have good sustained-release,magnetictargeting,and acoustic sensitivity.4 Conclusion4.1 The use of UV spectrophotometry to meet the requirements of body encapsulation rate,magnetic test and other content determinationrequirements;Under the established chromatographic conditions,usinghigh-performance liquid chromatography,whether it is blood concentration or microdialysis,in a certain concentration range Internally,the concentration of Ce6 has a good linear relationship with its peak area.The specificity,precision and stability of the method can meet the detection requirements of this study.High performance liquid chromatography can be used to quantitatively and accurately analyze Ce6.4.2 The entrapment efficiency,particle size,and electric potential of the liposomes prepared by the film dispersion method were all changed little after being freeze-dried,and their values were in line with the expected requirements.The in vitro release assay showed that Ce6 magnetic ultrasound-sensitive were significantly slowed down.Release and burstiness?ultrasonic excitation?.4.3 Differences between blood microdialysis and conventional blood drug concentration methods,the differences in relevant pharmacokinetic parameters are not significant,confirming the feasibility and reliability of the microdialysis method applied to the in vivo pharmacokinetics of Ce6;pharmacokinetic parameters of the two methods It was shown that Ce6 magnetic sound-sensitive liposomes can prolong the time of the drug in the body and have a good sustained-release effect.4.4 BALB/c nude mouse SPCA-1 human lung adenocarcinoma cell subcutaneous tumor model has a short model cycle and high tumorigenesis rate.It is suitable for microdialysis studies for local pharmacokinetics of tumors.4.5 The self-produced liposome of ehonnet e6 magnetic sound-sensitive liposome can rapidly accumulate to the target site under the condition of external magnetic field,and has high tumor targeting.After the radiosensitization of the liposome plus magnetic field Tmax,the local drug concentration of the tumor was significantly increased,indicating that the Ce6 liposome has good sonosensitivity?ultrasound can promote the release of Ce6 liposomes?.4.6 This project established a general research method of tumor local pharmacokinetics of a novel antitumor drug targeted drug delivery system based on microdialysis sampling technology,which laid the foundation for future research on local pharmacokinetics of tumors combined with sonodynamic therapy. |
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