| ObjectiveHydroxycamptothecin-phospholipid complex(HCPT-PC)was prepared and its physicochemical properties were characterized.Preparation and evaluation erythrocytes(ERs)loaded hydroxycamptothecin-phospholipid complex(HCPT-PC)as a novel drug carriers in vitro and in vivo.Methods1.Preparation and characterization of HCPT-PCThe particle size and morphology of HCPT-PC were characterized by malvern particle size potentiometer,scanning electron microscopy(SEM)and transmission electron microscopy(TEM).Its composite mechanism was investigated by X-ray powder diffraction and infrared spectroscopy.The solubility and antitumor activity were also investigated.2.Preparation and characterization of HCPT-PC-ERsHCPT-PC-ERs were prepared by hypotonic preswelling technique.The entrapment efficiency was used as an index to study the effect of the initial dose of the drug,the volume fraction of the drug and the red blood cell and the incubation time in the hypotonic osmotic loading to obtain a better package conditions.The cell recovery and encapsulation efficiency were measured under the best conditions.Morphological changes were examined by scanning electron microscopy.Transmission electron microscopy and confocal laser scanning microscopy were used to verify the presence of HCPT-PC in red blood cells.The changes of blood hematological parameters and osmotic fragility were investigated by hematology analyzer and osmotic pressure analyzer before and after erythrocytes were loaded.3.In vitro evaluation of HCPT-PC-ERsInhibitory effect of MTT on the growth inhibition of hepatoma cells in vitro was evaluated.The in vitro release behavior was investigated also.4.Pharmacokinetics of HCPT-PC-ERsThe concentration of HCPT in plasma of SD rats was determined by HPLC method and the method was validated.The pharmacokinetics of SD rats was studied and the pharmacokinetic parameters were calculated.5.In vivo distribution of HCPT-PC-ERsPharmacodynamics of KM mice bearing H22 hepatoma cells was studied.6.Pharmacodynamics evaluation of HCPT-PC-ERs in H22 liver tumor miceThe distribution of drugs in vivo was studied by small animal imaging.Results1.Preparation and characterization of HCPT-PCThe particle size of HCPT-PC was 145.08±18.37nm.Scanning electron microscopy and transmission electron microscopy revealed that HCPT-PC was uniformly distributed with a spherical shape.X-ray powder diffraction indicated that HCPT changed from crystalline to amorphous in HCPT-PC.Fourier transform infrared spectroscopy showed that there was a weak interaction between HCPT and PC.The solubility of HCPT-PC in water,PBS,ethanol and n-octanol was 21.91,20.36,1.42 and 6.32 times that of HCPT,respectively.The LogP of HCPT,physical mixtures and HCPT-PC in n-octanol/water systems were 2.39,2.34 and 2.03,respectively.2.Preparation and characterization of HCPT-PC-ERsBased on the hypotonic preswelling technique,HCPT-PC-ERs were successfully prepared by incubating with erythrocytes at a 1:1 ratio of 1 mg/ml for 30 min.76.15±6.72%survival rate,169.01 ± 5.26?g drug loading and 61.79 ±8.45%encapsulation efficiency were obtained.The shape of the erythrocytes changed from double-concave into spherical before and after drug administration.SEM and CLSM confirmed that HCPT-PC was successfully captured in ERs.The mean cell volume(MCV)values of HCPT-PC-ERs were significantly increased after drug loading,while the mean cell hemoglobin(MCH),the mean cell hemoglobin concentration(MCHC).The osmotic fragility decreased compared with normal erythrocytes and Sham-erythrocytes(Sham-ERs)at the osmotic pressure of 0.45%NaCl and 0.5%NaCl.3.In vitro evaluation of HCPT-PC-ERsAfter treated with HepG2,SMMC-7721 and H22 cells for 48 and 72 hours,the IC50 values of HCPT-PC were 3.57,11.14,2.79,37.26,21.23 and 24.49 times,the IC50 values of HCPT-PC-ERs were 2.05,5.21,3.04,1.42,2.53,3.11 times of HCPT,respectively.HCPT-PC-ERs showed a rapid release of 43 percent of HCPT-PC within the first 15 min,and the other 57 percent was retained inside erythrocytes for more than 36 h.4.Pharmacokinetics of HCPT-PC-ERsPharmacokinetic results showed that the commercially available HCPT was rapidly eliminated in vivo and almost no HCPT was detected after 4 h.The HCPT-PC-ERs group can delay the action time of HCPT,and the HCPT can be detected at 24h showing a sustained release effect.The distribution half-life,elimination half-life,the area under the curve and the mean residence time of HCPT-PC-ERs were 2.78,4.72,3.71 and 2.03 times that of HCPT,and HCPT-PC-ERs have obvious enhanced permeability and retention effect.5.In vivo distribution of HCPT-PC-ERsThe results of live imaging showed that the side effects of heart,lung and thyroid are lower than those of free Cy7-HCPT.HCPT-PC-ERs group was more.likely to accumulate in the liver and spleen than the free Cy7-HCPT and maintained a high level within 12 hours.6.Pharmacodynamics evaluation of HCPT-PC-ERs in H22 liver tumor miceThe results of pharmacodynamics showed that HCPT group and HCPT-PC-ERs group showed significant anti-tumor effect compared with the saline group.The tumor inhibition rates of HCPT and HCPT-PC-ERs were 68.95±7.18%and 89.26 ±1.47%.ConclusionsA high composite rate of HCPT-PC was prepared to overcome the problem of poor water solubility and provide the necessary basis for subsequent drug loading.Hypotonic preswelling technique was used to prepare HCPT-PC-ERs.The hematological parameters,osmotic fragility and morphological changes were within the acceptable range.In autologous plasma,the release of HCPT-PC-ERs in vitro showed rapid release early,late slow release characteristics.The bioavailability of HCPT-PC-ERs was 3.71 times that of HCPT.Cy7-HCPT-ERs are more abundant in the liver and spleen than the free Cy7-HCPT,and the side effects of the heart,lung and thyroid are lower.HCPT group and HCPT-PC-ERs group showed significant anti-tumor effect,HCPT-PC-ERs group had greater tumor inhibition than HCPT.HCPT-PC can be loaded into erythrocytes under appropriate loading parameters.Drug-loaded erythrocyte can be targeted to the liver to play its therapeutic role.The usage of erythrocytes as a novel drug carrier deliver HCPT-PC may be an effective approach for liver cancer targeting. |
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