Preparation And Characterization Of Vincristine-loaded Intact Human Erythrocytes

Part I Establishment of a modified method to prepare Vincristine-loaded intact human erythrocytesIn this part of study, a hypotonic preswelling and isotonic resealing method for preparation of drugs-loaded intact human erythrocytes was modified. We evaluated this modified method using Vincristine (VCR) as a model drug, and proved the feasibility of intact human erythrocytes as VCR delivery system. We explored the loading parameters, including loaded amount, efficiency of entrapment, and erythrocyte recovery rate, plotted the relationship curve of VCR-loaded amount in erythrocytes against concentration of VCR working solution, and summarized its calculation formula.Washing-packed erythrocytes were mixed with series of concentrations of hypotonic buffers at 1:4 of volume ratio (erythrocyte vs hypotonic buffer) for different incubation time. We observed that when washing-packed erythrocytes were suspended in hypotonic swelling buffer for 20min, erythrocytes swelled to the most and without marked visible hemolysis. Thus, we determined the concentration of hypotonic swelling buffer and the ratio of volume (erythrocyte vs hypotonic swelling buffer) and incubation time as the optimal erythrocytes hypotonic swelling system.The optimal encapsulation procedure of VCR was determined based on the loaded amount in erythrocytes: first, preswelling-packed erythrocytes were mixed with the same volume of VCR that dissolved in 0.9% or 0.6% saline respectively for the same time. The loaded amount that preswelling-packed erythrocytes mixed with 0.6% saline of VCR, which was 49.59g in erythrocytes, was significantly higher than other group. Thus, 0.6% saline was selected as the optimal dispensation solution of VCR; next, preswelling-packed erythrocytes were incubated with 0.6% saline of VCR for different time. The optimal VCR-loaded amount was displayed when erythrocytes were mixed with above VCR-solution for 20min; third, we mixed preswelling-packed erythrocytes withdifferent concentration of VCR in 0.6% saline for 20min, and then plotted the curve of the loaded amount in erythrocytes against VCR concentration. We found that the VCR-loaded amount of erythrocytes was linearly depended on VCR concentrations in the range of 0.05 to 2 mg/ml, followed the formula of y=0.0982x-2.4658, and correlation coefficient of which was 0.9961. The percentage of VCR in intracellular fluid of erythrocytes was (42.14+1.23)% of total VCR put in. Combined with VCR dosage in clinic application and our aim in further study, we determined 0.5mg/ml as the working concentration of VCR; finally, based on the amounts and mean corpuscular volume of whole blood, washing-packed, preswelling-packed, and VCR-loaded erythrocytes, we demonstrated the percentage of total VCR-loaded amount in erythrocytes was (61.42+3.69)%, and erythrocytes recovery rate was (92.15 + 3.82)%.The modified preparation method of drugs-loaded erythrocytes included following amelioration: first, 0.6% saline replaced routine distilled water as drug dispensing solution for its osmotic breakage to erythrocytes; next, it is difficult to observe the result that the point of erythrocyte lysis when preswelling-packed erythrocytes are mixed with a relatively small portion of drug water solution using traditional method repeatedly. The modified method make it easy to judge using equal volume of mixture system of preswelling-packed erythrocytes and drug saline; third, we resealed the drugs-loaded erythrocyte through 37 water-bath. Our modified method is easy to perform, and displayed certain stability in preparation of VCR-loaded erythrocytes. In the range of limited clinic dosage, the VCR-loaded amount in erythrocytes showed positive linear relationship with VCR concentrations, indicating it was a controllable method.Part The characterization of VCR-loaded intact human erythrocytes prepared by modified methodTo measure VCR-releasing rate, osmotic fragility, and stability after preservation of VCR-loaded erythrocytes prepared by modified method; to explore the methods and conditions of...