Using erythrocytes in drug delivery is a promising technology and is constantly expanding. To standardize the process of incorporating biologically active components (BAC) into erythrocytes automated devices are used. There are only two such devices from Erytech Pharma and Erydel companies around the world that are actively used at the present time. Each of them has its own limitations which reduce the efficiency of the erythrocyte loading process.

Aims

The aim of our work was to create an automated device for the incorporation of BAC into erythrocytes providing a high efficiency of incorporation.

Methods

The device is based on the flow dialysis method using commercial hemodialyzers and build-in automated cell processor for erythrocytes washing. The installation allows using both whole blood and pre-washed erythrocytes as a starting material. The process includes the stages of erythrocytes washing, concentrating the suspension of washed erythrocytes, hypoosmotic lysis of erythrocytes using flow dialysis and subsequent sealing of the obtained erythrocytes containing BAC by restoring the tonicity of the medium. The yield of cells, as well as relative (R) yield of BAC encapsulation were calculated and compared with the corresponding yields of known methods (where R was calculated as the ratio of the enzyme activity in the loaded erythrocytes to the enzyme activity in the initial suspension of erythrocytes)

Results

An automatic device was developed in which all stages of the encapsulation process such as washing and concentrating erythrocytes, dialysis, adding a drug to a suspension, incubating a suspension of erythrocytes are combined into a single device that works without operator intervention using software that controls pumps and valves. We used a standard erythrocyte washing system (APC 215) which was connected to our device with special lines. Distinctive features of this device are the erythrocytes concentration stage before the dialysis procedure, ensuring minimal dilution of the suspension, as well as creating a certain pressure (90-180 mm Hg) in the dialyzer during dialysis and concentrating stages. All this allowed us to obtain a higher efficiency of drug encapsulation relative to the existing analogues of this device. The relative efficiency of the enzyme encapsulation (R) was 51.8 ± 9.6% for L-asparaginase in our developed device, 30.0 ± 3.0% for Na-dexamethasone-21-phosphate (Erydel) and 29.8 ± 2.4% for L-asparaginase using device of Erytech Pharma. The cell yield was very similar for all the studied devices.

Conclusions

А prototype of an automatic device for loading drugs into erythrocytes with a high encapsulation efficiency was created. This high efficiency was reached both due to a selection of the optimal process conditions and due to the presence of concentration stage after erythrocytes washing. Pilot clinical trials of asparaginase-loaded erythrocytes obtained by this device were started.