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RESEALED ERYTHROCYTES: IN-VITRO CHARACTERISATION AND NOVEL SYSTEMS

M. Anitha Sri,Y11MPH448, I/II M. Pharmacy, II Semester, Industrial Pharmacy, Chalapathi Institute Of Pharmaceutical Sciences.

In-vitro Characterisation
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3. 4. 5. 6. 7. 8. 9. 10.

Drug Content In-vitro drug and Hb release Osmotic Fragility Osmotic Shock Turbulence Shock Morphology % Cell Recovery Entrapped Magnetite ESR ZSR

Drug Content:
Packed loaded cells (0.5mL)

Deproteinised with Acetonitrile (2mL)

Centrifuged at 2500-3000 rpm for 10 min.

Clear supernatant is analyzed for drug content

Retaining the entrapped magnetite:

Magnet placed adjacent to the base of centrifuge tube.

In-vitro Drug Content


Incubation Periodic withdrawl of samples

Deproteinisation with Acetonitrile


Centrifugation Supernatant was assayed for drug

Incubation of loaded normocytes at 37 0C in PBS (pH 7.4) at 50% haematocrit in a metabolic rotating wheel incubator.

Metabolic Shaker Incubator

0.8 spectropore membrane filter is fitted to a hypodermic syringe to withdraw the samples.

Hypodermic syringe with a membrane filter.

In-vitro Hb release:
5 % Hematocrit in PBS

Withdrawl of samples using 0.45 filter


Deproteinisation with methanol Centrifuge

Supernatant assayed for Hb at 540 nm

Osmotic Fragility
Exposing to solutions of varying tonicities

Isotonic to hypotonic
Centrifuge Supernatant assayed for drug and Hb

Based on resistance of cells to haemolysis

Change of shape due to osmotic imbalance

Osmotic Fragility

Osmotic Shock
Sudden exposure to non-isotonic environment Incubation of resealed erythrocytes

Centrifugation
Supernatant assayed for Hb release

Turbulence shock
Measure of simulating destruction of loaded erythrocytes during injection
Loaded cells passed through hypodermic needle

Flow rate 10 mL/min


Withdrawl of suspensions Centrifuge Supernatant assayed for Hb

Morphology & % cell recovery


Phase contrast optical microscopy Transmission electron microscopy Scanning electron microscopy

Haemocytometer

Magnetite concentration
Magnetite loaded cells treated with HCl

Heated at 60 0 C for 2 hrs


+ 20% w/v trichloroacetic acid Centrifuge

Supernatant assayed for magnetite

ESR & ZSR


ESR is the estimation of suspension stability in plasma Depends on the no. and size of RBC and other plasma proteins Normal Blood ESR: 0 to 15 mm/hr ZSR is a measure of the closeness with which RBC will approach each other

NOVEL APPROACHES
Erythrosomes

Nanoerythrosomes

Biotinylated erythrocytes

Erythrosomes:

Erythrosomes are specially engineered vesicular systems in which chemically cross-linked human erythrocyte cytoskeletons are used as a support upon which a lipid bilayer (phosphatidyl choline)is coated.

Nanoerythrosomes
Normal erythrocytes
Depletion of Hb

Preparation of erythrocyte ghost


Extrusion / Sonication / Electrical Breakdown

Nanoerythrosomes

Biotinylated erythrocytes
Biotin molecules are attached to the amino groups present on the RBC. Methods: 1. Direct interaction. 2. Using spacer arm.

References:
1. S.P. Vyas, R. K. Khar, Targeted and controlled drug delivery, Novel carrier systems, Pg No. 387 to 416. 2. Advances in controlled and Novel Drug Delivery, N. K. Jain, Pg No. 332 to 360. 3. Controlled and Novel Drug Delivery, N. K. Jain, Pg No. 256 to 291. 4. Progress in controlled and Novel Drug Delivery Systems, N. K. Jain, Pg No. 387 to 416.