resealed erythrocytes

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A Seminar on RESEALED ERYTHROCYTES

• BY
• T.SHIVA
• B.PHARMACY
• KOTTAM INSTITUTE OF PHARMACY

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Introduction

• Among the various carriers used for targeting of
  drugs to various body tissues, the cellular
  carriers meet several criteria desirable in
  clinical applications, among the most important
  being biocompatibility of carrier and its
  degradation products.
• Leukocytes, platelets and erythrocytes have been
  proposed as cellular carrier systems. Among
  these, the erythrocytes have been the most
  investigated and have found to possess great
  potential in drug delivery.

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Advantages

• They are the natural product of the body which
  are biodegradable in nature.
• Isolation of erythrocyte is easy and larger
  amount of drug can be encapsulated in a small
  volume of cells.
• The entrapment of drugs does not require any
  chemical modification of the substance to be
  entrapped.
• They are non-immunogenic in action
• They prolong the systemic activity of drug while
  residing for a longer time in the body

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• They protect the premature degradation,
  inactivation and excretion of proteins and
  enzymes
• They can target the drugs within
  reticuloendothelial system
• They facilitate incorporation of proteins and
  nucleic acids in eukaryotic cells by cell
  infusion with RBC.
• A longer life span in circulation as compared to
  other synthetic
• carrier
• Disadvantages
• They have a limited potential as carrier to
  non-phagocytic target tissue.
• Possibility of clumping of cells and dose dumping
  may be there.

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Entrapment methods

• 1) Hypo-osmotic methods.
• Dilution method.
• Dialysis.
• Presswell method.
• Isotonic osmotic lysis method.
• 2) Electrical break down.
• 3) Endocytosis.
• 4) Membrane perturbation method.
• 5) Normal transport.
• 6) Lipid fusion method.

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a. Dilution method

• Population of erythrocytes when exposed to
  hypotonic saline solution (0.4 NaCl) swells
  until it reaches a critical value of volume or
  pressure where membrane ruptures and becomes
  permeable to macromolecules and ions, therefore
  permitting the escape of cellular components.
• One volume of washed erythrocytes could be
  treated with 2-20 volumes of materials to be
  loaded in a hypotonic buffer at 0oC or 5 min.
• Further incubation at 25oC in an isotonic
  solution (0.9 NaCl) reseal them again. This
  method is rapid and simple for low molecular
  weight drugs.
• The entrapment efficiency is 1-8.

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Dialysis method
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Preswell dilutional technique
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• d. Isotonic osmotic lysis
• In order to avoid disadvantages of hypotonic
  hemolysis, efforts were made to develop resealed
  V under isotonic conditions. Hemolysis in
  isotonic conditions can be achieved both by
  physical and chemical means.
• 2. Electrical break down method
• Use of electrolysis to generate desirable
  membrane permeability for drug loading into RBC
  is the basis of this method. In this,
  electrically induced permeability changes at high
  membrane potential differences.

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Electrical breakdown method
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Endocytosis
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4. Membrane perturbation

• Antibiotics such as amphotericin B damage
  micro-organisms by increasing the permeability of
  their membrane to metabolites and ions. This
  property could be exploited for loading of drug
  into erythrocytes. Amphotericin-B was used to
  load erythrocytes with antileukaemic drug
  daunomycin. Amphoyericin-B interacts with the
  cholesterol of the plasma membrane of eukaryotic
  cells causing change impermeability of the
  membrane.

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Membrane perturbation
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5. Normal transport mechanism

• It is possible to load erythrocytes with drug
  without disrupting the erythrocyte membrane in
  any way by incubating the drug and erythrocytes
  for varying period of time. After infusion the
  drug would, exit from the cell following kinetics
  compared to those observed for entry.
• 6. Lipid fusion method
• Lipid vesicles containing drug can be directly
  fused with human erythrocytes leading to exchange
  of lipid entrapped drug. This technique was used
  for loading inositol hexaphosphate into resealed
  erythrocytes. This method gives very low
  encapsulation efficiency (1).

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Characterization of resealed erythrocytes

• 1. Drug contain determination
• To determine drug contain, packed loaded cell
  are de-protenized with acetonitrile after
  centrifugation at 3000 rpm for a fix time
  interval.
• The clear supernatant is assayed for drug
  contain.
• 2. In vitro Drug and Hemoglobin release
• Normal and loaded erythrocytes are incubated at
  370 C in phosphate buffer saline at 50
  haematocrit in a metabolic rotating wheel
  incubator bath.
• The samples are withdrawn with the help of a
  hypodermic syringe fitted with a 0.8 µ
  spectrophore membrane filter. The samples are
  then deprotenized with the acetonitrile and can
  be estimated amount of drug release.

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5. Turbulence shock

• The parameter indicates the effects of shear
  force and pressure by which resealed erythrocytes
  formulations are injected, on the integrity of
  the loaded cells.
• Loaded erythrocytes are passed through a 23
  gauge hypodermic needle at flow rate of 10
  ml/min. After every pass, aliquot of suspension
  is withdrawn and centrifuged for 15 min and
  hemoglobin content leached out are estimated
  spectrophotometrically.

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6. Morphology and percent cellular Recovery

• Phase-contrast optical microscopy, transmission
  electron microscopy and scanning electron
  microscopy are the microscopic methods used to
  evaluate the shape, size and the surface features
  of the loaded erythrocytes.
• Percent cell recovery can be determined by
  assessing the number of intact erythrocytes
  remaining per cubic mm with the help of a
  haemocytometer.

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Applications

• 1. Treatment of lysosomal storage disease
• Resealed erythrocytes have been proposed to
  deliver lysosomal enzymes to lysosomes of the
  erythrophagocytic cells, thus resulting in
  replacement of the missing enzyme. Ex
  ß-glucoronidase, ß-galactoronidase and
  ß-glucosiade.
• 2. Treatment of Gauchers disease
• Gauchers disease is due to accumulation of
  glucocerebroside from catabolised erythrocytes
  and leukocytes in spleen, liver and bone marrow.
  This disease was treated by encapsulating
  glucocerebroside in erythrocyte.

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• 3. Treatment of liver tumors
• Anticancer agents like bleomycin, adriamycin,
  L-asparaginase, doxorobucin and methotrexate are
  encapsulated in erythrocyte to treat hepatic
  carcinomas.
• 4. Erythrocytes as circulating carriers
• Various bioactive agents are encapsulated in
  erythrocytes for their slow release in
  circulation for treatment of parasitic diseases
  in cattle. Ex homidium bromide is encapsulated
  in erythrocytes to treat trypanosomiasis.

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• 5. In enzyme delivery
• To eliminate or minimize the problems related to
  immunologic responses and toxicity, encapsulated
  enzyme administration is suggested
• 6. Prevention of thromboembolism
• Encapsulated heparin is liberated from
  circulating erythrocytes at the site of thrombus
  formation thus reducing the risk of further
  thrombus growth.

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• 7.Slow drug release.
• Erythrocytes have been used as circulating depots
  for the sustained delivery of antineoplastics,
  antiparasitics, veterinary antiamoebics ,
  vitamins , steroids , antibiotics , and
  cardiovascular drugs .
• 8.Treatment of hepatic tumors.
• Hepatic tumors are one of the most prevalent
  types of cancer. Antineoplastic drugs such as
  methotrexate , bleomycin , asparginase , and
  adriamycin have been successfully delivered by
  erythrocytes.

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• 9. Treatment of parasitic diseases.
• The ability of resealed erythrocytes to
  selectively accumulate within RES organs make
  them usefultool during the delivery of
  antiparasitic agents. Parasitic diseases that
  involve harboring parasites in the RES organs can
  be successfully controlled by this method.
  Results were favorable in studies involving
  animal models for erythrocytes loaded with
  antimalarial (17), antileishmanial (17, 23, 51),
  and antiamoebic drugs (13, 38).

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• 10. Removal of RES iron overload.
• Desferrioxamine-loaded erythrocytes have been
  used to treat excess iron accumulated because of
  multiple transfusions to thalassemic patients
  (13, 51). Targeting this drug to the RES is very
  beneficial because the aged erythrocytes are
  destroyed in RES organs, which results in an
  accumulation of iron in these organs.

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CONCLUSION

• I concluded among the various carriers used for
  targeting of drugs to various body tissues, the
  erythrocytes have been the most investigated and
  have found to possess great potential in drug
  delivery.

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REFERENCES

• Gilbert s Banker. Modern Pharmaceutics. 4 th
  edition.
• S.P. Vyas and R.K. Khar. Targeted and Controlled
  drug delivery. 1 st edition.
• N.K. Jain. Controlled and Novel drug delivery. 1
  st edition.
• Y.W. Chien. Novel Drug Delivery Systems.
• Binghe Wany, Teruna Siahaan, Richard A Soltao.
  Drug Delivery Principles and Applications.
• Patel RP, Patel MJ and Patel NA. an overview of
  resealed erythrocyte drug delivery. J Pharm Res.
  2009 2(6) 1008-1012.
• Gothoskar AV. Resealed erythrocytes an overview.
  www.Pharmatech.com 140-54.

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• THANK YOU
• SHIVA.PHARMACIST_at_GMAIL.COM