Faculty of Pharmacy, Nursing and Health Professions

Master in Industrial Pharmaceutical Technology

Red blood cells as a novel drug delivery system

MSc Degree seminar 1 Proposal

By Maysoun bali

Introduction
Red blood cells (RBCs), or erythrocytes, have been promising endogenous candidates
for drug delivery for more than four decades. Technically, there are two main
approaches to combine drugs with erythrocytes. The first one is by means of forming
transient pores in the erythrocyte membrane, thus allowing the drug to enter the
erythrocyte. The second strategy consists of coupling the active drug to the
erythrocyte membrane, thus exposing it at the surface of the cell.

Objectives
1- Analyzing methods of engineering of erythrocytes as a drug delivery system.
2- Presentation of methods of isolation and encapsulation.
3- Characterization of engineered erythrocytes.
4- Displaying the possible rout of administration, cross-linking, stability, survival
in vivo, storage, mechanism of drug release, toxicity, immunological
consideration and potential for targeting.
5- Reviewing of recent advances, and pharmaceutical application of engineered
erythrocytes.

Methods of drug loading

1- Hypo-osmotic lyses method:
a) Dilution method
b) Dialysis method.
c) Preswell method.
d) Isotonic osmotic lyses method.
2- Electro-insertion/Electro encapsulation method.
3- Endocytosis method.
4- Membrane perturbation method.
5- Lipid fusion method.
I propose to apply the following method in order to complete the seminar
As the most of the methods now days in use that allow the encapsulation of drugs in
erythrocytes are based on a high-haematocrit dialysis procedure. In brief, washed
erythrocytes are placed in dialysis tubing together with the substance to be
encapsulated. Then dialyse the suspension against a hypotonic solution until the
erythrocytes have been lysed. Once in equilibrium with the exogenous substance, the
dialysed cells are resealed by restoring the physiological isotonicity, then washed and
resuspended in a physiological saline solution or in plasma.
Several apparatuses have been developed for the
dialysis of erythrocytes, but only one is available for the
large scale entrapment of drugs under blood-banking
conditions. This apparatus is based on a continuous-
flow dialysis procedure and allows the processing of a
blood unit (450 ml of blood). The process is easy to
perform, is reproducible and can be completed in 2 h.
This approach can be applied to the delivery and
targeting of drugs in human patients.
Data analysis

Statistical analysis of data corresponding to drug

encapsulation, osmotic fragility and the haematological
parameters can be performed using SPSS 15.0 statistical
software.

Analysis of variance (ANOVA) can be performed, with

dialysis time, the initial drug concentration and dialysis bag
volume as the three independent variables.

Advantages and limitations

1- They are the natural product of the body which
are biodegradable and their isolation is easy and
large amount of the drug can be encapsulated in
a small volume of cell.
2- The entrapment of the drug does not require
chemical modification of the substance to be
entrapped.
3- They prolong the systemic activity of the drug while residing for a longer time
in the day.
4- They protect the premature degradation, inactivation and excretion of proteins
and enzymes and act as a carrier for a number of drugs.
5- They can target the drug within the reticule endothelial system and facilitate
incorporation of proteins and nucleic acid in eukaryotic cells by cell infusion
with RBCs.
6- The techniques for collection and storage are very well known.
7- They have limited potential as carrier to non-phagocytic target tissue and
possibility for cells and dose dumping.

Routes of administration
The carrier cells are injected mainly intravenously or intra-arterially, however,
intraperitonial and subcutaneous routes can also be utilized.
Release mechanisms
The various mechanisms proposed for drug release include
1- Passive diffusion
2- Specialized membrane associated carrier transport
3- Phagocytosis of resealed cells with senescent antigen by macrophages of RES,
subsequent accumulation of drug into the macrophage interior, followed by
slow release.
4- Accumulation of erythrocytes in lymph nodes upon subcutaneous
administration followed by hemolysis to release the drug.

In vitro characterization of resealed erythrocytes

1- Physical Characterization
a) Shape and Surface Morphology: The morphological examination of
these ghost erythrocytes is undertaken by comparison with untreated
erythrocytes using transmission (TEM), scanning (SEM) electron
microscopy or phase-contrast optical microscopy.
b) Drug Release: It is determined by Diffusion cell or dialysis.
c) Percentage Encapsulation or Drug Content: The cell membrane of
packed loaded erythrocytes are first deproteinized using methanol or
acetonitrile and after centrifugation at 3000 rpm for a fixed time
interval, the clear supernatant is assayed for drug content, radiolabeled
markers or magnoresponsive markers such as magnetite.
d) Electrical Surface Potential and Surface pH: Electrical surface
potential and surface pH is measured by Zeta potential measurements
and pH sensitive probes respectively.
2- Cellular Characterization
a) In Vitro Drug Release and Haemoglobin Content: In vitro release of
drug(s) and haemoglobin are monitored periodically from drug loaded
cells. The cell suspension (5% hematocrit in PBS) is stored at 4 ˚C, in
amber colored glass container. Periodically the supernatant are
withdrawn using a hypodermic syringe, deproteinized using methanol
and filtered through 0.45 μm filter. They are then estimated for drug or
hemoglobin content. Another parameter used to evaluate hemoglobin
disposition after the resealing, is mean corpuscular haemoglobin. It is
the mean concentration of Hb per 100 ml of cells and is an index,
which is independent of the red cell and therefore, it is true expression
of their Hb content.
b) Percent Cell Recovery: Percent cell recovery can be determined by
counting the number of intact cells per cubic mm of packed
erythrocytes before and after loading the drug. Haematological
analyser and Neubeur’s chamber are used for this purpose.
c) Osmotic Fragility: The osmotic fragility of resealed erythrocytes is an
indicator of the possible changes in cell membrane integrity and the
resistance of these cells to osmotic pressure of the suspension medium.
This test is carried out by stepwise incubation with isotonic to
 hypotonic saline solutions and estimation of drug and Hb In most
cases, osmotic fragility of resealed cells is higher than that of the
normal cells because of increased intracellular osmotic pressure.
d) Osmotic Shock: For osmotic shock study, erythrocytes suspension is
diluted with distilled water and centrifuged at 3000 rpm for 15 min.
The supernatant is estimated for drug and Hb spectrophotometrically.
e) Turbulence Shock: The turbulence fragility is yet another characteristic
that depends upon changes in the integrity of cellular membrane and
reflects resistance of loaded cells against hemolysis resulting from
turbulent flow within circulation. It is determined by passing cell
suspension through a 23-gauge hypodermic needle (10ml/min) and
estimation of residual drug and Hb. The turbulent fragility of resealed
cells is found to be higher than normal erythrocytes.
f) Erythrocyte Sedimentation Rate (ESR): It is an estimate of the
suspension stability of red blood cells in plasma and is related to the
number and size of the red cells and to the relative concentration of
plasma proteins. It is measured by ESR apparatus.

3- Biological Characterization.
It includes Sterility testing, which is carried out by using aerobic and
anaerobic cultures, pyrogenicity testing by rabbit fever response test or
limulus amoebocyte lysate (LAL) test and Animal toxicity testing.

Novel approaches
1- Erythrosomes: These are specially engineered vesicular system in which
chemically cross-linked human erythrocytes cytoskeletons are used as support
upon which a lipid bilayer is coated. This process is achieved by modifying a
reverse-phase evaporation technique. These vesicles have been proposed as
useful encapsulation systems for macromolecular drugs
2- Nanoerythrosomes [NES]: They are patented nano-vesicle (average diameter
of 100nm) derived from red blood cell membranes through extrusion,
sonification or electrical break down method.
Conclusion
Human erythrocytes, owing to the remarkable ability of their membrane to be opened
and resealed, provide an extraordinary vehicle for the dissemination of drugs in
circulation. While most RBC-based drug delivery systems are still in proof-of-concept
stage, some have been proved to be effective and safe in preclinical studies and will
likely be promising clinical treatments. Among potentially useful biomedical
applications, RBC-mediated intracellular delivery of drugs including replacement
therapies, imaging probes and toxic agents into RES macrophages and other cells
exerting robust endocytic uptake seems as a reasonably promising goal.
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