Faculty of Pharmacy, Nursing and Health Professions

Master Program of Industrial Pharmaceutical Technology

Seminar Paper Submitted for the Seminar:

Erythrocytes as novel drug delivery system

Submitted by: Maysoun Bali 1185273

Supervisor: DR. HANI SHTIEH
MIPT830, Seminar 1
Submission date: 5/2020
  Erythrocytes as a novel drug delivery system

Abstract
An erythrocyte based drug delivery system have been extensively studied
for their capability as a carrier to prolong the drug’s action by its slow
release or as a biological device for targeted drug delivery to target organs,
also to enhance the bioavailability, pharmacokinetics and eliminates
toxicities of the drugs. The concept in this review is to investigate the
methods of drug encapsulation in erythrocytes with experimental details and
loading conditions. Focusing only on drugs loaded inside erythrocytes using
techniques such as hypotonic pre-swell dilution, endocytosis and by Normal
transportive mechanism, and reviewing some in vitro studies for drug
delivery of metformin as parenteral slow release depot formulation and
propranolol as bioadhesive system.

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 Erythrocytes as a novel drug delivery system

Table of content
1. INTRODUCTION......................................................................................................................4
2. DRUG LOADING OF CARRIER RBCS.......................................................................................4
3. PREPARATION OF RESEALED ERYTHROCYTES.......................................................................5
3.1 ERYTHROCYTES ISOLATION..................................................................................................5
3.2 DRUG ENCAPSULATION..........................................................................................................5
3.3 ESSENTIALS FOR ENCAPSULATION........................................................................................5
4. OSMOSIS BASED METHODS......................................................................................................5
4.1 DILUTION METHOD................................................................................................................6
4.2 HYPOTONIC DIALYSIS.............................................................................................................6
4.3 PRE-SWELL DILUTION............................................................................................................7
4.4 ISOTONIC OSMOTIC LYSIS...................................................................................................7
5. ENTRAPMENT BY ENDOCYTOSIS.............................................................................................8
6. IN-VITRO CHARACTERIZATION OF RESEALED ERYTHROCYTES..........................................9
6.1 DRUG CONTENT QUANTIFICATION........................................................................................9
6.2 IN-VITRO DRUG RELEASE AND HEMOGLOBIN CONTENT STUDY..........................................9
6.3 PERCENT CELL RECOVERY AND MORPHOLOGICAL STUDY................................................9
6.4 OSMOTIC FRAGILITY AND OSMOTIC SHOCK STUDY............................................................9
6.5 TURBULENCE SHOCK STUDY...............................................................................................10
6.6 ERYTHROCYTE SEDIMENTATION RATE (ESR)..................................................................10
6.7 SHELF LIFE AND STABILITY AND CROSSLINKING OF RELEASED ERYTHROCYTES.........10
6.8 MECHANISM OF DRUG RELEASE FROM RESEALED ERYTHROCYTE................................10
6.9 STORAGE..............................................................................................................................11
7. ROUTE OF ADMINISTRATION.................................................................................................11
8. ERYTHROCYTES FOR TARGETED DRUG DELIVERY............................................................11
8.1 HUMAN ERYTHROCYTES AS CARRIER FOR METFORMIN...................................................11
8.1.1 COLLECTION OF ERYTHROCYTES................................................................................11
8.1.2 LOADING OF METFORMIN IN RBC..............................................................................11
8.1.3 METHOD OF ENDOCYTOSIS..........................................................................................12
8.1.4 NORMAL TRANSPORTIVE MECHANISM........................................................................12
8.1.5 CHARACTERIZATION OF METFORMIN LOADED RESEALED ERYTHROCYTES............12
8.1.6 RESULTS AND DISCUSSION..................................................................................14
8.2 ERYTHROCYTE BASED BIO ADHESIVE SYSTEM FOR NASAL DELIVERY OF PROPRANOLOL18
8.2.1 ERYTHROCYTE SEPARATION........................................................................................18
8.2.2 LOADING OF PROPRANOLOL HCI................................................................................18
8.2.3 BIOADHESION PREPARATION.......................................................................................18
8.2.4 RESULTS AND DISCUSSION............................................................................................19
9. CONCLUSION..........................................................................................................................20
10. REFERENCES......................................................................................................................21

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  Erythrocytes as a novel drug delivery system

degradation within erythrocytes, no chemical or

physical interaction with erythrocyte membrane, and
1. Introduction well known pharmacokinetic and pharmacodynamics
Red blood cells or erythrocytes are part of the novel properties.[2][3][4]
drug carrier system, that Characterized as natural
compartments capable of protecting encapsulated 2. Drug loading of carrier RBCs
cargos and this allows them to be a natural long- The Drugs can be encapsulated within RBCs or
circulating delivery vehicles. They have flexibility, conjugated to their surface. The process of RBCs
elasticity, biconcavity and nucleated structure with encapsulation comprise of enzyme replacement
diameter near of 7.3µm and thickness of 2.2µm that therapies, antigens to modulate immune response,
are suited for intravascular delivery, RBCs are antimicrobial and anti-viral agents, and anti-
biodegradable, biocompatible and nonimmunogenic. inflammatory drugs. The encapsulated drugs activity
In addition, their semipermeable membrane affords within RBC can be manipulated by attaching to
sustained release to small molecule drugs, with the hemoglobin, interaction with nitric oxide-donors,
ability to retain large proteins while providing them oxidation catalyzed by iron, or by catalytic reactions.
access to the substrates. The aim here is to load The encapsulation enzymes inside carrier RBCs can
natural RBCs with therapeutic agents without be used to neutralize or detoxify of diffuses
compromising the structural integrity and biological substrates through the cell membrane.[5]
functions of the RBCs, improve the pharmacokinetics All of the methods used for encapsulation in
of the drug, increase its half-life and reduces its erythrocyte utilizes the impressive capacity of cell to
adverse effect. be reversibly deformed and change in their shape
There are several way for RBCs to be used as a drug under stress permitting the formation of large pores
carrier including for targeting specific tissue/organ, for external intercross of macromolecules.[6]
for sustain release of drug, to target The risk of losing RBC biocompatibility is a major
reticuloendothelial system (RES), as therapy of liver challenge in drug loading, a methods based on
deficiency, removal of toxic agents, treating parasitic membrane resealing and transient osmotic shock
disease,as a treatment of hepatic tumour, for antiviral have provided an accepted biocompatibility, the
drug delivery. encapsulation of small molecules and therapeutic
There are two main procedures to couple the drugs
with erythrocytes. First is by forming transient pores
in the erythrocyte membrane, permitting the drug to
get in the erythrocyte. This loading method is used to
enhance the half-life of the drug and to prevent
hypersensitive reactions by concealing the drug from
the immune system. The other strategy attaching the
drug to the erythrocyte membrane, thus displaying it
at the surface of the cell.[1]
There are many techniques to combine drugs with
RBCs including Hypotonic Hemolysis methods
(Dilution, Pre-swelling Dilutional, Dialysis Methods
and isotonic osmotic lysis), Osmotic Pulse Method,
Chemical perturbation of the membrane, Electro-
insertion or electro encapsulation and Entrapment by
endocytosis. As mentioned in fig1.
For the efficient entrapment the drug it must have a proteins like dexamethasone and enzymes, have been
good degree of water solubility, must resist the successful when tested in animal models.[7][8]

Figure 1 Main procedures for the entrapment of drugs in erythrocytes.[7]

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 Erythrocytes as a novel drug delivery system

3. Preparation of resealed erythrocytes This is necessary for ensuring the appropriate

Resealed erythrocytes are simply prepared by durability and circulation of the loaded erythrocytes.
obtaining blood samples from the mammalian of The most widely used method for encapsulation in
interest and are centrifuged to separate the erythrocytes and for the appropriate delivery of
erythrocytes from the plasma, followed by entrapping drugs, enzymes or peptides are osmotic methods by
the drug by forming transient pores in the erythrocyte encapsulation using reduced osmotic pressure state
membrane, under osmotic conditions (hypotonic like hypotonic dilution, hypotonic preswelling,
buffer) permitting the drug to get in the erythrocyte, osmotic pulse, hypotonic hemolysis and hypotonic
finally resealing the resultant carrier erythrocytes dialysis.
using isotonic condition (isotonic buffer, or From all the methods, hypotonic dialysis is the
hypertonic buffer). To restore isotonicity. The encapsulation strategy most frequently used. This is
process depends on the cells response under osmotic because it is best preserves the characteristics
conditions. The basics of the preparation Procedure (biochemical and physiological) of the erythrocytes
involves. produced. [3]
3.1 Erythrocytes isolation 3.3 Essentials for encapsulation
In order to use erythrocytes as a drug delivery system Multiple bioactive molecules (5000-600,000 Daltons
mammalian erythrocytes have been taken in in size) can be encapsulated in erythrocytes They
advantage for drug loading, resealing and used for should be hydrophilic, polar, resist degradation
delivery of drug, enzyme and other bioactive agents’. inside erythrocytes, compatible with the
Majority of erythrocytes are obtained from red blood erythrocyte membrane with defined
cells of human being, mice, cattle, pigs, dogs, sheep, pharmacokinetic and pharmacodynamics
goats, monkeys, chicken, rats and rabbits. In order to characteristics. Since the encapsulation of charged
avoid antigen-antibody reaction, the evaluation blood molecules maintained longer than uncharged
group and compatibility parameters should be molecules, the encapsulation of both polar and non-
performed. polar molecules have been achieved. Sucrose is used
Fresh intact blood should be used for the isolation of as an indicator for encapsulation applications.
erythrocytes; the blood is taken using a syringe Hydrophobic molecules can be entrapped by getting
containing a drop of anticoagulant, from cardiac/ absorbed over other molecules; non-polar molecules
splenic puncture in small animals and from veins in can be entrapped in their various salts. The molecules
large animals. Then transferred into heparinized or intended for encapsulation must be compatible with
EDTA-coated tubes, and immediately congealed to the erythrocyte cell structure and components.[10]
4°C and reserved for less than 2 days, the blood [11]
sample centrifuged at 2500 rpm for 5 minutes at
4±1ºC in a refrigerated centrifuge and washed thrice 4. Osmosis based methods
with phosphate-buffered saline (PBS, pH 7.4). This
Erythrocytes are capable to go through reversible
step is usually repeated with isosmotic solution to
swelling and changing their shape in a hypotonic
remove other blood components. Then store in acid-
solution or osmotic stress. Erythrocytes can grow
citrate-dextrose buffer at 4°C up to 48 hr. before use.
in volume up to 25-50% resulting to a primary
As described below [9][10]
alteration in the shape from biconcave to
spherical increasing in volume at the same time
3.2 Drug encapsulation maintaining the surface area constant.
The several methods of encapsulation available offer
In general, when placing the erythrocytes in solutions
an enhanced function of the substance being
up to 150 milliosmol /kg they maintain their integrity,
encapsulated, at the same time ensuring that the
if above result in rupture of the membrane and large
erythrocytes endure the least possible alterations, so
pores formation (200-500 nm in diameter) just before
that it will function similarly to a normal erythrocyte.
cell lysis resulting in escape of the cellular contents.

 Page 5
Figure 2 Schematic illustration of hypotonic hemolysis drugs loading
method. [7]
  Erythrocytes as a novel drug delivery system

Erythrocyte ghost is the left over parts after

membrane rupture and escaping of cellular
contents. For membrane resealing step by increasing
the concentration of salt to its early level, the resealed
erythrocytes can retain their original shape and their
original impermeability characteristics and
maintaining almost the same life interval in
circulation. At the time that the intracellular
substances discharged in hypotonic hemodialysis, we 4.2 hypotonic dialysis
can externally added molecules required for
encapsulation. After re- sealing, these substances are
trapped into erythrocytes. Figure 2 summarizing the
basic steps.[12]

The basic steps of the hypotonic processes starts with

detachment of RBC and plasma followed by
Removal of platelets and leukocytes then multiple
washings of erythrocytes suspension then Hypotonic
lysis followed by Resealing of the cells and final
washings of lysed and resealed erythrocytes finished
with Re-suspension of the cells in phosphate buffered
saline (PBS) solution or plasma. The methods used Figure 5 Pre-swell dilution hemolysis and isotonic resealing method [5]
for Hypotonic Hemolysis in details are described
below.[6][7] Figure 3 Dilution method [4]

4.1 Dilution method

Since erythrocytes have a limited capacity to
withstand the increase in volume, when they are set
in the hypotonic (0.4% NACL), the volume of
erythrocytes increases by 50-75% leading to rupture
in cell membrane allowing liberation of cellular
content until the balance is attained within 1min,
which arise in swelling up to 1.6 time its original
volume. 2-20 volumes of hypotonic buffer or water
containing the compounds to be loaded are added for
one volume of washed erythrocytes. Then the cell
suspension is kept at 0°C for 5 min. This process is Figure 4 erythrocyte dialysis apparatus [14]
simple and fast, but with low encapsulation
efficiency (1-8%) and losing of cytoplasmic This method based on a dialysis bag (semipermeable
composition during osmotic lysis. drugs like s- s- membranes) containing isotonic media plus the
galactosidase and glycosidase with low MW can be substance for encapsulation in which the erythrocytes
encapsulated As shown in fig3. [13][4][14] at a hematocrit range from 5 to 80% (the ratio of the
volume of red blood cells to the total volume of
blood) are immersed in. Then the bag is filled with
air and sealed so that 75% of the erythrocytes volume
occupying the internal volume. During dialysis, an
adequate mixing is crucial for maximum

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 Erythrocytes as a novel drug delivery system

encapsulation. Then deeply place the bag in a

hypotonic solution (5times the volume of erythrocyte
suspension) with continuous stirring. If the molecular
weight of the substance to be encapsulated is larger
than the cut-off of the dialysis bag, ( pore size
distribution and permeability of the membranes), then
add the substance before dialysis. In cases if the
substances quickly dialyzable then it is added to the
outer dialyzing buffer or added after the dialysis
process by incubating dialyzed RBC with the
substance directly in a tube. As illustrated in fig4
The composition of the hypotonic dialysis medium is
5volumes of diluted PBS (6volumes of PBS with
not essential due to the high buffering capacity of
5volumes of water) to achieve salt concentration of
hemoglobin (resist changes in pH) in the bag; the
0.6% NACL. A centrifugation at a low voltage (1000
medium could be water for lysis, diluted PBS or 10
rpm for 10 min at 0-4˚C) is performed after 5 min to
mM phosphate, pH 7.0, with 2 mM glucose and 0.5
obtain packed RBCs. Discard supernatant and Collect
mM CaCl2. Lysis time depends on the factor of
packed RBCs then repeat the swelling procedure. The
hematocrit percentage in the dialysis bag e.g. 50%
second step starts with collecting the swelled cells,
hematocrit total lysis achieved in 45 min, 80%
the supernatant is discarded and applying a very
hematocrit total lysis achieved in 75 min.
hypotonic lysing solution including the drug to be
To obtain isotonicity for resealing of erythrocytes we encapsulated, the volume of hypotonic solution added
can add 1.54 mM KCL or applying Reverse dialysis is equal to the volume of packed cells. Following
against isosmotic buffer, or include 4 mM MgCl2, 10 intense vortex mixing for cells re-suspending, the
mM glucose, 2 mM adenosine which is the best for lyse conditions take place for 10 min at 0°C. This
preservation of metabolic energy in the cells. The method is simple, fast, with good retention of
optimal encapsulation conditions are a combination cytoplasmic components, minimum damage to the
of low temperatures (4°C) for lysis and high cell and high drug encapsulation (72%) and good
temperature (37°C) for resealing. Typical cell survival in-vivo. This method applied for
recovery is 50 to 90%, so the highest encapsulation encapsulation of several drugs such as methotrexate,
percentage for RBC at 70%. The encapsulation insulin, and metronidazole. The pre-swell dilution
percent is calculated hemolysis process is clarified in Figure 5.[2][5]
This method relies mainly on passive diffusion but
using transmission electron micrographs shows that a 4.4 Isotonic osmotic lysis
fraction of the substances encapsulated by a process Also known as osmotic pulse method, here the cells
of endocytosis (up to 20% of the cells display are presented to a short and intense duration of
endocytic vacuoles). osmotic stress. In this method the dimethylsulfoxide
Hypotonic dialysis allows the encapsulation of all (DMSO) (own high transerythrocytic membrane
types of molecules, drugs, enzymes and peptides with permeability) is employed to construct a transient,
molecular weight up to 50,000 Da, with high large osmotic gradient through the RBC membrane,
entrapment efficiency 30-40%. [6][15][14][7] thus permitting passage of molecules. The process
including several steps of DMSO incubation, isotonic
dilution using molecules to be entrapped, incubation
4.3 Pre-swell dilution and resealing to the initial shape.as shown in fig6
This method based on initiatory controlled swelling The process starts with the addition of DMSO
of RBCs without lysis in hypotonic-buffered solution. isotonic solution to RBC, the solute will diffuse into
The suspension is prepared in two steps start by to the cells and set up an elevated intracellular and
taking one volume of washed cells and suspend in extracellular osmolality (~1500 mOsm for 8%
DMSO). Followed by rapid mixing of the suspension

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  Erythrocytes as a novel drug delivery system

with the isotonic solution that contains the

molecules required for encapsulation, then the
dilution step using isotonic solution to lower the
DMSO concentration leading to develop a gradient
concentration of DMSO and osmolality through the
RBCs membrane until equilibrium is achieved by
DMSO diffusion out of the cells,
The gradient will lead to inflow of water and
cellular swelling, followed by higher membrane
Figure 6 Isotonic osmotic lysis technique [5]
permeability and the movement of the molecules to
be entrapped into the cells and the hemoglobin out
of the cells. Since the DMSO has abandoned the
cells, the osmotic equilibrium is reestablished and
the cells retreat to the initial shape. The DMSO This method comprise of preparing 1:5 v/v of washed
technique easily applied within one day and is more packed erythrocytes to buffer containing 2.5 mmol
suited for low volumes of blood.[5][6][16]

5. Entrapment by endocytosis

Endocytosis is the process in which external fluid

including dissolved substances, proteins, and ions get
entrapped (plasma membrane invagination) forming
intracellular vesicle.
Endocytosis can be achieved by exposing
erythrocytes to a variety of amphipathic cations,
having both hydrophilic and hydrophobic parts
Figure 7 Entrapment by endocytosis method [6]
(specific membrane activated drugs) that can alter the
RBCs cell membrane producing stomatocytosis
(coffee beans appearance), that will progress to form
continuous membrane edge, which spontaneously
curls, cuts, and unite the surface of the membrane to
form single or centered vesicles as clarified in fig 7. MgCl2 , and 1mmol CaCl2 , 2.5 mmol ATP
These protein-led membrane fusion serve as (supplied as 100 mM aqueous solution, titrated to pH
substitute to membrane fusion mechanisms that based 7.3-7.5 with NaOH, act as intracellular energy
on phospholipid interactions, which is relevant to transfer), and low concentrations of primaquine (to
endocytosis process stabilize membranes against hypotonic hemolysis)
Various classes of drugs can be entrapped by this next step is incubation at room temperature for 2 min.
method include primaquine this phenomenon such as The created pores resealed with addition of 154 mM
8-amino quinolones, Chlorpromazine, Vinblastine, of NACL and incubation at 37C˚ for 2 min. The
Phenothiazine’s, propranolol and tetracine encapsulation of material take place by endocytosis.
hydrocortisone and vitamin A, resulting in formation The endocytosised material are separated by vesicle
of endocytic vacuoles in which the entrapped membrane from cytoplasm, which protects it from
substance could be dissolved in the extracellular the erythrocytes and.
fluid.

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 Erythrocytes as a novel drug delivery system

The internalization process through erythrocyte

membrane depends on drug concentration,
temperature and pH. The ideal temperature for
vacuole creation is around 37°C, while no vacuoles
appeared at temperatures less than 23°C and their
appearance is lowered even at temperature higher
than 45°C. The ideal pH for vacuole forming was
constructed to be 7.9-8.1 and at pH’s lower than 6.4-
6.5 vacuole disappeared.[16][6][17] [3]

6. In-vitro Characterization of Resealed

Erythrocytes
The following characterizations are crucial to
guarantee their in-vivo efficiency and therapeutic
benefits. Moreover, mentioning Various
characterization parameters and method employ for Table 1 Various characterization parameters and method employ for Resealed erythrocytes.
Resealed erythrocytes in tabe1. [3] [3]

6.1 Drug content quantification

To evaluate the drug load, loaded RBCs are
deproteinized (to remove protein molecules that 6.3 Percent cell recovery and Morphological
would interfere in the analysis) with acetonitrile or study
methanol followed by centrifugation at specific rpm By using electron microscope or Phase contrast
for a fixed time interval. The supernatant liquid
to count the intact cells number contained in each
pipetted out and analyzed spectrophotometrically.[3]
mm of consigned erythrocytes before and after
[9]
entrapment.[9]

6.2 In-vitro drug release

For periodically monitoring of in-vitro release of
hemoglobin and drug from drug-loaded cells, first
store the cells suspension of hematocrit in PBS 5% at 6.4 Osmotic fragility and Osmotic shock
4˚C in a dark colored amber glass container, then by study
using a hypodermic syringe fitted with 0.45 µ filter
withdraw the clear supernatant at predetermined To test the cells resistance to hemolysis in low
programmed time intervals. Followed by adding concentration of hypotonic saline.
methanol supernatant for deprotenization and used The solutions with different tonicity can affect the
for drug content estimation, then the sample is osmotic balance of RBCs leading to change their
centrifuged and collect sample supernatant to assay. shape. Incubate the loaded erythrocytes in normal
the relationship between the rate of hemoglobin and saline solution for 10 minutes at 37˚C then
the rate of drug release helps in interpreting the centrifuge for 10 min at 2000 rpm. To do osmotic
mechanisms involved in the release of the drug shock study, the suspension of resealed erythrocytes
encapsulated in erythrocytes.[3] [16] dispersed in distilled water followed by
centrifugation for 15 min at 300 rpm, the hemoglobin
release percent is determined spectrophotometrically
by supernatant analysis.[9][3]

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  Erythrocytes as a novel drug delivery system

6.5 Turbulence shock study recognizable by RES macrophages. Most Rh

To define the influence of shear and pressure, antibodies are IgG, when antibody (IgG type) utilised
produced by injection of resealed erythrocyte for coating, the targeting to spleen is favoured; if IgM
formulas, and its effect on the stability of the loaded type used, the liver targeting is preferred. A fourth
cells. exploited method is by Pre-exposing the resealed
erythrocytes to thermal shock, or by Using enzymes
The simulation of loaded cell destruction while such as neuraminidase, proteolytic enzymes and
injection is done by insertion of both normal and antibiotics also improved cell stability.
loaded cells (10% haematocrit,5 ml) in a 23 gauge
hypodermic needle with a flow rate of 10 ml/min Here we begin with treating the erythrocytes using
similar to blood flow rate followed with several Glutaraldehyde (0.2%) (For protein immobilization it
processes of sampling, centrifugation (2000 rpm for is oily liquid that is soluble in water and alcohol and
10 minutes), determination of the hemoglobin in the organic solvents and available as acidic aqueous
samples. The loaded erythrocytes shows less solutions (pH 3.0–4.0), ranging in concentration from
resistance to disturbance demonstrating cell less than 2% to 70% (w/v)). in a glass funnel
destruction when shaking.[3] followed by filtration and dry vacuum (200mm Hg)
for 10 hr. followed by lyophilisation using a
laboratory lyophilizer in vials at 4˚C, the resulted dry
powder are filled in dark colour amber vials then
6.6 Erythrocyte Sedimentation Rate store for one month. The shelf life of loaded
(ESR) erythrocytes was improved when cell storing was at
powder form and all set to be reconstituted at 4C˚.
To detect the stability of RBCs suspension in [19][11][2][20]
plasma which is relevant to size and Number
of red cells and the concentration of plasma 6.8 Mechanism of Drug Release from
protein. Resealed Erythrocyte
This test is accomplished by calculating the There are three main possible ways available for a
blood cells ESR in a classic tube of ESR device. drug to discharge from erythrocyte carriers. Diffusion
[3][18]. through the cell membrane, Phagocytosis and by a
specific transport system. Using standard blood bag
for encapsulation and storage.[18]

6.7 Shelf life and Stability and

Crosslinking of Released Erythrocytes
Several methods have been utilized to improve 6.9 Storage
stability (RES targeting) of carrier erythrocytes. First
method is by exposing the drug-loaded erythrocytes For storage, hank's balanced salt solution [HBSS] and
to membrane stabilizing agents, acid–citrate–dextrose were used as a storage media at
4˚C for two weeks in order to maintain the integrity
Using band 3 cross-linking agents such as of loaded erythrocytes. The cells survived for two
Glutaraldehyde, which decreases the deformability of weeks. Similar results obtained when suspending the
the cells and increases the osmotic fragility, stability encapsulated cells in oxygenated HBSS,(This salt
and sensitivity towards RES. Second method is biotin solution was charged with oxygen in a small pressure
Surface alteration with phenylhydrazine and N- chamber so that the liquid contained as much oxygen
hydroxysuccinamide increases the macrophage as air), containing 1% soft bloom gelatin followed by
uptake of carrier erythrocytes in-vivo and in-vitro. gentle rotation for the tubes at 4˚C, tubes are set and
Third methods is by Coating the carrier cells by anti- stored in a refrigerator for 2 weeks. To prepare for
Rh antibodies, to produce the erythrocytes use, place the tubes in a water bath at 37°C to liquefy

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 Erythrocytes as a novel drug delivery system

the gel, followed by centrifugation and washed in White crystalline powder of metformin hydrochloride
HBSS before use. The benefits of storing in gel is the C4H11N5•HCl, USP have a MW of 165.62.
ease in dispersing after liquefaction and the Metformin hydrochloride 2.0 g is soluble in 20 mL of
prevention in clumping (a draw back in liquid water. The PKa of metformin is 12.4. The pH of a
media). To boost the cells circulation survival time 1% of metformin hydrochloride in aqueous solution
upon reinjection, purine nucleosides or calcium- is 6.68. It is freely soluble in water [19][25]
chelating agents are added.
The storage conditions and characteristics varies and
8.1.1 Collection of erythrocytes
depends on the type of preservatives used and the RBC was obtained from the Amrita Institute of
container for the finished product; the most common Medical Sciences blood bank, Kochi. The clinic has
utilized containers are plasticized polyvinyl chloride an identified approval from donators that their blood
bags (compatible and non-extractable in blood). usable for medical research. Using phosphate
Loaded erythrocyte should administered briefly after buffered saline (PBS) (pH 7.4) for RBCs washing to
production to skip storage-induced RBC degradation. get rid of the remains of heparin because it performs
Saline, adenine, glucose, and mannitol are the most as an analytic interferential in the study as the heparin
widely utilized preservatives solutions for resealed and metformin absorption maxima is 232 nm. The
erythrocytes. [21][9][22] [23] resilient buffer was excluded by aspiration then by
centrifugation to obtain packed volume cell. Care
was taken for using PBS 7.4 Instead of distilled water
for washing erythrocyte because distilled water is a
7. Route of administration haemolysing agent.[19]
The intravenous route is the most common rout of
administration next is subcutaneous, intranasal, oral
and intraperitoneal in which 25% of resealed cell
8.1.2 Loading of metformin in RBC
survived in circulation for 14 days.[24]
A set of metformin in water concentrations varying
from one to ten mg/ml was prepared. Then the
8. Erythrocytes for Targeted Drug
mixture added to the suspended RBC in PBS and
Delivery allowed to set for one hour. The supernatant liquid
This review focuses on the studies of tow potential was retreated, and percentage of hemolysis was
routs of administration of carrier erythrocytes; slow calculated for each metformin concentration. The
release depot parenteral and bio adhesive system for hemolysis percentage was determined
nasal delivery.[19] spectrophotometrically using the equation no.2.
Absorbance of sample−absorbance of blan
% hemolysis=
Absorbance of positive control
A positive control test of supernatant was
8.1 Human erythrocytes as carrier for prepared; in order to obtain the supernatant the
metformin addition of the nonionic detergent triton X to
Metformin is a biguanide class drug used as first-line erythrocyte suspension as positive control was
drug of choice for the treatment of type 2 diabetes used. 1% v/v triton X/water with a haemolysing
and in cases of polycystic ovary syndrome. The capacity of 100% (The rate of haemolysis
objective in this review is to slow down the release of elevated with the increase in detergent
metformin using carrier erythrocyte to achieve a slow concentration, by elevating membrane
release depot parenteral formulation.
permeability to KCL and colloid osmotic
The endocytosis method was used for metformin haemolysis).
encapsulation in carrier erythrocytes in order to retard The blank supernatant is prepared adding normal
release of the drug. saline to the erythrocyte suspension. By using

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  Erythrocytes as a novel drug delivery system

ultraviolet (UV) spectrophotometrically the Data of the amount(µg) of metformin loaded in RBCs
absorbance determined at 540 nm. using endocytosis method versus normal transportive
The metformin concentration to be loaded to mechanism at four different concentrations is shown
erythrocytes were optimized by detecting the in table2. [19]
percentage haemolysis. Drug loading was
performed by the method of normal transportive
mechanism and endocytosis.[19][26] 8.1.5 Characterization of metformin
loaded resealed erythrocytes
 Encapsulation efficiency
8.1.3 Method of endocytosis
To evaluate the drug content, packed loaded RBCs
One ml of the desired concentration of membrane are deproteinized with methanol after
metformin/water (range: 1-10 mg/ml) is added to five centrifugation at 3000 rpm for 15min ( mechanical
ml of buffer consisting of ATP 2.5 mmol, CaCl2 1 stress leading to RBCs lysis and release their
mmol and MgCl2 2.5 mmol. Followed by the content), 1 ml of clear supernatant liquid is pipetted
addition of erythrocytes 0.65 ml to the solution and out and analyzed using UV-visible spectrophotometer
incubation step for 15 min at 0˚C. The resealing step at 232 nm. For comparison, the evaluation carried out
was performed using sodium chloride 0.9% solution for both methods of encapsulation endocytosis and
at 37°C to obtain the required suspension. Then normal transportive mechanism. The blank
washing tow times using cold PBS to discard the supernatant is prepared by adding normal saline to
metformin bounded on surface membrane of RBCs, the erythrocyte suspension. The encapsulation
then final centrifugation for 15 min at 5000 rpm.[19] efficiency is calculated using the equation no3 below.
[27] [19]
Table 2 Data table of the amount of metformin(µg) loaded in RBCs using endocytosis method versus normal transportive mechanism at four different
concentrations.[19]

Encapsulated drug
Encapsulation efficiency= × 100
8.1.4 Normal transportive mechanism Total drug
This method involves the encapsulation of the drug
into erythrocyte by incubation without destruction the
erythrocyte membrane.  In vitro drug release
One ml of the desired concentration of Using orbital shaking incubator(applied as
metformin/water (range: 1-10 mg/ml) is added to mechanical stress to induce drug release from RBCs),
five ml of PBS( pH7.4), which then added to withdraw one ml of the supernatant at different time
erythrocytes solution 0.65 ml and incubated for intervals followed by deprotenization by methanol
15min at 0˚C. The resealing step was performed and inserted through syringe filter 0.22 µm to clear
using equal volume of sodium chloride 0.9% solution the precipitated protein. Then determine the amount
at 37°C. . Then washing tow times, using cold PBS to of metformin released in every time interval at 232
obtain the required suspension then final nm using UV spectrophotometry, a plotting of the
centrifugation for 15 min at 5000 rpm to discard the obtained data in different kinetic models are done in
metformin bounded on surface membrane of RBCs. order to study the release kinetics. This study

 Page 12
 Erythrocytes as a novel drug delivery system

performed for the concentration with highest loading

efficiency for 12 days.[19]

 Effect of cross-linking agent on drug release

For the evaluation of the effect of cross-linking agent
concentration on drug release, the carrier erythrocytes
loaded with the drug were treated using
Glutaraldehyde cross linker agent, Glutaraldehyde
treated preparations also studied for In vitro drug
release using different concentrations ranged from
0.2-0.8% v/v focusing on treating the loaded calculated, the number of insertions can be plotted
erythrocytes with optimum metformin concentration against hemolysis percentage for both loaded and
10mg/ml. The main function of Glutaraldehyde as normal erythrocytes.[19]
cross-linking agent is to provide better chemical
stability and improved thermostable cross-links with  Cell counting and cell recovery
protein better than aldehyde cross linkers.[20][19] This test include counting the number of intact cells
per cubic mm of packed erythrocytes before and after
loading of the bioactive compound with the help of
 Osmotic fragility test haemocytometer. Phase contrast or electron
To measure erythrocyte resistance to hemolysis while microscope may be used for normal and drug loaded
being exposed to varying levels of phosphate erythrocytes.
buffered saline (PBS). The test was performed for The calculation of RBCs recovery based on the
both loaded and normal erythrocytes. A range of variations in the volume of the suspension and
2.5ml sodium chloride concentrations ( 0.1% to 0.9% hematocrit of erythrocytes prior to and after loading.
w/v ) was prepared then add 25µl of erythrocytes Cell recovery defined as the cells retained number
(loaded and unloaded) independently to each sodium followed by loading. Percentage of cell recovery was
chloride various concentrations, (took a place in a test evaluated by calculating the number of sound cells
tube), followed by centrifugation and permitted to set contained in each cubic mm of congested
aside for 1 hr. at 37°C. Withdraw the supernatant and erythrocytes prior to and after loading. According to
evaluate the absorbance percentage released the equation below.[19]
hemoglobin at 540 nm in a UV-visible NOof cells before loading−NOof cells a
spectrophotometer. Usually the resealed cells have % cell recovery=
higher osmotic fragility than the normal cells due to
NOof cells before loading
their increased intracellular osmotic pressure.
The maximum hemolysis (complete lysing) was
determined as the amount of hemoglobin released  Morphological studies
into distilled water. A special type of scanning electron microscope
The amount of hemoglobin released in the test was equipped with a digital camera was used to estimate
expressed as a percentage in correlation to a the quality and the load correlated to Morphology
completely lysed sample.[28][11][19] and structure variation of normal intact erythrocytes
and the loaded ones.
 Turbulence shock studies
By placing the sample in buffered Glutaraldehyde
In order to evaluate the stability of loaded and draining the aldehyde medium, rinsing of cells is
erythrocytes a study was performed by passing the performed using phosphate buffer three times for 5
erythrocyte suspension through a 22-standard of min, and then placed in osmium tetroxide for 1 hr. A
calibration needle multiple times with a flow rate of second rinsing step is performed by purified water,
10 ml/minutes and hemolysis percentage was for 10 min, the sample is study
obtained and fixed on studs
Figure8 hocking result between metformin and hemoglobin[19]

 Page 13
  Erythrocytes as a novel drug delivery system

splutter-cover with gold, then examined by SEM

(scanning electron micrograph).[19]

 Stability studies
There is not a specific guideline for the estimation of
resealed erythrocytes stability, in this study; the
storage of resealed erythrocytes must be in
lyophilized state. Lyophilized erythrocyte
formulation stability was evaluated by calculating the
hemolysis percentage following re-suspending the
formulation in PBS. [19]
indicates exclusively a fragile interaction among
metformin and hemoglobin. Docking results of
8.1.6 RESULTS AND DISCUSSION
 Physical interaction of RBC with metformin
The study focused on physical interaction of
metformin with hemoglobin and metformin with
RBC, the metformin with RBC physical interaction
was evaluated by having FTIR spectra of lyophilized
RBC, metformin and physical combination of
lyophilized RBC and metformin stored for 7 days.
Docking human oxy hemoglobin chain (a) with
metformin is a process used to evaluate the physical
interaction of metformin with hemoglobin.
Docking is the molecular design method established Metformin with hemoglobin was viewed in Fig 8.
for the search of the ligand-binding site in the
objected protein movable site and the followed by
calculation of the amount, which permits the
evaluation protein-ligand binding free energy. [19]  Characterization of metformin loaded
resealed erythrocytes
 Encapsulation efficiency
Various concentrations of 1, 4, 7, 10 mg/ml of
metformin were applied for encapsulation. The load
of encapsulated in erythrocytes of these
concentrations of Metformin displayed in Fig. 9. The
chart indicates that when we used concentration of 10
mg/ml the load of metformin encapsulated is higher.
 Physical interaction of metformin with The equivalent encapsulation efficiency of various
hemoglobin concentration of metformin was determined. Fig 10
displaying the encapsulation efficiency at four ranges
The docking study shows the existence of hydrogen of Metformin. Fig 11 displays a correlation between
bonding among the drug and amino acids LYS127 encapsulationFigure 10 the encapsulation
efficiency efficiency at loaded
of metformin four different
byconcentration by
the method of endocytosis. [19]
and ASP126. The binding score was −3.33, means normal transportive mechanism with endocytosis
the lack of other interactions such as pication method at different concentration.
interaction or cation-cation interaction, which

Figure 9 the amount encapsulated in erythrocyte at four different

Page 14 of metformin by the method of endocytosis.[19]
concentrations
 Erythrocytes as a novel drug delivery system

Fig 11 displays data as mean ± standard deviation,

using three samples in every group n=3. 2×4. Two-
way factorial analysis of variances was performed.
The comparison between methods of endocytosis
with normal transportive mechanism showed that the
process of drug loading not only depend on
endocytosis, some load of encapsulation arise due to
the diffusion effect also.
When the endocytosis loading mechanism is used the
metformin will locate inside the endocytic vacuoles,
while in the normal transportive mechanism (simple Figure 11 Comparison of encapsulation efficiency of metformin loaded by
diffusion), the metformin will attach by forming endocytosis method with normal transportive mechanism at 4 different
concentration. Data are expressed as mean±standard deviation, 3 samples
hydrogen bond to hemoglobin as shown in fig12.[19] in each group n=3. 2×4.
Two-way factorial analysis of variances was carried out followed by post
 In vitro drug release hoc Tukey HSD test. p<0.0001 suggests that method of endocytosis had
significant role in metformin loading [19]
From the loading efficiency results showed that the
10 mg/ml concentration of metformin loaded by the
endocytosis was producing the highest loading
efficiency and so used for in vitro drug release study.
Glutaraldehyde cross linker agent of concentrations
0.2%, 0.4%, 0.6%and 0.8% v/v were added to the
metformin-loaded erythrocytes (with the highest
loading concentration 10mg/ml) to get F1, F2, F3 and
F4 formulations and F0 without the addition of
Glutaraldehyde. Cumulative Percentage of drug
release of F0 Formulation was viewed in fig 13. It
took 312 hrs. (12 days) to release 98.33% of drug.
Data are expressed as mean±standard deviation, 3
samples in each group n=three. Effect of
Glutaraldehyde concentration on the % cumulative
 Kinetic modelling of release rate
drug release data are provided in the table3.[19]
Erythrocytes are vesicular bodies made up of
phospholipid bilayer so in order to figure their release

 Effect of cross-linking agent in drug release

To evaluate the effect of Glutaraldehyde
concentration on the release rate, the Glutaraldehyde
treated formulation were studied for metformin In
vitro release. The results shows a reverse relation
between the cross-linking agent concentration and the
release rate. The data of release were studied just for
12 days due to the short in vitro stability of resealed pattern in vitro Kinetic model utilized. The results
erythrocyte at 37°C. After 12 days, the percentage of shows that 98.34% of drug was liberated from F0
recovered cells significantly decreased and was formulation within 12 days as viewed in fig14.
sensitive to contamination.[19] Figure 13 Percentage cumulative drug release of F0 formulation against
time. [19]

 Page 15
  Erythrocytes as a novel drug delivery system

R-squared values are best-fitted regression line in

zero order kinetics and higher than the first order
kinetics; the graph shows that the release of
metformin from erythrocytes followed zero order
kinetics. Korsmeyer-Peppas model and Higuchi plot
was applied to describe the drug dissolution from
loaded erythrocytes. In Higuchi plot, the data
obtained plotted as cumulative percentage of drug
release versus square root of time. In Korsmeyer-
Peppas model, data obtained from in vitro drug
release studies plotted as log cumulative percentage
drug release versus log time. The release exponent (n
value) of Korsmeyer-Peppas graph was >0.5 which influenced by permeability of the cell membrane and
indicates a non-Fickian diffusion (dissolution surface-to-volume ratio. This test indicates the
followed by diffusion). The weak interaction among
metformin and hemoglobin is the reason for the
initial delays in the release rate as the docking energy
score −3.33 of hydrogen bonds among the drug and
amino acids ASP126 and LYS127. It is required to
achieve a breaking in hydrogen bond for the release
of metformin.

Figure 14 (a and b) Kinetic data modeling for F0 formulation. [19]

metformin-loaded erythrocytes
Table 3 Effect of Glutaraldehyde concentration on the percentage
cumulative drug release within 12 days.[19]
(with the highest loading metformin
concentration 10mg/ml)
F0 F1 F2 F3 F4
 Osmotic fragility Glutaraldehy 0.0% 0.2 0.4 0.6 0.8
This test evaluates the ability RBCs to resist the de % % % %
destruction in hypotonic solutions. RBC are concentration
Figure 14 (a and b) Kinetic data modeling for F0 formulation. [19]
conferred to a solution of salt water (NaCl) with v/v
accelerating dilution. % cumulative 98.34 84.6 76.0 70.3 58.9
drug release % % % %
The preceding blood destruction occurs, the more %
within 12
easily to see breakage in quality of RBC by osmosis. days
Hypotonic (0.9% NaCl) leads to a RBCs burst. A
membrane break appears, in hypotonic conditions,
permitting (a portion of the oxygen hemoglobin to
leave the cells. By determining the concentration of
hemoglobin, the destruction percentage of blood at
various NaCl concentrations can be evaluated. Figure 15The osmotic fragility test of normal and loaded red blood
cell. Percentage hemolysis plotted against concentration of sodium
Usually hemolysis begins at 0.45-0.5% NaCl and chloride. Data are expressed as mean±standard deviation, 3
conclude at 0.3-0.33% NaCl. Osmotic fragility samples in each group n=three. [19]

 Page 16
 Erythrocytes as a novel drug delivery system

potential modifications in cell membrane (reliable MCV values suggest macrocytic (large RBC size
and average). The reference range for MCV in
acceptable human quality/integrity or completeness) Adult/elderly/child is 80-96 FL/red cell. The normal
and the cells resistance to osmotic pressure of the erythrocyte gave an MCV value of 93 FL and
medium. Applying Paired t-test (to determine metformin loaded erythrocytes gave an MCV value
whether the mean difference between two sets of of 95 FL expressing a normal average RBC size in
observations is zero), shows that that the variance in spite of the loading stress.[19]
osmotic fragility of both unloaded and loaded is not
statistically critical, and the unloaded erythrocyte has  Stability studies
less osmotic fragility than the loaded erythrocyte. In
fig 15 percentage, hemolysis plotted versus To evaluate the resealed erythrocyte stability, they
concentration of sodium chloride. Data are have been stored in lyophilized state at 4°C. SEM
demonstrated as mean±standard deviation, three image showed a deformity in RBC shape and that F0
samples in every group n= 3.[19] formulas without Glutaraldehyde cross-linking were
Figure 16 Scanning electron micrograph image of normal erythrocytes. [19]

only stable for 28 days at 4°C in erythrosol solution.

 Cell counting and cell recovery The stability of erythrocytes was improved when cell
The recovered percentage of cells was calculated and storing was at powder form and all set to be
to be 73-78%, which concludes that the loading
procedure was barely abrasive.
Reconstituted at 4C˚. Investigations are still in
 Morphological studies progress for using freeze-drying of blood product.
Using JEOL JSM-6390 LA scanning electron [19][29][30][31]
microscope a SEM image was taken for normal and
loaded erythrocytes to figure out the morphological  Results
variations induced by endocytosis method.
Speculated RBCs (echinocytes, form when the The developed formulation displayed 98.34% of drug
surface area of the outer lipid monolayer increases release within 12 days. The data from in vitro release
relative to the inner monolayer.) were observed in revealed that zero order kinetic graph was the
unloaded RBCs this might be related to storage best-fit graph. Percentage cell recovered detected to
lesion. Echinocytes were limited number correlated be 73-78% indicating that the loading method was
to the normal shaped minor destructive.

discoid RBCs (discocytes). The SEM analysis results

8.2 Erythrocyte based bio adhesive system
indicate that RBCs have maintained their bio
concavity morphology after loading, suggesting that for nasal delivery of propranolol
endocytosis has limited destructive influence. The In this study, a characterization and evaluation of
SEM image of normal RBC is shown in Fig16.[19] Glutaraldehyde treated erythrocytes as carrier for
[29][30] propranolol Based on hypotonic pre-swelling, to be
delivered through the nasal route. Propranolol
hydrochloride is a beta-blocker, which characterized
 The mean corpuscular volume (MCV) to be fairly well absorbed through the nasal mucosa.
MCV expresses the red cells average volume in the [32]
sample. Its reading values are increased or decreased
in depending on the average size of the red cell. The 8.2.1 Erythrocyte separation
small average of RBCs size will produce low MCV Albino rat’s blood was obtained and anti-coagulated
values (microcytic), normal MCV (normocytic) using heparin. Followed by centrifugation for 5 min
obtained in normal cell size average, and elevated at 3000 rpm, 4°C. Remove the buffy coat and plasma
(supernatant), wash the isolated erythrocytes three

 Page 17
  Erythrocytes as a novel drug delivery system

time using isotonic phosphate buffered saline pH 7.4

composed of NACL, K2HPO4, and KH2PO4. Then mix
with PBS to (sufficient volume) to achieve
hematocrit 50%.[32]

8.2.2 Loading of propranolol HCI

Based on hypotonic preswelling method. By
centrifugation, recover 1ml of packed erythrocytes
followed by addition of 0.6% sodium chloride
solution 4ml.
Do second centrifugation at for 5 min 2000 rpm to
extract the swollen cells then lysing normal
erythrocytes in water 1: 1 to attain 100µl of cell
haemolysate, then add 10 mg/ml in water propranolol
HCL solution ( take 100µl aliquot) to the swollen  In vivo performance evaluation
cells placed in glass tube and gently mixed, Keep The performance of loaded-treated erythrocytes was
adding the solution to attain the point of lysis. in vitro evaluated for albino rats of weighing range
To restore the isotonicity attained by adding about 250g. Three groups of nine rats utilized for the
hypertonic saline solution. Incubate the cells for 15 evaluation. The rats were given pentobarbitone
min at 37 a˚ C followed by three time washing with intravenously as anesthesia (50 mg/kg body weight).
isotonic PBS to get rid of excess drug. The 1 mg of propranolol was given for every treatment.
propranolol-loaded erythrocytes stabilized by treating First treatment; for the first group 0.2 ml of
with Glutaraldehyde.[32] propranolol hydrochloride solution was given
intravenously through the caudal vein, second
treatment; for the second group of rats was given
8.2.3 Bioadhesion preparation propranolol HCL solution (0.43 ml in 0.9% saline )
nasally. The third treatment for the third group
Obtain a freshly cut 5-6 cm long piece of rat small
propranolol loaded erythrocyte suspension
intestine and washed with isotonic saline to be
Glutaraldehyde treated (0.43 ml; 0.2%) was given
cleaned, cut open the piece to expose the mucosal Figure 17 In vitro diffusion profile of propranolol across rat
nasally. The instillation of the suspension was drop
intestine from glutaraldehyde v/v: ◊ 0.1%, ○ 0.3%, • 0.5%)-
surface. Glutaraldehyde treated erythrocytes 0.2 ml,
wise using a syringe over in a duration
treated erythrocytes (▪control). of
Bars10at min
data to
points indicate +
30% HCT was evenly spread on the mucosal surface,
permit the bioadhesion
SD. [32] to take a place.
the piece was preserved in a desiccator for 30 min at
80% RH , then the piece get weighed, then flow a
specific volume of isotonic PBS overtop the
intestinal piece at approximate rate of 5 ml/ min.
collect and weigh the perfusate.
The percentage of bioadhesion was calculated by the
ratio of utilized amount to adhered erythrocytes.

8.2.4 Results and discussion

 Page 18
 Erythrocytes as a novel drug delivery system

After a wash out extent of seven days. Samples of

The blood were extracted repeatedly from the
femoral aorta. Then centrifuge 0.2 ml of plasma was
tested for propranolol content fluorimetrically.
As illustrated in table.4 Propranolol-loaded
Glutaraldehyde-treated erythrocytes were showed to
have a good bioadhesive characteristic, by the
elevating degree of crosslinking there was a small
decline in bioadhesion percentage. This result are
related to crosslinking density elevation of which
leads to reduction in active sites accessible for survey showed regulated release of propranolol from
mucoadhesion. erythrocytes and regulated absorption of drug
throughout the nasal mucosa. The recorded treatment
 In vitro release profiles of hemoglobin showed no primary or secondary maxima that linked
As shown in fig18 insignificant hemoglobin loss with oral administration. Which means drug shielding
detected from treated cells when stored for 24 h, and from first pass metabolism. [32][5][33][34]
for non-treated cells, 40% of hemoglobin detected to
be extracellular. Figure 18 In vitro hemoglobin loss from treated (• ) and non- treated (∆ )
erythrocytes. Bars at data points indicate + SD. [32]

 In-vitro across-the-intestine diffusion profile

As shown in fig17, 95% of the drug had diffused In
the case of control, at the end of 90 min. The drug
discharging out of the cross-linked erythrocytes
detected to diffuse gradually. The percent of diffused
drug decreased consecutively by the increasing
degree in crosslinking.

 in-vivo performance estimation of stabilized 9. Conclusion

erythrocytes
This review aimed to discuss in summery a novel
group1, propranolol maximum plasma concentration drug delivery system using erythrocytes as natural
(Cmax) observed to be 1920 + 22 ng/ml, and gradually biological carriers for effective, safe and efficient
decreased. The alteration in drug levels detected to be targeting of diverse bioactive molecules. Covering a
minimal (p< 0.05). group2, the nasal control, plasma revision of two studies, the first study intended to
survey was detected to be almost analogous to the develop a sustain release metformin loaded
I.V. survey. A lower Cmax (1206k 34 ng/ml) obtained. erythrocytes by the method of endocytosis, from the
Group3 the nasal administration detected to be study it was revealed that some amount of metformin
358±20 ng/ml at 120 min. The plasma levels detected encapsulated by the mechanism of diffusion. The
to be stable around the C max for 8-10 h. The plasma Table 4 Bioadhesion of propranolol loaded erythrocytes* and ** indicate
significantly different data (p<0.05). [32]

 Page 19
  Erythrocytes as a novel drug delivery system

loaded erythrocyte possessed all hematological,

criterions close to normal erythrocytes, so would stay
in the circulation with approximately the identical
half-life of normal erythrocyte, and the delay in the
release of metformin would reduce the dosing
repetitiveness. The second study aimed to develop a
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