Loaded red blood cells as natural micro carriers for drug delivery

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Loaded red blood cells as natural micro carriers for drug delivery
Sternberg N, Georgieva R, Abdallah Z, Müller A and Bäumler H
BCRT, Charité- Universitätsmedizin Berlin, Germany
* supervisor # contact email [email protected]
The development of suitable drug delivery systems offers a huge number of advantages in contrast
to conventional administration of therapeutics: reduction of toxicity and adverse side effects by
keeping the blood concentration within the therapeutic window, protection from premature
inactivation and loss of its systemic activity (Rossi L, 2005). A number of attempts have been made
to develop controlled drug transport systems using biodegradable and non-immunogenic carriers,
through which therapeutic substances can be disseminated throughout the body via the circulating
blood stream. Additionally, modifications of the carrier’s surface allow direct targeting of specific
tissues and organs. Red blood cells (RBC) represent a potential natural drug carrier system due to
the ability of their membranes to be opened and resealed (Brähler M, 2006). The morphological,
mechanical and biological properties make the RBC an ideal vehicle for a broad spectrum of drugs,
enzymes and any other kinds of biologically active substances (DeLoach JR, 1986). The presence
of a huge amount of functional groups on the cell surface is a great additional advantage giving
many possibilities for attachments of specific peptides or antibodies for targeting the desired site of
action (Muzykantov V, 1994). The matured cells are highly specialised for the transport of oxygen
and carbon dioxide between the lung and tissues whereas they cover a distance of 250 km in the
cardiovascular system. RBC are biconcave shaped cells with a mean diameter of 7 to 8 µm and a
thickness of approx. 2 µm. Due to their large surface to volume ratio (140 µm2 / 90 µm3) they are
very elastic and thus able to pass the smallest capillaries. This extraordinary flexibility, the size,
shape, integrity and durability of the RBC membrane is controlled by proteins of the cytoskeleton
(Anderson RA, 1981). Human erythrocytes have a definite life span of about 120 days. The RBC
membrane consists of 20% phospholipids which are asymmetrically distributed across the bilayer.
Whereas phosphatidylcholin and sphingomyelin are essential parts of the outer leaflet,
phosphatidylethanolamin and first of all phosphatidylserin (PS) are mainly found on the inner
surface. RBC damage or normal cell aging causes a translocation and presentation of PS on the
outer membrane to designate the cell for phagocytosis (Bratosin D, 1998). Hence, controlled
artificial aging of the erythrocyte membrane allows specific targeting of the RES whereas the
circulation time of carrier RBC in most cases depends on the membrane integrity and thus on the
loading procedure (Magnani M, 2002).
Here we present studies focussed on optimization of the loading conditions in order to improve a
long circulation time. For this we studied the effect of different loading procedures on the integrity
of the cell membrane encapsulating model substances (BSA) and nanoparticles (QD, AmphBnanosuspension). Subsequently, surface modification by the model peptide insulin was performed
in order to provoke an uptake of the surface modified and loaded RBC by RBE4 cells in cell
Human RBC were loaded with FITC-labeled bovine serum albumin (FITC-BSA), quantum dots
(QD) and AmB- nanosuspension using several hypotonic dilution methods (preparations A-F). The
Poster P74 – page 1
XVIIth International Conference on Bioencapsulation, Groningen, Netherlands ; September 24-26, 2009
loading was carried out in hypoosmotic buffer (e.g. with osmolartity at 50% rate of hemolysis,
Figure 1) at 4°C. The general loading procedure is schematically illustrated in Figure 2. Freshly
drawn whole blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) was used to isolate
the RBC by centrifugation. To produce white ghosts (preparation A) the RBC were incubated in
20mOsm/kg lysing buffer (NaH2PO3/Na2HPO3) at pH8 and washed till no hemoglobin was left in
the cells. Red ghosts were only washed once (preparation B). We implemented a pre-swelling
procedure for the samples C and E. RBC were incubated with a drug of interest in the hypoosmotic
environment till cell swelling and pore opening has taken place. In preparations A and B the
isotonicity was reconstituted in with phosphate buffered saline (PBS, pH8). For preparations C and
D the cells were lysed at an osmolarity of 70mOsm/kg with PBS and 1M potassium chloride was
used for resealing. In preparations E and F the resealing buffer was PIGPA-C (containing adenine,
glucose, inosine and pyruvate). After reconstitution of isotonicity and resealing of the membranes,
the drug has been entrapped inside. The membrane damage was quantified by Annexin V binding
intensity in which loaded and control RBC were incubated with the FITC-labeled protein.
Successful drug encapsulation, loading efficiency and RBC membrane integrity was investigated by
means of confocal laser scanning microscopy (CLSM), freeze-fracture scanning electron
microscopy (SEM) and flow cytometry (FACS).
hem olysis [% ]
XVIIth International Conference on Bioencapsulation, Groningen, Netherlands ; September 24-26, 2009
human RBC
osmotic swelling
and pore opening
NP loaded
osmolarity [osm/kg]
Figure 1: rate of hemolysis
resealing at
Figure 2: RBC loading procedure
Figure 3: Surface modification of RBC
After loading, the RBC surface was doped by
fluorescently labeled insulin (Figure 3).
Therefore insulin (4) was first conjugated with
hydroxysuccinimide ester of the dye PKPF488 or -633 (5). Then the peptide was linked
to sulfo-hydroxysuccinimide-biotin (2). In
parallel, the amino- groups of the RBC
surface were biotinylated the same way and
then conjugated with avidin (3). Finally, RBC
(1) and insulin were linked via the biotinavidin bridge.
The insulin-modified und natural RBC were incubated with a rat brain endothelial cell culture
(RBE4). The cells were grown in DMEM with 10% FCS and 1% penicillin/streptomycin inside of
an Ibidi µ-Slide 8well chamber and incubated for 24 hours with RBC. The surface modification of
erythrocytes and interaction with endothelial cells were studied by CLSM. The immune response
caused by carrier-RBC was analysed using Phagotest® and Phagoburst® assays.
Poster P74 – page 2
XVIIth International Conference on Bioencapsulation, Groningen, Netherlands ; September 24-26, 2009
Human RBC were loaded with BSA, NPs or phosphate buffered saline (PBS) as a control sample
using different loading strategies (A-F). For examination of the membrane integrity affected by the
loading procedure, an Annexin V binding test was performed and analysed by FACS. The diagram
(Figure 4) shows the fraction of Annexin V labelled RBC in dependence on the used loading
method (A: white ghosts; B: red ghosts; C, D: KCl-resealing; E, F: PIGPA-C-resealing). As
illustrated the red and white ghost preparations evoke the most intense binding of Annexin V due to
the strongest disturbance of the cell’s integrity during loading. However, white ghosts (sample A)
show the highest loading efficiency of about 99,7%. Samples C and E (subjected to a pre-swelling
procedure) show a higher PS translocation in comparison to the corresponding samples without preswelling (D, F). In contrast to preparation A, sample C only shows a loading efficiency of about
85%. Sample F demonstrates the lowest Annexin V binding rate at all with an effective amount of
FITC-BSA encapsulated similar to that achieved in samples A (about 96%). Additionally, this
sample arouses less than 1% phagocytosis or burst activity by granulocytes and monocytes in
heparinised whole blood samples. Consequently, this drug loading procedure (F) appears as the
most gentle and favourable loading procedure possibly achieved by re-energizing of the RBC with
glucose and inosine of the PIGPA-C reversal buffer.
Annexin V Test
XVIIth International Conference on Bioencapsulation, Groningen, Netherlands ; September 24-26, 2009
complement system in vivo and would no longer exhibit a useful carrier system for targeted drug
delivery. In addition, the sterical accessibility of the residual peptide-binding sites will be lost too.
The consequence would be a decrease in the insulin-binding capacity. Therefore, an optimal surface
density of the biotin-avidin bonds is essential for the success of a targeted drug delivery via loaded
The peptide attached to the surface was tested concerning its ability to cause uptake of the carrier
erythrocytes by endothelial cells. Figure 6 illustrates RBE4 cells incubated with FITC-BSA loaded
RBC (a) and with FITC-BSA loaded RBC additionally modified by attachment of insulin-PKPF633
(b). Whereas surface-modified erythrocytes appear to be incorporated and destructed by endothelial
cells, plain FITC-BSA loaded RBC can only be found in the surrounding. Thus, surface
modification with insulin seems to be an eminent tool for targeting and enhancing uptake of carriererythrocytes into endothelial cell.
RBC were successfully loaded with diverse model substances such as AmB- nanosuspension, QD
and FITC-BSA. Different loading strategies were used to achieve the appropriate surface
characteristics according to the desired particular application. The surface of loaded erythrocytes
was additionally modified with insulin via the biotin-avidin pair. The insulin-modified surface of
the carrier RBC provoked an uptake by endothelial cells confirming the possibility of specific cell
targeting by this strategy.
Annexin+cells [%]
Loading procedures
Figure 4: Annexin V test
Figure 6 : cell-cell interaction
Figure 5 : loaded and modified RBC: a)-c)
CLSM micrographs; d)-f) Freeze-fracture
Figure 5 displays some examples of differently loaded and modified RBC: rat RBC with insulinPKPF633 doped cell membrane (a) and with additional FITC-BSA loading (b); human RBC loaded
with QD and coated with insulin-PKPF488 (c); (a-c CLSM images); freeze-fracture scanning
electron microscopy of pure AmB- nano-suspension (Staedke V, 2009) (d), unloaded (e) and AmBloaded human RBC (f;). The CLSM pictures show normally shaped RBC, where the labelling of the
membrane was successful. An optimal amount of bound peptide without any deformation of the cell
by linking the surface to biotin and avidin could be observed. A biotin overload together with
subsequent attachment of avidin caused a strong shape transformation from discocytes to
echinocytes. Under conditions of biotin excess on the membrane multivalent binding of avidin
occurs. This leads to alterations and damages of the RBC membrane and finally to a deformation of
the cell. Such modified RBC would be immediately eliminated by macrophages and by the
Poster P74 – page 3
• Anderson RA et al. (1981) Erythrocyte membrane sideness in lectin control of the Ca2+-A23187mediated discocyte-echinocyte conversion. Nature 292; 158-161
• Brähler M et al. (2006) Magnetite-Loaded Carrier Erythrocytes as Contrast Agents for Magnetic
Resonance Imaging. Nano letters 6 (11) 2505-2509.
• Bratosin D et al. (1998) Cellular and molecular mechanisms of senescent erythrocyte
phagocytosis my macrophages. A review. Biochimie 80, 173-195.
• DeLoach JR. et al. (1986) Carrier erythrocytes. Med Res Rev. 6(5) 487-504.
• Magnani M. et al. (2002) Erythrocyte-mediated delivery of drugs, peptides and modified
oligonucleotides. Gene Therapy 9; 749-751.
• Muzykantov V. et al. (1994) Attachment of biotinylated antibody to red blood cells: antigenbinding capacity of immunoerythrocytes and their susceptibility to lysis by complement. Anal
Biochem. 223(1):142-8.
• Rossi L. et al. (2005) Erythrocyte-based drug delivery. Expert Opin Drug Deliv. 2(2) 311-322
• Staedke V. et al. (2009) In vitro Inhibition of Fungal Activity by Macrophages Mediated
Sequestration and Release of Encapsulated Amphotericin B - Nanosupension in Red Blood Cells.
Small – submitted.
Poster P74 – page 4

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