IN
VITRO
STUDIES
OF
AMIKACIN-LOADED
HUMAN
CARRIER
ERYTHROCYTES.
Carmen Gutiérrez Millán1, Bridget E. Bax2, Aránzazu Zarzuelo Castañeda1, María
Luisa Sayalero Marinero1, José M. Lanao1*
1.- Department of Pharmacy and Pharmaceutical Technology. Faculty of Pharmacy.
University of Salamanca. Salamanca. Spain.
2.- Carrier Erythrocyte Research Laboratory, Child Health, Department of Clinical
Developmental Sciences, St. George´s University of London. London. United Kingdom,
SW17 0RE.
* Corresponding author. Prof. José M. Lanao. Department of Pharmacy and Pharmaceutical
Technology. Faculty of Pharmacy. University of Salamanca. 37007. Salamanca. Spain. Email: jmlanao@usal.es. Phone: 34-923-294536. Fax: 34-923-294515.
1
Abstract
Erythrocyte-encapsulated antibiotics have the potential to provide an effective therapy against
intracellular pathogens. The advantages over the administration of free antibiotics include a
lower systemic dose, reduced toxicity, a sustained delivery of the antibiotic at higher
concentrations to the intracellular site of pathogen replication, and an increased efficacy. In
this study the encapsulation of amikacin by human carrier erythrocytes prepared using a
hypo-osmotic dialysis was investigated. The effects of the initial amikacin dialysis
concentration and hypo-osmotic dialysis time on the encapsulation efficiency of amikacin
were determined, and the osmotic fragility and haematological parameters of amikacin-loaded
carrier erythrocytes were measured. The efficiency of amikacin entrapment by carrier
erythrocytes was dependent on the initial dialysis concentration of the drug. Statistically
significant differences in the osmotic fragility curves between control and carrier erythrocytes
osmotic fragility curves were observed which were dependent on dialysis time and the
dialysis concentration of amikacin. Mean haematological parameters were evaluated and
compared to unloaded, native erythrocytes; the MCV (mean corpuscular volume) of amikacin
loaded-carrier erythrocytes was statistically significant smaller. Amikacin demonstrated a
sustained release from loaded erythrocytes over a 48-hour period suggesting a potential use of
the erythrocyte as slow systemic release system for antibiotics.
Keywords: Amikacin, hypo-osmotic dialysis, human carrier erythrocytes, antibiotics,
monocyte macrophage system.
2
Running head: In vitro evaluation of amikacin-loaded erythrocytes
Abbreviations: RES, reticulo-endothelial system; MPS, mononuclear phagocytic system;
HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome; ATP,
adenosine 5´-triphosphate; HPLC, high performance liquid chromatography; EDTA,
ethylenediaminetetraacetic acid; OPA, o-phthaldialdehyde; TCA, trichloroacetic acid; IU,
international units; PBS, phosphate buffered saline; MCV, mean corpuscular volume; MCH,
mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; RSD,
relative standard deviation; ANOVA, analysis of variance.
3
1. Introduction
Erythrocytes have been used as carriers for drugs, enzymes and other
macromolecules.1-3 This has been a research field in constant development due to its
important potential use.4-7
The advantages of these cellular carriers in comparison with other traditional carriers
are their biocompatibility and lack of toxicity, their potential to selectively transport the drug
to the monocyte-macrophage system (previously known as the reticulo-endothelial system,
RES) of the liver, spleen and bone marrow, and their ability to behave as circulating
bioreactors in a sustained fashion.8-14
Several methods have been employed to encapsulate drugs and other molecules, such
as enzymes and peptides, within erythrocytes and the method of encapsulation and conditions
employed have been shown to affect the in vitro and in vivo properties of the erythrocyte
carrier. The hypo-osmotic dialysis procedure has been shown to be preferable in terms
preserving the biochemical and physiological characteristics of the erythrocyte. 3, 10-12, 15-18
Within the field of antibiotics, apart from modifying the pharmacokinetic behaviour of
the encapsulated drug, the carrier erythrocyte has the potential to specifically target antibiotics
to the mononuclear phagocytic system (MPS), that can act as a reservoir for facultative
intracellular pathogens,19 such as Mycobacterium.20 Pathogens located in the intracellular
compartment, or phagosome, typically receive a lower concentration of antibiotic than the
concentration which is administered systemically, and therefore
for optimal efficacy
relatively high concentrations are required, which often lead to adverse side effects.
Among different pathogens residing within the monocyte-macrophage system, HIV
must be emphasized, due to the important consequences of the AIDS latency. Different anti-
4
viral agents have been encapsulated within carrier erythrocytes, achieving optimistic results
for the targeting of drugs to macrophages.21-23
In previous studies we optimized the conditions for the encapsulation of the
aminoglycoside antibiotic, amikacin, in rat erythrocytes, using a hypo-osmotic dialysis
procedure.24 We have demonstrated that rat loaded carrier erythrocytes have haematological
and morphological properties similar to the unloaded native erythrocytes, and that the loaded
erythrocytes demonstrated a moderate change in osmotic fragility, and exhibited a sustained
in vitro release.
Further in vivo studies with rats revealed changes in the pharmacokinetics of the
amikacin when it is administered within carrier erythrocytes, with a greater accumulation of
the antibiotic in RES organs in comparison with the administration of free drug,
demonstrating the potential use of this system in intracellular infections treatment.25
Here we report studies on the encapsulation of amikacin by human carrier
erythrocytes; the encapsulation efficiency of amikacin using different hypo-osmotic dialysis
times and different initial concentrations of antibiotic were investigated; the haematological
parameters and osmotic fragility of the loaded carrier erythrocytes were evaluated and the in
vitro release of amikacin from carrier erythrocytes was measured over time.
2. Materials and Methods
2.1. Materials
The chemicals used in this study were as follows: NaCl (Panreac), KCl (Merck),
Na2HPO4·12H20 (Panreac), KH2PO4 (Panreac), MgCl2·6H2O (Panreac), MgSO4·7H2O
(Sigma), glucose (Panreac), NaH2PO4·2H2O (Panreac), NaHCO3 (Panreac), ATP (Sigma),
reduced glutathione (Sigma), amikacin sulphate (Sigma), sodium pyruvate (Sigma), inosine
5
(Sigma), adenosine (Sigma), methanol for HPLC grade gradient (Merck), mercaptoethanol
(Sigma),
isopropanol
(Panreac),
K3-EDTA
(Panreac),
o-phthaldialdehyde
(Sigma),
trichloroacetic acid (TCA) (Panreac), methylene chloride (Merck), NaOH (Panreac),
anhydrous sodium carbonate (Panreac).
2.2. Instrumentation
Centrifugations at 4ºC were performed in a Denley BR401 Refrigerated Centrifuge,
and those at room temperature were performed using a Beckman GPR Centrifuge.
Incubations for the hypo-osmotic dialysis and resealing steps were made in specially
modified LabHeat refrigerated incubators (BoroLabs Ltd, Berks, U.K.) set at 4oC and 37oC,
respectively.
Absorbance values for the osmotic fragility studies were determined using an
Ultrospec 3100 pro (UV/Visible Spectrophotometer) and osmolalities were measured using an
Osmometer automatic Roebling.
High performance liquid chromatography were performed using a Shimadzu liquid
chromatograph LC-10 AD with a SCL-10 A system controller and a SIL-10 AXL auto injector.
Fluorescence was analyzed with RF-10 AXL fluorescence detector and a column oven 480
Kontron was used to guarantee the 37ºC temperature.
2.3. Blood collection and preparation
Whole blood was collected from healthy individuals into tubes containing heparin (10
IU/mL) and used within a few hours of collection. Informed consent was obtained from all
donors. The blood was centrifuged for 10 minutes at 4oC at 1,100 x g. After removal of
plasma and the buffy coat, erythrocytes were washed twice in cold (4oC) phosphate buffered
6
saline (PBS; 136.89 mmol/L NaCl, 2.68 mmol/L KCl, 8.10 mmol/L Na2HPO4, 1.47 mmol/L
KH2PO4, pH 7.4) with centrifugation for 10 minutes at 1,100 x g.
2.4. Carrier erythrocyte preparation
Energy-replete carrier erythrocytes were prepared using the hypo-osmotic dialysis
technique of Bax et al.
10,12
Briefly, seven volumes of washed and packed fresh erythrocytes
were mixed with three volumes of cold PBS containing a pre-determined concentration of
amikacin; a 5-mL aliquot of this cell suspension was then placed in a dialysis bag (molecular
mass cut-off of 12000 Da, Medicell International Ltd.).
Dialysis was performed against 150 mL of hypo-osmotic buffer pH 7.4 (5 mmol/L
KH2PO4, 5 mmol/L K2HPO4) containing different concentrations of amikacin (0.2, 1, 2 and
10 mg/mL), at 4ºC with rotation at 6 r.p.m. for 90, 120 and 180 minutes (St. George’s
Erythrodialyser, LabHeat, BoroLabs Ltd, Berks, U.K.). The lysed erythrocytes were resealed
by transferring the dialysis bag to a container holding 150 mL of PBS supplemented with 5
mmol/L adenosine, 5 mmol/L glucose and 5 mmol/L MgCl2 and continuing rotation at 6
r.p.m. at 37ºC, for 60 minutes. The energy-replete carrier erythrocytes were washed three
times in 3 volumes supplemented PBS, with centrifugation at 100 x g for 15 minutes.
Experiments were performed in triplicate.
2.5. Measurement of haematological parameters
Mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular
haemoglobin concentration were determined using an AcT 8 Coulter counter.
7
2.6. Osmotic fragility
The osmotic fragility of native erythrocytes and amikacin-loaded carrier erythrocytes
was determined according to the Dacie´s method. 26,27
Twenty microlitres of washed and packed cells were incubated in 0.5 mL NaCl
solution of varying concentrations (0 to 0.9% w/v) for 30 minutes at room temperature
(25oC). The suspension was centrifuged at 1,100 x g for 10 minutes and the supernatant
assayed spectrophotometrically at 540 nm for haemoglobin. The amount of haemoglobin
released into distilled water was taken as 100% haemolysis and the haemoglobin released in
the NaCl solutions was expressed as a percentage of this maximal haemolysis.
An osmotic fragility index was defined for native and amikacin-loaded erythrocytes.
This index was calculated as the NaCl concentration (% w/v) necessary for obtaining 50% of
haemolysis in the conditions described above.
Osmotic fragility was studied for each dialysis, and was determined in triplicate for
each dialysis time and initial amikacin concentration.
2.7. In vitro release
To study the in vitro release of amikacin from carrier erythrocytes, loaded erythrocytes
were resuspended in autologous plasma to a final haematocrit of 30%. The suspensions were
incubated at 37ºC with gentle stirring, removing aliquots at the following times: 5, 15, 30, 60,
120 minutes, 24 and 48 hours. The aliquots were centrifuged at 1,100 x g for 10 minutes and
the supernatant removed from the erythrocytes. The concentration of amikacin released into
the plasma was determined as described below.
8
In vitro release studies were performed in triplicate with carrier erythrocytes prepared
using 180 minutes of hypo-osmotic dialysis time and 10 mg/mL of initial amikacin
concentration.
2.8. Quantification of carrier erythrocytes encapsulated amikacin.
Amikacin concentration in loaded carrier erythrocytes and in autologous plasma were
determined using a high performance liquid chromatography (HPLC) technique based on precolumn derivatization with o-phthaldialdehyde (OPA) and fluorescence detection. 28
Treatment of the samples, prior to their chromatographic separation was as follows:
100 L of sample plus 100 L of TCA (20%) were mixed and vortexed for 30 seconds and
then centrifuged for 5 minutes at 5,000 x g. Following this, the supernatant was removed, 100
L of 1 M NaOH plus 1 mL of phosphate buffer (pH 11) were added and the mixture was
stirred for 30 seconds. Two mL of methylene chloride were then added, the mixture shaken
for 10 seconds, centrifuged for 5 min at 5,000 x g, and the aqueous phase collected. To the
latter, 1 mL of OPA reagent was then added, the mixture shaken, and 500 mg of anhydrous
sodium carbonate added, and then shaken for 30 seconds. Finally, 500 L of isopropanol was
added to extract the derivatized amikacin; after centrifuging for 5 min at 5,000 x g, 20 L of
the isopropanol extract was injected into the chromatograph. Chromatographic analysis was
performed using a Shimadzu LC 10 AD chromatograph using an ion-pair technique and a
LiChroCART® 55-4 (Purospher RP-18e, 3 m) column.
The mobile phase was methanol (67 %) and EDTA aqueous solution (2.2 g/L, 33 %).
Flow rate was set to 1 mL/min. Fluorescence detection was accomplished at excitation and
emission wavelengths of 360 and 435 nm, respectively. The technique was linear, exact
(recuperation 100.80 %  3.58) and accurate (RSD<10%) for the range of 50-1000 g/mL of
9
amikacin. Higher concentration samples were conveniently diluted with isotonic saline for
analysis.
2.9. Data analysis
Statistical analysis of data corresponding to amikacin encapsulation, osmotic fragility
and haematological parameters was performed using a two-way ANOVA using the SPSS
statistical software. 29
3. Results
Table 1 shows the mean encapsulated amikacin concentration in carrier erythrocytes
as a function of initial dialysis antibiotic concentration and dialysis time. A higher
encapsulation efficiency of amikacin was associated with higher concentrations of drug in the
dialysis bag (p<0.01).
This dependence is also showed in figure 1, where the surface-plot of the encapsulated
antibiotic concentration simultaneously represented versus dialysis concentration and dialysis
time makes clear the simultaneous influence of both variables on the efficiency entrapment of
amikacin.
Figure 2 shows the osmotic fragility of native erythrocytes and carrier erythrocytes
obtained using 90, 120 minutes and 180 minutes of hypo-osmotic dialysis. A two-way
analysis of variance demonstrates a simultaneous dependence of the osmotic fragility with
dialysis time (p<0.05) and initial concentration of amikacin (p<0.01). This dependence is
more obvious in figure 3, where the amikacin concentration and the dialysis time are
10
simultaneously represented for a specific NaCl concentration (0.50 %, w/v). This surface-plot
of the measured haemolysis versus dialysis amikacin concentration and dialysis time shows
even more clearly the dependence of the osmotic fragility of carrier erythrocytes on dialysis
time and initial amikacin concentration.
Table 2 also shows the osmotic fragility index (NaCl concentration required for 50%
haemolysis) for carrier erythrocytes obtained with different dialysis conditions, this being the
index for native erythrocytes 0.45 ± 0.01 % (w/v). A statistically significant decrease (p<0.01)
in the osmotic fragility index was observed which was both dependent on the initial amikacin
concentration and dialysis time.
Table 3 shows the mean haematological parameters of the amikacin loaded carrier
erythrocytes obtained with the different dialysis conditions in comparison with values for
these parameters in the same cells before undergoing the encapsulation procedure. Compared
to unloaded, pre-dialysis erythrocytes, the amikacin loaded carrier erythrocytes had a
statistically reduced mean corpuscular volume (MCV) (p<0.05) dependent on initial amikacin
concentration.
As the main aim of this work was to demonstrate the usefulness of carrier erythrocytes
as a sustained delivery system, studies of in vitro release were performed.
Taking into consideration the encapsulation yield, 180 minutes of hypo-osmotic
dialysis and a dialysis amikacin concentration of 10 mg/mL were chosen for the preparation
of carrier erythrocytes for in vitro release studies.
Figure 4 shows the curve of amikacin release from loaded erythrocytes into autologous
plasma, over a 48-hour incubation period.
An initial rapid leakage of 3 % of the total amount encapsulated, followed by
sustained release of the antibiotic in a 48-hour period was observed.
11
4. Discussion
In earlier studies of amikacin encapsulation within rat erythrocytes, we have achieved
a moderate encapsulation yield with slight modifications on haematological parameters.24
This work here was performed in order to conduct a more in depth study of the potential of
human red cell as a delivery vehicle for amikacin.
Table 1 and figure 1 show the encapsulation of amikacin in carrier erythrocytes as a
function of hypo-osmotic dialysis initial concentration of amikacin and the hypo-osmotic
dialysis times. Considering that the amikacin uptake by carrier erythrocytes is a passive
process, we can conclude that the conditions in which the hypo-osmotic dialysis is performed
may influence the encapsulation yield, and a dependence on the initial concentration of drug
and dialysis time was observed.
Osmotic fragility is not a measure of the strength or mechanical properties of the
erythrocyte membrane. The main factor in this procedure is the shape of the erythrocyte,
which is dependent on the volume, surface area and functional state of the erythrocyte
membrane. The altered profile of the osmotic fragility curve for carrier erythrocytes is
presumably due to a reduction in the MCV of these cells.
As it can be seen in figure 2 for the different NaCl concentrations tested, a dependence
of the release of haemoglobin with the initial concentration of the drug (p<0.01) and the hypoosmotic dialysis time (p<0.05) was observed. Osmotic fragility index is higher for native
12
erythrocytes than the index estimated for amikacin-loaded erythrocytes and dependent both
on the initial concentration of amikacin and dialysis time.
These results suggest that during the loading procedure (dialysis, resealing, washings,
etc.) the most fragile cells are removed and the most robust carrier erythrocytes remain, as it
has been already observed in previous studies for other molecules encapsulated.12
The haematological parameters evaluated for the different dialysis conditions
employed revealed human carrier erythrocytes had only significant changes in mean
corpuscular volume in comparison with native cells, implying a good in vitro haematological
behaviour for the amikacin-loaded erythrocytes.
The in vitro release of amikacin from carrier erythrocytes in autologous plasma reveal
the existence of a rapid release of a small amount of antibiotic, 0.15 ± 0.02 mg per 1010 cells
(3 % of the encapsulated drug), maintaining a similar amount for the first 2 hours. The rapid
release of a fraction of the encapsulated drug could be attributed to the drug remaining on the
surface of the cell membrane. This rapid leakage of part of the encapsulated drug has been
reported previously for other drugs such as actinomycin D from human erythrocytes and for
amikacin from rat carrier erythrocytes. 24, 30
Most of the encapsulated amikacin (80 % approximately) remains retained in
erythrocytes for long periods of time, in 48 hours there was 1.52±0.34 mg per 10 10 cells in the
supernatant, and this allows the prediction of a sustained release of the antibiotic from the
erythrocytes with a half-life of 22.15 hours. It could also suggest a higher RES targeting of
the drug when the loaded erythrocytes still containing the most of the amikacin encapsulated
are phagocyted by cells of the monocyte-macrophage system. Alteration of the carrier
erythrocyte membrane to promote an enhanced uptake by the monocyte-macrophage system
could potentially achieve higher intracellular concentrations of amikacin. The use of the
specific targeting would allow a more effective antimicrobial therapy against specific
13
intracellular pathogens such as M. tuberculosis. The initial slow release of the antibiotic
would be due to amikacin being a polar drug and that, once entrapped inside the erythrocyte,
would not be able to diffuse across the cell membrane. The release must occur as a result of
cell lysis, as it has been observed with other aminoglycosides.31 These results demonstrate the
suitability of human carrier erythrocytes use as a sustained release system for amikacin
administration and as a potential delivery system to target the antibiotic to the monocytemacrophage system. The potential systemic toxicity which may arise from an intravascular
release however would need to be investigated.
Acknowledgements
The authors wish to express their appreciation to the volunteers at St. George’s
Hospital, London, who kindly donated blood for this study. The support of Department of
Health and Human Sciences of London Metropolitan University with HPLC is also
acknowledged.
This paper was supported by the National Research and Development Plan (Projects
SAF 2001-0740 and PI 06152) from the Spanish Government. These studies were supported
also by a discretionary grant held by Dr Bridget Bax.
14
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18
Figure legends
Figure 1: Surface-plot of encapsulated amikacin in carrier erythrocytes versus initial amikacin
concentration and dialysis time.
Figure 2: Osmotic fragility of native erythrocytes and carrier erythrocytes obtained by 90
minutes of dialysis time, 120 minutes and 180 minutes. Statistically significant differences for
initial amikacin concentration (p<0.01) and dialysis time (p<0.05).
Figure 3: Surface-plot of carrier erythrocyte haemolysis versus initial amikacin concentrations
and dialysis time, using 0.50% NaCl solution.
Figure 4: Mean released amikacin versus time from human loaded erythrocytes.
19