Formulation and Drug Loading Features of Nano-Erythrocytes
© The Author(s). 2017
Received: 13 December 2016
Accepted: 6 March 2017
Published: 17 March 2017
Nano erythrocyte ghosts have recently been used as drug carriers of water-soluble APIs due to inherit biological characteristics of good compatibility, low toxicity, and small side-effect. In this study, we developed a novel drug delivery system based on nano erythrocyte ghosts (STS-Nano-RBCs) to transport Sodium Tanshinone IIA sulfonate (STS) for intravenous use in rat. STS-Nano-RBCs were prepared by hypotonic lysis and by extrusion methods, and its biological properties were investigated compared with STS injection. The results revealed that STS-Nano-RBCs have narrow particle size distribution, good drug loading efficiency, and good stability within 21 days. Compared with STS injection, STS-Nano-RBCs extended the drug release time in vitro and in vivo with better repairing effect on oxidative stress-impaired endothelial cells. These results suggest that the nano erythrocyte ghosts system could be used to deliver STS.
KeywordsSTS Erythrocyte ghosts Drug delivery Repairing effect
To date, numerous nanoparticles (NPs) delivery has been used to deliver drugs, including polymeric NPs, liposome, microspheres, cell and cell derivatives . As potential drug delivery systems, cell and cell derivatives have been widely researched recently, such as erythrocyte (RBC), tumor cell, stem cell, macrophage, dendritic cell. [2–4]. Among them, erythrocyte widely attracted the attention of researchers. Comparing with other cell drug carriers, erythrocyte is easier to be separated, with better biological compatibility as well as the inherent biological degradation. Besides that, it also has the remarkable long life span of about 3 months in the body . Therefore, small-molecule drugs (doxorubicin , dexamethasone sodium phosphate ), enzyme (pegademase, adenosine deaminase , L-Asprraginase ), nucleoside (FdUMP , antisense oligodeoxynucleotides ), and nanoparticles  have been encapsulated in erythrocyte. In recent years, nanoparticles derived from erythrocyte (Nano-RBCs), have strongly attracted the attention of investigators due to the virtue inherited from their parent cells. Besides that, nanoparticles contain therapeutic molecule which could camouflaged themselves with erythrocyte membrane to reduce toxicity . Researches revealed that polymeric nanoparticles loaded in Nano-RBCs showed longer circulation time in blood than PEG-medicated nanoparticles . Besides, coating with Nano-RBC membrane could protect the original nanoparticle from being uptake by macrophage . 1–1.5 × 109 erythrocyte ghosts can split into 1.6 × 1012 nanoparticles without considering the loss of erythrocyte membrane. Nanoparticles derived from erythrocyte have high surface area-volume ratio, and its diameter can be small to 100 nm, which helps them easily transmit in vivo . The popular method of Nano-RBC preparation is using hypotonic medium to make erythrocyte change to erythrocyte ghost and then split to smaller vesicles with ultrasonic bath, finally extrude the vesicles through polycarbonate membrane from different pore sizes to unified size gradually [14, 15]. Besides, tip sonicator could also split the Nano-RBCs to around 100 nm directly . So far, a variety of nanoparticles [14, 15] and a small-molecule weight drug (Fasudil, paclitaxel, and doxorubicin) [17, 18] have been encapsulated into Nano-RBC, such as vaccine encapsulated in polymeric nanoparticles and coated with Nano-RBC was developed to prevent the melanoma . Besides, the Nano-RBC with gold nanocages was prepared to improve the efficiency of the photothermal therapy .
In present study, we developed a novel STS-loaded nano-erythrocyte system which was extracted from Sprague dowley (SD) rats. The influence factors of preparation process were investigated to optimize the manufacture process. The stability was tested with the index of particle size and loading efficiency. In vitro and in vivo release behaviors were analyzed to predict its pharmacological effect. Since STS has a protecting function against the oxidative stress induced injury , an oxidative stress-impaired model was established with EA.hy926 endothelial cells to assess the repair efficiency of STS-Nano-RBCs and STS injection.
STS, heparin sodium, 30% hydrogen peroxide solution (H2O2, 30 wt.% in water), ethyl p-hydroxybenzoate, acetonitrile, and DMSO were made by Aladdin (Shanghai, China). Red blood cell lysis buffer, kaumas blue, cell lysis buffer for western and IP, Nitric Oxide Assay Kit, Total Superoxide Dismutase Assay Kit with WST-8, Lipid Peroxidation MDA Assay Kit, and Reactive Oxygen Species Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). Amicon Ultra-4 Centrifugal Filter (30 kDa) and Nuclepore Track-Etched Membrane were purchased from Millipore (Boston, USA). 0.5% Triton X-100, 3-(4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide (MTT), 1,1-Dioctadecyl −3,3,3,3-tetramethylindodicarbocyanine (DiD), were purchased from Keygen Biotech (Nanjing, China). FITC-dextran (70 kDa) was purchased from TdB Consultancy (Ferndown, UK). Dialysis cassettes (3500 Da) were purchased from Viskase Companies (Houston, USA). 1 × PBS and 10 × PBS (pH 7.4) were purchased from HyClone (USA), fetal bovine serum (FBS), penicillin (10 kU/ml), streptomycin (10 μg/ml) (Penicillin-Streptomycin Solution, 100×), 0.25% Trypsin-EDTA (1×), and Gibco Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Invitrogen (USA). The dialysis tube (width is 10 mm, molecular cut-off is 3500 Da) was purchased from Viskase (USA).
Animals and Ethics
The Sprague Dawley rats, 8 weeks of age, were purchased from the Experimental Animal Center of Southern Medical University of China (SCXK (Guangdong) 2012–0015) and were raised under specific pathogen-free (SPF) condition in the Animal Center of Guangdong Pharmaceutical University. The animal experiments involved in present study were consistent with the guidelines set by the National Institutes of Health and were approved by the Guangdong Experimental Animal Ethics Committee.
The human umbilical vein cell line, EA.hy926, was established by fusing primary human umbilical vein cells with a thioguanine-resistant clone of A549 by exposure to polyethylene glycol (PEG). The EA.hy926 cells used in present study were donated by professor Li Ming of the Foundation College of Guangdong Pharmaceutical University.
HPLC Analysis Method
HPLC method was established by our lab and was used for quantitative determination of STS. The experiment was performed with a Shiseido-ODS C18 column (4.6 × 150 mm); the mobile phase was consisted of methanol and phosphate buffer (pH 3.5) (60:40 v/v); the flow rate was 1 ml/min, and the UV detected wavelength was 271 nm. The temperature of column heater was maintained at 40 °C, and 20 μl sample solution was injected for analysis. The limit of detection (LOD) of STS by HPLC method was 0.02 μg/ml, and the limit of quantitation (LOQ) was 0.06 μg/ml.
Preparation of STS-Nano-RBCs
STS-Nano-RBCs were prepared with the method reported previously . To prepare erythrocyte ghosts, firstly, blood collected from SD rat put into tube with heparin sodium. Then centrifuged to discard serum and buffy coat, washed RBC three times with 1 × PBS (pH 7.4) carefully to obtain packed RBC. After that, 1 volume packed RBC was incubated with 4 volumes hypotonic 0.1 × PBS at 4 °C for 15 min to obtain erythrocyte ghosts. The erythrocyte ghosts were centrifuged at 2000 rpm for 10 min and washed 3 times with 0.1 × PBS until the supernatant was colorless.
To prepare STS-Nano-RBCs, 1 volume STS (dissolved in 0.1 × PBS) is added into the erythrocyte ghosts and is incubated at 4 °C for 1 h with constant shaking by table concentrator to load STS. Subsequently, 10 × PBS was added to reseal the erythrocyte ghosts at 4 °C for 1 h. The resealed erythrocyte ghosts were washed three times and were resuspended in 1 × PBS. The particle size of drug loaded erythrocyte ghosts was reduced with extrusion method. The erythrocyte ghosts contained STS were extruded 21 cycles by the extruder (ATS engineering Canada) with different size of Nuclepore Track-Etched Membrane (800, 400, and 200 nm). The free STS was removed by ultrafiltration using Amicon Ultra-4 Centrifugal Filter at 5000 rpm for 30 min and was washed with 1 × PBS to obtain the final STS-Nano-RBC system.
In order to screen the optimal drug loading conditions, different influence factors were investigated including the STS concentration (0.2–2.5 mg/ml), loading temperature (4 and 37 °C), loading volume ratio (1:2–3:1), osmotic pressure (0.1 × PBS and 0.3 × PBS) as well as STS loading time.
Characterization of STS-Nano-RBCs
To test whether the erythrocyte external aqueous phase could enter into the internal phase when erythrocyte ghosts sealing in hyperosmotic solution, two different fluorescent dyes were used for observation with fluorescence microscope (Carl Zeiss Jena, Germany). Red fluorescent dye DiD (10 μg/ml) and green fluorescent dye FITC-dextran (70 kDa, 100 μg/ml) were incubated with erythrocyte ghosts for 30 min to stain the membrane and cytoplasm, respectively. The structure of STS-Nano-RBCs was examined with a transmission electron microscope. A drop of the sample solution was deposited onto a glow-discharged carbon-coated grid. 5 min later, the grid was rinsed with 10 drops of distilled water. A drop of 1% uranyl acetate stain was added to the grid. The grid was subsequently dried and visualized under a HITACHI H-7650 microscope.
Particle Size and Zeta Potential
Beckman Coulter (LS13320, USA) was employed to measure the particle size, size distribution, and surface zeta-potential of STS-Nano-RBCs. The test sample was prepared by diluting STS-Nano-RBCs with 1 × PBS into proper concentration, and the tests were performed in triplicate at room temperature.
To investigate the content of membrane protein in erythrocyte and the STS-Nano-RBCs, red blood cell lysis buffer was used to lyses the test samples and was extracted the total protein. SDS-PAGE method was used to analyze the type of proteins. In short, erythrocyte and STS-Nano-RBCs were prepared in SDS sample buffer. The samples were then ran on a polyacrylamide gel electrophoresis apparatus (BioRad, USA) at 100 V for 2 h. Finally, the gel was stained with kaumas blue for 1 h then was visualized using electrophoretic imaging system (Aplegen Omega Lum G, USA).
STS Loading Efficiency Assay
Where, M in = the entrapment amount of STS in STS-Nano-RBCs, M total = the total amount of STS in STS-Nano-RBCs.
The centrifugal stability of STS-Nano-RBCs was assessed by centrifuging samples 10 min with different speed (2000–12000 rpm). The turbulence stability of STS-Nano-RBCs was also evaluated. In short, STS-Nano-RBC was passed through a 4.5-gauge needle with the flow rate of 10 ml/min, which was comparable with the blood flow rate in vivo. The number of passes was varied (10–30 times). Next, 10 K MWCO Amicon Ultra-4 Centrifugal Filters were used to isolate free STS for HPLC analysis. STS leakage rate was used as the index to evaluate the system stability. Samples were stored at 4 °C for 21 days to investigate its storage stability, and at given time point, indexes including appearance, drug leakage rate, particle size, and zeta potential were used to evaluate the short-term stability of STS-Nano-RBCs.
In Vitro Release
The drug release behavior of STS-Nano-RBCs system was evaluated with dialysis method in PBS (pH7.4) at 37 °C. 1 ml concentrated STS-Nano-RBCs was placed into dialysis tube and immersed in 250 ml PBS. The dialysis tube is 10 mm wide, and the molecular cut-off is 3500 Da. At each given time point, 1 ml released solution were collected and were replenished with equal volume of PBS solution. For comparison, the release of STS injection was also tested. All collected solution was analyzed with HPLC method described before.
In Vivo Pharmacokinetics and Biodistribution Study
Pharmacokinetic characteristics of STS-Nano-RBCs were conducted in adult SD rats. 12 SD rats were divided into 2 groups, and test samples were intravenously injected through rat tail vein with the dose of 5 mg/kg; the concentration of STS-Nano-RBC and STS was 1.1 mg/ml. At each time points, the blood was collected and centrifuged (2000 rpm for 10 min) to separate the plasma. After that, 50 μl ethyl p-hydroxybenzoate (1 μg/ml) and 1 ml acetonitrile were added into plasma to extract STS. The mixture was vortex-mixed for 1 min followed by centrifugation at 10000 rpm for 10 min. The supernatant was evaporated under nitrogen atmosphere condition and was redissolved with 200 μl mobile phase before HPLC assay. The pharmacokinetic parameters were calculated from the plasma concentrations of STS with DAS2.0 software system. The pharmacokinetic parameters estimated were maximum plasma concentration (C max), half-life (T 1/2), the area under the plasma concentration-time curve (AUC), and the mean residence time (MRT).
Next, we investigated the tissue distribution of STS-Nano-RBC in SD rats to further evaluate its potential as a delivery vehicle. We performed the biodistribution study on 8 week-old SD rats (190–210 g, n = 12 per group, half male, half female). SD rats were injected STS-Nano-RBC or STS via tail vein, each rat was injected with approximately 1.0 mg of STS injection or STS-Nano-RBC. At each time point (12, 24, and 36 h) post-injection of drug, blood was collected from the rat eye vein plexus, four rats of each group were euthanized, and their organs (hearts, livers, spleen, lungs, kidney) were extracted and weighed. STS content in each group was measured with HPLC method described before.
MTT assay was conducted to identify the biocompatibility of STS-Nano-RBCs. EA.hy926 cell was seeded at the density of 10000 cells per well in 96-well plate and with 100 μl DMEM. When 80% cells confluence was observed, the growth media were replaced with equal volume of test samples (STS, Nano-RBCs and STS-Nano-RBCs, all dissolved in DMEM) with various concentrations for 24 h. Then, the media were refreshed and were incubated with 20 μl MTT (5 mg/ml in PBS) for another 4 h. Finally, the culture medium was removed, 100 μl of DMSO was added, and the absorbance was measured at 570 nm with a microplate reader (BioRad, USA).
Protective Effect on Oxidative Stress-Impaired Cell
In order to study the repairing effect of STS-Nano-RBC on oxidative stress impairs EA.hy926 cell, we adopted the method published before . In short, an oxidative stress-impaired model was established with 750 μM H2O2 to evaluate the protective and to repair effect of STS-Nano-RBCs on oxidative stress-impaired cell. For nitric oxide (NO), superoxide dismutase (SOD) and MDA assay, EA.hy926 cells were seeded in 96-well plate with suitable density. When 80% cell confluence was observed, the growth media was replaced with 100 μl H2O2 (750 μM) and was incubated for 12 h. Then the media was replaced with various concentration of STS-Nano-RBC. 12 h later, the supernatant (or cell lysis solution) was collected and analyzed with Nitric Oxide Assay Kit, Total Superoxide Dismutase Assay Kit, and Lipid Peroxidation MDA Assay Kit. The repair efficiency of STS-Nano-RBCs was also observed with fluorescence microscope.
All experimental data in this study were shown as mean ± standard deviation (SD). Statistical comparisons were calculated by using Student’s t test, p values of <0.05 was taken to indicate statistical difference.
Results and Discussion
Preparation and Characterization of STS-Nano-RBCs
One of the most important advantages to use erythrocytes as a drug delivery system is that the functional components contain immunosuppressive proteins on erythrocyte membranes, which could inhibit macrophage uptake and therefore prolonged the circulation time . As shown in Fig. 4, SDS-PAGE analysis result revealed that in both samples, erythrocyte ghosts contain all major protein fractions: α-spectrin, β-spectrin, actin, glyceraldehyde-6-phosphate dehydrogenase, stomatin-tropomyosin, and peroxiredoxin and reduced globin chains. However, the result also revealed that several major bands of protein color in Nano-RBC group was weaker than in RBC group, which means the preparation process might not cause the loss of protein species, but could cause the loss of some protein content.
Stability of STS-Nano-RBCs
The parameters of storage stability of STS-Nano-RBCs
Drug loading efficiency (%)
Zeta potential (mv)
160.2 ± 4.5
163.2 ± 5.7
166.9 ± 3.3
174.4 ± 3.4
In Vitro and In Vivo Drug Release and In Vivo Biodistribution
Pharmacokinetics parameters calculated by DAS2.0 software
C max (μg/ml)
T 1/2 (h)
AUC0−∞ (μg/ml · h)
Where A = 2.423, B = 1.687, α = 0.856, β = 0.093.
Subsequently, we used SD rats to conduct the in vivo biodistribution study. Figure 7c, d showed the STS content per gram of tissue, at 12 h after the injection, STS-Nano-RBC showed hepatic and splenic uptake of 7.5 and 6.1 μg/g tissue, respectively, as compared to 6.4 and 5.2 μg/g tissue by STS injection. After accounting for the tissue mass, it can be observed that with the injection time extended, STS-Nano-RBC observed in the liver and spleen was increased, while in blood was decreased. Liver and spleen, as the two primary organs of RES, contained the highest amount of STS-Nano-RBCs, which also explained the fast blood elimination of STS-Nano-RBC.
It is well known that the cycle time of erythrocyte in rats is about 1 month, compared with autologous RBC, the sustained release effect of STS-Nano-RBC was quite limited. Combined with Fig. 4, it can be speculated that the surface protein of erythrocyte, not only the protein species but also the content, played an important role on its cycle time. The surface glycoproteins of erythrocyte, such as CD47, as the signal molecule, could protect RBCs from damage and elimination from body by inhibiting the phagocytosis of macrophage through binding to SIRPα. Loss or just decrease of surface glycoproteins of erythrocyte, such as CD47, could inhibit the “do not eat me” signal, which will cause STS-Nano-RBC to be phagocytosed by macrophage . Besides, Nano-RBC, as the derivative of RBC, destructed the integrity of mother cell, and the process of drug encapsulation typically require multistep manipulations involving cell isolation, incubations for several times, and washings, which could harm the cell membrane and thus lead to transposition of PS from the inner leaflet of the plasma membrane to the RBC surface, which might accelerate elimination of Nano-RBC in rats . In addition, the effect of polymeric nanoparticles on biocompatibility of carrier erythrocyte has been investigated before; the result indicated that non-covalent adsorption of model NPs (200 nm) to mouse and human RBC could affect their sensitivity to osmotic stress, low level shear stress, vigorous mechanical insult, and agglutination. Although the result showed non-covalent adsorption of model NPs to mouse and human RBC is not detrimental at ratios of and below NP/RBC 200:1, it inspired us to assume that whether Nano-RBC attached on RBC in rats increases the RBC susceptibility to complement-mediated lysis .
Cell Viability Assay
Application safety is a prerequisite for drug delivery system, and here, the cell cytotoxicity was tested to evaluate the biocompatibility of STS-Nano-RBCs. EA.hy926 cells were chosen to assess the cell viability of STS-Nano-RBC with MTT method. As shown in Fig. 8b, the concentration of STS-Nano-RBCs was ranged from 10 to 500 μg/ml. The MTT results indicated Nano-RBCs showed little cytotoxicity to EA.hy926 cells. Cell viability of both STS injection group and STS-Nano-RBC group were decreased with the increase of STS concentration. It was worth noting that when the concentration of STS was higher than or equal to 100 μg/ml, STS-Nano-RBCs group showed more cytotoxicity than STS group. This might be due to the difference of STS concentration in cells at a given time. It is well known that the way in which small-molecule weight drug and nanoparticles enter cells is different, which might cause different concentration of STS at a given time and thus result in different cell viability. Since cell survival rate was less than 50% when STS-Nano-RBC concentration was greater than or equal to 200 μg/ml, we selected 10–150 μg/ml as the concentration range to examine the protection of STS against oxidative stress injury cells.
Repairing Effect of STS-Nano-RBCs on Oxidative Stress Impairs EA.hy926 cell
In this study, we developed a drug carrier system of nano erythrocyte ghosts to deliver water-soluble drug STS and to investigate its biological characteristics. STS loaded into nano erythrocyte ghosts was found to exert several benefits including good stability, sustained drug releasing behavior, as well as bioavailability improvement. It could also extend in vivo circulation time. Based on the pharmacological experiment, it is evidence that STS-loaded nano erythrocyte ghosts could upgrade the repairing effect of damaged endothelial cells and can serve as a drug delivery system to deliver STS.
- AUC0−∞ :
Area under the curve
- C max :
Maximum plasma concentration
Mean retention time (h)
Nanoparticles derived from erythrocyte
Reactive oxygen species
Sodium Tanshinone IIA sulfonate
- t1/2 :
Half-life of STS in plasma
Vascular endothelial growth factor
We would like to thank the Sun Yat-sen University Test Center for its assistance in this study.
This work was financially supported by Guangdong Natural Science Foundation (2014A030310362), Science and Technology Program of Guangzhou (201508010036) and the IAR Collaborative Innovation Project of Guangzhou (201605131249066).
LHQ and XTD designed the experiments. XTD carried out the experiments, analyzed the data, and wrote the paper. YWN and YD analyzed the data, YWN, YMW, WL, and JLZ assisted in the preparation and characterization of STS-Nano-RBC. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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