Nanoerythropoietin Is 10-Times More Effective Than
Regular Erythropoietin in Neuroprotection in a Neonatal
Rat Model of Hypoxia and Ischemia
Han Chen, MD; Frédéric Spagnoli, MS; Michael Burris, BS; William B. Rolland, BS;
Adriel Fajilan, BS; Huanyu Dou, PhD; Jiping Tang, MD; John H. Zhang, MD, PhD
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Background and Purpose—Erythropoietin (EPO) has been demonstrated to possess significant neuroprotective effects in
stroke. We determined if the nano-drug form of human recombinant EPO (PLGA-EPO nanoparticles [PLGA-EPO-NP])
can enhance neuroprotection at lower dosages versus human recombinant EPO (r-EPO).
Methods—Established neonatal rat model of unilateral ischemic stroke was used to compare r-EPO, PLGA-EPO-NP and
phosphate-buffered saline, given by daily intraperitoneal injections, followed by infarction volume and Rotarod
Performance Test assessment.
Results—PLGA-EPO-NP significantly reduced infarction volumes 72 hours after injury compared with the same
concentrations of r-EPO. Functional deficits were significantly reduced by 300 U/kg PLGA-EPO-NP versus controls,
with deficit attenuation apparent at significantly lower dosages of PLGA-EPO-NP versus r-EPO.
Conclusions—PLGA-EPO-NP is neuroprotective and beneficial against deficits after brain ischemia, at significantly
reduced dosages versus r-EPO. (Stroke. 2012;43:884-887.)
Key Words: erythropoietin 䡲 focal ischemia 䡲 functional recovery 䡲 hypoxia 䡲 neonatal
E
rythropoietin (EPO), a key endogenous cytokine in
hypoxic physiological response, enhances oxygen delivery and attenuates brain injury in vitro and in vivo,1–5
promoting cell survival via the bcl antiapoptotic gene subfamily6 via limited-carrier blood– brain barrier passage, delaying critical timing in clinical stroke.3,7 Nano-medicine
enhances brain drug delivery8 up to 50 times,9 bypassing
usual blood– brain barrier routes and increasing clinical viability. With successful demonstration of significant neuroprotective function after systemic administration, it was noted
that elevated doses of EPO were needed for optimal protection against brain injury for EPO to pass in substantial doses
across the blood– brain barrier. EPO, a large glycosylated
molecule, is unable to pass directly across the blood– brain
barrier, requiring specific carrier transport and/or endocytosis.10 This type of transport is limited and critical time points
in clinical management may be lost during the delay of EPO
brain entry after systemic administration. Because recombinant EPO (r-EPO) has been previously demonstrated to have
long-term beneficial effects on the brain,11 the purpose of this
study was to compare the exact same r-EPO in nano forms
and non-nano forms for the relative effects on infarction and
behavior at different concentrations.
Materials and Methods
Human r-EPO, sharing 80% homology with rodent EPO, without report
of immunologic complications,6,12 was epoetin-alfa (Centocor Orth
Biotech). Poly-DL-lactide-coglycolide (PLGA; 3.07 g) and fluoresceinamine in 30 mL of acetonitrile with 0.0408 g 1-ethyl-3-(3Dimethylaminopropyl)-carbodiimide hydrochloride underwent 24 hours of
incubation, lyophilization, precipitate centrifugation, and water washing.
PLGA-EPO nanoparticles (PLGA-EPO-NP) nanoprecipitation
uses r-EPO double-emulsion solvent evaporation, primarily w/o emulsion of first aqueous phase (EPO 200 ␮L) with sonication of organic
phase 100 mg PLGA in ethyl acetate (5 mL), emulsification with
secondary aqueous phase (20 mL polyvinyl acetate; 1.5% weight/
volume phosphate-buffered saline) forming secondary water-in-oil-inwater emulsion, and continuous stirring at 1800 rpm. Nanoparticle
washing followed frozen dryer evaporation by ethyl acetate and water.
Perinatal Hypoxia-Ischemia Exposure Model
Procedures followed those of Rice-Vannucci12 with approval by the
Loma Linda University Institutional Animal Care and Use Committee.
Thirteen litters of Sprague-Dawley dams (Harlan Laboratories, Livermore, CA) at 10 days postnatal age (n⫽156) under isofluorane 1%, 0.7
L/min room air, 300 mL/min O2, and perioperative 38°C warming
underwent right common carotid artery 7-0 silk suture ligation (Ethicon)
around microscissor transection, all completed at 4 minutes, minimizing
anesthesia.13 After 1-hour recovery, mice underwent 75 minutes at 3.5
L/min humidified 8% O2, 92% N, and 36°C, preceded by 75 minutes of
4.0 L/min; 12.8% (20 of 156) of pups died during hypoxia.
Received August 24, 2011; accepted September 26, 2011.
From the Departments of Physiology and Pharmacology (H.C., F.S., M.B., W.B.R., A.F., J.T., J.H.Z.), Department of Neurosurgery (J.H.Z.),
Department of Anesthesiology (J.H.Z.), Loma Linda University, Loma Linda, CA; and Center of Excellence for Infectious Disease (H.D.), Department
of Biomedicine Sciences, Texas Tech University Health Sciences Center, El Paso, TX.
Correspondence to John H. Zhang, MD, PhD, Department of Neurosurgery, Loma Linda University, Loma Linda, CA 92324. E-mail
[email protected]
© 2011 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.111.637090
884
Chen et al
Neuroprotection in a Neonatal Rat Model
885
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Figure 1. A–C, Seventy-two hours after
injury (n⫽8): 30 U/kg poly-DL-lactidecoglycolide– erythropoietin nanoparticles
(PLGA-EPO-NP) averaged 20.2% vs
control (9.4%); 100 U/kg PLGA-EPO-NP
(12.6%) outperformed control (29.4%;
n⫽8; P⬍0.001); 300 U/kg PLGAEPO-NP (7.5%) outperformed 300 U/kg
r-EPO (23.9%) and control (29.4%) (n⫽8;
P⬍0.001); 300 U/kg PLGA-EPO-NP
approximated 5000 U/kg recombinant
EPO (r-EPO; 6.8%).
Experimental Groups
Results
100 U/kg r-EPO and vehicle (P⬍0.001; Figure 1B); and 300
U/kg PLGA-EPO-NP (7.5%) outperformed control and 300
U/kg (P⬍0.001; Figure 1C), approximating 5000 U/kg r-EPO
(6.8%; P⫽0.574). Rotarod 21 days after injury (n⫽8; Figure
2A, B), from 5 rpm (Figure 2A), 300 U/kg r-EPO, and
PLGA-EPO-NP outperformed control (P⫽0.009 and
P⫽0.004), with 300 U/kg PLGA-EPO-NP (41.5 s) approaching 5000 U/kg r-EPO (41.9 s). From 10 rpm (Figure 2B), 300
U/kg PLGA-EPO-NP outperformed 300 U/kg r-EPO
(P⫽0.011), approximating 5000 U/kg r-EPO and sham
(P⫽0.508 and P⫽0.214).
Brain weight ratios (ipsilateral/contralateral side to injury)
28 days after injury (n⫽8) showed 300 U/kg PLGA-EPO-NP
(Figure 3) approximated 5000 U/kg r-EPO. Three-hundred
U/kg PLGA-EPO-NP and 5000 U/kg r-EPO reduced loss
(15.1% and 12%) versus control (35.2%; P⬍0.001).
Thirty U/kg PLGA-EPO-NP (Figure 1A) averaged 20.2%
versus vehicle (29.4%) and 30 U/kg r-EPO (29.5%;
P⫽0.092); 100 U/kg PLGA-EPO-NP (12.6%) outperformed
Intravenous 100 000 IU r-EPO proved therapeutic in clinical
stroke.15 Capillary sludging risk prompted studies of EPO,
r-EPO and PLGA-EPO-NP, in 10 mmol/L phosphate-buffered saline
and 0.1% bovine serum albumin were injected intraperitoneally 1
hour after hypoxia and during 2 24-hour intervals, and were
randomly assigned across 9 groups (n⫽8): vehicle; 30 U/kg r-Epo
(n⫽7) or PLGA-EPO-NP; 100 U/kg r-Epo or PLGA-EPO-NP; 300
U/kg r-Epo or PLGA-EPO-NP; 5000 U/kg r-Epo; and sham.
Seventy-two hours after injury, 2-mm brain slices in triphenyltetrazolium chloride at 37°C were analyzed by Image J (National
Institutes of Health, Bethesda, MD).
For longer-term evaluation of comparative effect of nano forms
and original forms of the same r-EPO, Rotarod14 (9 groups of 8
animals) 21 days after injury involved analysis of 3 falling latency
trials accelerated from 5- or 10-rpm velocities. Data expressed as
mean⫾standard error of the mean were analyzed using SigmaStat
(SyStat),with P⬍0.05 as statistically significant.
Infarction Volumes
Discussion
886
Stroke
March 2012
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Figure 3. Twenty-eight days after injury. Y-axis: hemispheric
weight ratios of ipsilateral vs contralateral to injury (n⫽8); 300
U/kg PLGA-EPO-NP and 5000 U/kg r-EPO (15.1% and 12%)
outperformed control (35.2%).
Conclusions
Figure 2. A and B, Twenty-one days after injury (n⫽8), 5 m/s
starting: 300 U/kg recombinant erythropoietin (r-EPO) and polyDL-lactide-coglycolide– erythropoietin nanoparticles (PLGA-EPONP) outperformed vehicle (P⫽0.009 and P⫽0.004); 10 m/s starting: 300 U/kg PLGA-EPO-NP outperformed 300 U/kg r-EPO;
300 U/kg PLGA-EPO-NP (33.4 s) approximated 5000 U/kg
r-EPO (36.8 s) and sham (39.9 s). P⫽0.508 and P⫽0.214.
novel EPO forms,1 and EPO receptors to develop neuroprotective forms lacking hematopoietic functions. 1– 6
Nanoformulation stabilizes EPO and facilitates blood–
brain barrier crossing with controlled release, improving
efficacy. PLGA-EPO-NP was effective at 16-times lower
dosage (300 U/kg) than 5000 U/kg EPO.3,7 PLGA-NP
delivery includes siRNA, proteins, antibodies, antibiotics,
and cancer treatments.8,9 The present study demonstrates
that the beneficial effects of EPO are enhanced at lower
dosages when loaded to a polymeric NP carrier. EPO has
been previously demonstrated to offer neuroprotective
value against several types of brain injury, including
ischemic stroke, neuronal degeneration, and apoptosis.16 –18
Presently, dosages of up to 400 IU/kg EPO are administered as clinical treatment for neonatal anemia.19 Elevateddose r-EPO has been successfully used in a clinical acute
stroke trial with significant long-term recovery benefit
seen at 1 month involving intravenous administration of
100 000 U of r-EPO over 3 days.15
In summary, the benefits we have observed here are the same
as those of r-EPO previously published in the literature,
except that in this case the same r-EPO has an attached
nano-carrier, PLGA-EPO-NP, which allowed for observation
of the beneficial effects at much lower dosages of the same
r-EPO, with 300 U/kg PLGA-EPO-NP having significant
effect comparable to 5000 U/kg without the nano-carrier.
Disclosures
None.
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Nanoerythropoietin Is 10-Times More Effective Than Regular Erythropoietin in
Neuroprotection in a Neonatal Rat Model of Hypoxia and Ischemia
Han Chen, Frédéric Spagnoli, Michael Burris, William B. Rolland, Adriel Fajilan, Huanyu Dou,
Jiping Tang and John H. Zhang
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Stroke. 2012;43:884-887; originally published online December 8, 2011;
doi: 10.1161/STROKEAHA.111.637090
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