Single intranasal administration of 17ß-estradiol loaded gelatin nanoparticles confers Neuroprotection in the post-ischemic brain
Elizabeth Joachim, Radwa Barakat, Benjamin Lew, Kyekyoon (Kevin) Kim, CheMyong Ko, Hyungsoo Choi
PII: S1549-9634(20)30100-3
DOI: https://doi.org/10.1016/j.nano.2020.102246
Reference: NANO 102246
To appear in: Nanomedicine: Nanotechnology, Biology, and Medicine Revised date: 11 May 2020
Please cite this article as: E. Joachim, R. Barakat, B. Lew, et al., Single intranasal administration of 17ß-estradiol loaded gelatin nanoparticles confers Neuroprotection in the post-ischemic brain, Nanomedicine: Nanotechnology, Biology, and Medicine (2020), https://doi.org/10.1016/j.nano.2020.102246
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© 2020 Published by Elsevier.
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Single Intranasal Administration of 17ß-Estradiol Loaded Gelatin Nanoparticles Confers Neuroprotection in the Post-Ischemic Brain
Elizabeth Joachima, Radwa Barakatb,c, Benjamin Lewd, Kyekyoon (Kevin) Kim*a,d, CheMyong Ko*b, Hyungsoo Choi*d
Author Affiliations
a Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; b Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; c Department of Toxicology and
Forensic Medicine, Faculty of Veterinary Medicine, Benha University, Qalyubia, 13518, Egypt;
d Department of Electrical and Computer Engineering, University of Illinois at Urbana- Champaign, Urbana, IL, 61801, USA
Word count (abstract): 150/150 Word count (manuscript): 3316/5000 Figures: 4
References: 41 Tables: 1
*Corresponding authors:
Kevin Kim and Hyungsoo Choi
Thin Film and Charged Particle Research Lab 306 N Wright St
Urbana, IL 61801 Phone: 217-333-7162
Email: [email protected], [email protected] CheMyong Ko
College of Veterinary Medicine 2001 S Lincoln Ave
Urbana, IL 61801 Phone: 217-333-9362 Email: [email protected]
Funding: This work was supported in part by the American Heart Association.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflict of interest.
This work was presented in part as a poster abstract at the American Heart Association Scientific Sessions 2018.
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Abstract
Globally, ischemic stroke is a leading cause of death and adult disability. Previous efforts to repair damaged brain tissue following ischemic events have been hindered by the relative isolation of the central nervous system. We have developed a gelatin nanoparticle-mediated intranasal drug delivery system as an efficient, non-invasive method for delivering 17-estradiol (E2) specifically to the brain, enhancing neuroprotection, and limiting systemic side effects.
Young adult male C57BL/6J mice subjected to 30 min of middle cerebral artery occlusion (MCAO) were administered intranasal preparations of E2-GNPs, water soluble E2, or saline as control 1 h after reperfusion. Following intranasal administration of 500 ng E2-GNPs, brain E2 content rose by 5.24 fold (p<0.0001) after 30 min and remained elevated by 2.5 fold at 2 h (p<0.05). The 100 ng dose of E2-GNPs reduced mean infarct volume by 54.3% (p<0.05, n=4) in comparison to saline treated controls, demonstrating our intranasal delivery system’s efficacy.
Key words: gelatin nanoparticles, estrogen, MCAO, intranasal delivery
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Background
Neurologic disorders, such as ischemic stroke, take a significant toll on their victims, as well as burden society as a whole. An estimated 6.2 million people died from stroke in 2015, making it the second leading cause of death globally.1 In the United States, ischemic stroke is a leading cause of severe adult disability and 5th leading cause of death with an estimated total annual cost of $33.9 billion in 2012.2 As the population continues to age, the annual cost of stroke is
projected to be $184.1 billion by 2030.2 Interestingly, loss of wages is anticipated to be the single biggest contributor to the total economic burden of stroke, indicating that merely increasing stroke survival is insufficient; preventing long-term disability should be of paramount concern.
Clinical data indicating that pre-menopausal women have lower stroke risk, but that elderly women have more severe strokes, poorer recovery, and greater long-term disability,3 spurred intense interest in the potential neuroprotective effects of estrogen. Physiological doses of estrogens have shown powerful neuroprotective abilities in numerous animal models of stroke, including transient and permanent ischemia, global and focal ischemia, and even subarachnoid hemorrhage (SAH).4-6 However, seemingly in direct contrast with the animal model data, clinical trials have shown higher cardiovascular risk and worse stroke outcomes for women on estrogen replacement therapy (ERT).3 Ultimately, differences in timing and route of administration seem to be at the root of this disparity between clinical and preclinical results.5 For example, previous studies in rodents have found that increasing the time between ovariectomy surgery, which stops endogenous estrogen production, and 17-estradiol (E2) pretreatment decreased E2’s protective effects in the post-ischemic brain.7 The hypothesized mechanism for such decreases in efficacy is a combination of tissue specific changes in estrogen receptor (ER) expression7 and sensitivity8
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following the termination of endogenous estrogen production. Therefore, development of an estrogen therapy that can be used in the acute phase of stroke treatment, regardless of
endogenous estrogen status, would be beneficial. We propose that using the intranasal (IN) route would confer the neuroprotective benefits seen with other E2 administration routes while being safe, patient-friendly, and, therefore, clinically relevant in the acute stages of stroke treatment. IN drug delivery is under investigation as a convenient method to rapidly administer therapeutics to the central nervous system (CNS) by allowing for the passage of materials along the trigeminal and olfactory nerves effectively bypassing the blood brain barrier (BBB).9-11
Gelatin, a natural polymer of hydrolyzed collagen, is a biocompatible and biodegradable
hydrogel that can be formed into mucoadhesive nanoparticles.12, 13 Gelatin nanoparticles (GNPs) can be used to encapsulate a variety of therapeutics with the release profiles manipulated by changing particle size and crosslinking.14, 15 Because E2 is nonpolar and hydrophobic, it does not complex well with GNPs in the aqueous environment required for IN administration. GNPs can instead be loaded with water-soluble 17-estradiol (WS-E2), which contains 40-55 mg E2 per gram of solid 2-hydroxypropyl--cyclodextrin (-CD). The cyclic oligosaccharides composing
-CD form a hydrophilic exterior shell with a hydrophobic interior cavity that can complex with
hydrophobic drugs and improve their solubility in an aqueous nanoparticle environment.16 -CD can also be conjugated to free amine groups on gelatin before forming GNPs, allowing for direct loading of pure E2. Both methods of E2 loading were investigated here.
Methods
Fabrication and in vitro analysis of GNPs
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β-CD-conjugated GNPs (β-CD-GNPs) with 5 or 10 w/w% β-CD were prepared from Type A gelatin using a modified desolvation method at 50°C and pH 2.5, as previously described.13, 17 GNPs were crosslinked with 0.0625% glutaraldehyde for 2 hours at 25°C. The nanoparticle morphology and size were characterized by scanning electron microscope (SEM) and dynamic light scattering (DLS), respectively. Zeta potential of blank particles was also measured with Malvern Zetasizer (n=3). Fourier transform infrared (FTIR) spectra of raw gelatin, 5% β-CD- GNPs, and β-CD were recorded with Nicolet Nexus 670 FTIR spectrophotometer (n=3). For the in vitro release study, 5% and 10% -CD-GNPs were loaded with E2 in a ratio of 1:60 for 2 h at
25°C. As a comparison, unmodified GNPs were loaded with water-soluble E2 (WS-E2) to form WS-E2-GNPs. Loading efficiency was determined by UV-vis absorption at 280 nm (n=3). After a 2 h loading period, samples were immersed in 1 mL PBS with collagenase (1.24 µg/mL) and incubated in a shaking incubator at 37°C. At each time point, supernatant samples were collected and replaced with fresh PBS. The amount of released E2 in PBS was determined by UV-Vis absorption at 280 nm (n=4).
Quantification of endogenous and delivered estrogen
Baseline E2 content in the brain and gonads of young adult male wild type (WT), global
aromatase knockout (Ar -/-), and heterozygote (Ar +/-) mice on a standard diet or after at least 2 weeks on a soy-free diet (Teklad 2920X, Envigo) was determined by ELISA (DRG Int.).18 For the biodistribution study, we measured the E2 concentration in the brain, serum, gonads, lung, liver, and spleen of WT male mice with intact neurovasculature at 30 and 120 min after IN administration of E2 loaded -CD-GNPs (E2-GNPs), with untreated control animals designated as 0 min. For each mouse, 500 ng E2 in 1 µL EtOH is added to 50 µg of -CD-GNPs and
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allowed to dry for 30 min before hydrating with 5 µL PBS for 2 h prior to IN administration.
Mice were euthanized by CO2 fixation and blood samples drawn directly from the heart before harvesting the remaining tissues. All samples were flash frozen in liquid nitrogen and stored at - 80°C until bulk processing. The total lipids were extracted from solid tissue samples
as previously described with few modifications19 and then 17-estradiol content was determined by ELISA (DRG Int.) with a reportable range of 0–200 pg/mL.18
MCAO surgery
Mice were housed on a 12 h light-dark cycle with ad libitum access to food and water. Animal procedures were performed with strict adherence to all national and institutional guidelines and were approved by the University of Illinois’ Institutional Animal Care and Use Committee (protocol #15152). For the stroke model, 3-4 month old male C57BL/6J mice (wild type, WT) from the Jackson Laboratory and age matched male estrogen receptor knockout mice
(ERKO) bred in house (average weight 27.95 ± 2.12 g) underwent 30 min of transient MCAO using a modified Koizumi technique, as described previously.20 Pre- and post-operative blood glucose measurements were made by sampling the lateral tail vein. Post-operatively, mice were housed individually in heated cages with food pellets, water, 15% glucose solution, and food mush freely available and analgesia (0.5 mg/kg carprofen in 1 mL warm saline, subcutaneous) was administered daily. The sham procedure and care are identical except the filament is not inserted.
Intranasal administration
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Minimally anesthetized mice were placed in a supine position in a custom, tube-shaped holder that exposes the nares while inhibiting other movements 1 h after reperfusion. Once restrained, a plastic pipette tip was used to administer 2.5 µL drops to alternating nares.17 Intranasal
treatments in wild type mice were 50, 100, and 500 ng of E2 loaded into 25 µg of -CD-GNPs (E2-GNPs) in 5 µL PBS, 100 ng of WS-E2 in 5 µL PBS, or 5 µL PBS as control with sham mice receiving PBS. ERKO mice received 100 ng E2-GNPs in 5 µL PBS or 5 µL PBS as control.
Infarct volume measurement
Mice were euthanized by CO2 asphyxiation and cervical dislocation at 48h post-MCAO. Six 1 mm coronal sections were collected from each brain using fresh razor blades (Lord Super Stainless) and a pre-chilled mold (acrylic, Zivic Instruments). Slices were immediately stained by immersion in 1% 2,3,5-triphenyl tetrazolium chloride (TTC) at 37℃ for 10 min and fixed in 4% paraformaldehyde. Infarct volumes were automatically determined from images of scanned brain slices using the R programming language (www.r-project.org).
Behavioral testing
Sensorimotor and cognitive tests were performed over the course of the experiment to assess motor coordination. Training and recording of baseline performance on behavioral tasks began one day before MCAO surgery; animals were then tested 24 and 48 h post-MCAO. Three tests, the open field, y-maze, and pole tests, were performed as described previously.21, 22 Briefly, the pole test is a useful method for evaluating the mouse’s movement disorder caused by any cortical damage. The mice were placed on the top of the pole and observed as they make their way down to the cage. A sliding or falling mouse indicates motor impairment. In addition, open field test
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was used to measure anxiety and locomotor activity. The open-field apparatus consisted of a box (50×50×22 cm) with the floor divided into 16 squares. The “border” was defined as the 12 outer periphery squares and the “center” as the four inner squares, as described previously.23 Each mouse was placed at the center of the apparatus and allowed to move freely for 5 minutes. The open field was cleaned with 70% ethanol to remove odors from the previous mouse between trials. It is well known that rodents show natural avoidance of open surfaces.24 The time taken before the first entry to the center squares and the number of entries to the center square area were evaluated as an index of anxiety. The Y-maze spontaneous alteration test was used to evaluate the spatial memory performance and locomotor activity. The Y-maze was composed of three equal arms with walls15 cm high. Each mouse was placed in one of the arm compartments (A, B, or C) and was allowed to move freely for 5 minutes. The sequence of arm entries was manually recorded as A, B, or C. The percentage alternation is calculated as [(actual alternations /maximum alternations) x 100].22 The total number of arm entries was also recorded as an indication of general locomotor activity of the mice. Testing and training were always done in the first half of the light cycle to reduce the impact of circadian rhythms on performance.
Additionally, subjects were allotted a rest period of at least 10 min between the different tests.
Statistical analysis
Two-sample comparisons were performed using the Student’s t-test and multiple comparisons by one-way or two-way analysis of variance (ANOVA) followed by Tukey post hoc testing
implemented with R (www.r-project.org). Results are presented as mean±SEM and statistical difference was accepted for p-values <0.05.
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Results
In vitro analysis of GNPs
The unaltered GNPs, 5% β-CD-GNPs, and 10% β-CD-GNPs exhibited spherical morphology (Figure 1A and B) with an average diameter of 298.3 ± 99.4 nm, 315.7 ± 96.8 nm, and 362.3 ± 151.4 nm, respectively. Zeta potentials of unaltered GNPs and 10% β-CD-GNPs were 25.3 ± 4.42 mV and 8.13±4.80 mV, respectively. FTIR indicated that β-CD was successfully conjugated with gelatin as the 5% β-CD-GNP sample had the same set of peaks as those in β-CD at 1028, 1070, 1150 and 3371 cm-1 (Supplementary Figure 2). These peaks represent CO stretching vibration in primary alcohols, cyclic alcohols, glucosidic bonds, and the symmetric and
antisymmetric OH stretching modes of pure β-CD, respectively.25-27 The loading assessment in Table 1 indicates significantly higher E2-loading and loading efficiency for 5% and 10% β-CD- GNPs (85.2% and 95.5% efficiency, respectively) compared to unaltered GNPs (17.4%
efficiency).
Figure 1C shows the in vitro release profiles for WS-E2-GNPs, 5% β-CD-GNPs, and 10% β-CD- GNPs. Initial burst release was observed for all samples within the first few hours as un-
complexed E2 rapidly diffused out of the GNPs. WS-E2-GNPs had complete E2 within 12 hours; while only about 72% and 63% of E2 were released from 5% and 10% β-CD-GNPs, respectively. As we hypothesized, WS-E2 was rapidly released from GNPs under aqueous conditions, whereas incorporating -CD into the GNP matrix extended E2 release to over 48 h with full release not occurring until 120 h. As -CD content did not impact the release rate, we used 5 w/w% -CD-GNPs for all further studies.
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Endogenous and delivered estrogen content
Estrogen content in the brain and gonads of WT mice was not statistically different in animals on a standard or estrogen free diet (Figure 2A). Quantification of brain and gonad samples in WT and Ar+/- animals demonstrated no differences between genotypes (Figure 2B), indicating that a single copy of CYP19 is sufficient for normal aromatase enzyme activity and estrogen
production.
In the IN E2-GNP biodistribution study, we achieved a 5.24 fold increase (p<0.0001) in brain E2 concentration at 30 min post IN administration of E2-GNPs in comparison to baseline E2
concentration (Figure 3). Brain E2 levels were still elevated after 120 min (2.5 fold increase over baseline, p<0.03). Interestingly, if the olfactory bulbs are excluded from analysis, the
concentration of E2 in the brain is the same at 30 and 120 min (0.64 ± 0.01 and 0.62 ± 0.02 pg/mg tissue, respectively). The elevated lung E2 levels observed at 30 min (2.18 fold increase, p<0.05) were shown to return to baseline by 120 min with no accumulation in other tissues, indicating a limited capacity for off-target effects.
Effect of E2-GNPs on infarct volume
The E2-GNP dose response study resulted in a 54.3% reduction (p<0.05, n=4) in mean infarct volume for 100 ng E2-GNPs in comparison to MCAO (Figure 4A & D). In contrast, treatment with 100 ng of WS-E2 had no effect in WT mice (Figure 4C & D). Remarkably, 100 ng E2- GNPs were unable to reduce infarct volume in ERKO mice (Figure 4B).
Behavioral analysis
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The behavioral battery showed little difference in post-ischemia performance between treated and untreated animals with the primary effect seen at 24 h post-MCAO (Supplementary Figure 1). In the pole test, both the WS-E2 and E2-GNP groups were slower than the sham, but not different from MCAO controls at 24 hr. The number of entries in the open field test and alternation percent in the y-maze showed a significant difference between sham and MCAO animals at 24 h (p<0.05), but not between treated or untreated MCAO animals. Interestingly, at 48 hr post-MCAO, the E2-GNP group had a trend towards more entries than the untreated MCAO group on the y-maze test, indicating faster recovery of motor function and ability. Out of the 16 animals tested, nearly half (1 MCAO, 2 E2-GNPs, and 3 WS-E2) were incapable of completing the behavioral battery 24 h after surgery, thereby drastically reducing the effective sample size. Interpretation of results at 48 h post-MCAO was similarly impaired: only one mouse in the WS-E2 group was able to perform all three behavioral tasks.
Discussion
Intranasal (IN) drug administration is advantageous for the treatment of CNS disorders; multiple studies have shown increased brain targeting with IN delivery in comparison to intravenous administration.28-30 Use of a nanocarrier, especially a mucoadhesive carrier such as gelatin nanoparticles, can increase delivery to the brain following IN administration in comparison to drug solution alone.29 Additionally, the use of GNPs as a delivery vehicle passively targets damaged brain regions due to upregulation of matrix metalloproteinases (MMPs) 2 and 9, also known as gelatinase A and B, in damaged areas of the brain following an ischemic event.31 As we previously demonstrated with intranasal iNOS-siRNA GNPs,17 the present results confirm that E2-GNPs localize to the brain after IN administration with little potential for off-target
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effects. Our observation that the olfactory bulb contained the majority of the administered E2- GNPs in neurovascularly intact mice aligns with previous IN uptake studies.32, 33 A more detailed exploration of E2-GNP distribution in pre- and post-ischemic brains is warranted to better
untangle the neuroprotective effects of E2.
Similarly, the 53.7% reduction in infarct volume between E2-GNPs and WS-E2 (p=0.037, n=4) is consistent with that found from our previous work using an osteopontin peptide13 and siRNA17 as model drugs, and provides further evidence for the neuroprotection enhancing abilities of IN GNPs. Interestingly, when infarcts were calculated using the indirect method (percent of control hemisphere) the trends were the same but statistical significance was lost. As the indirect method is designed to correct for the effects of cerebral edema on infarct volume, we hypothesize that one of estrogen’s neuroprotective effects in the post-ischemic brain is to reduce edema.
Recent studies have reported that estrogens synthesized de novo in the post-ischemic brain confer neuroprotection,34 indicating a beneficial effect of timely delivery of estrogens in stroke victims. Interestingly, previous studies have shown that pretreatment, but not post-injury
treatment with E2 causes reductions in infarct volume that are dependent on ER.35 Conversely, a paradigm involving repeated post-ischemia dosing of an ER specific agonist demonstrated increased functional recovery without reducing infarct volume.36 And yet, Suzuki et al. found the ability of E2 pre-injury treatment to stimulate neurogenesis in the post-ischemic brain to be dependent on both ER and ER.34 While the roles of ER, ER, and aromatase have been investigated with E2 pretreatment, the effects of E2 in post-injury treatment, which is more relevant as a treatment regimen for stroke victims, have been scarcely studied.36, 37 Previous
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studies indicate that ER provides neuroprotection in permanent MCAO models whereas ER is more important in transient MCAO models.35, 38, 39 Therefore, with our stroke model we
predicted that E2-GNPs would reduce infarct volume in ERKO mice due to their retained ERactivity. Because of the failure of E2-GNPs to reduce infarct in ERKO mice in the current study, further investigation in ERKO mice is necessary to determine if these receptor effects are species, KO model, or delivery method mediated.
Although infarct volumes are known to fully form by 24 h post-MCAO and remain stable for 7 days,40 the time-course for development and recovery of functional deficits in rodent models of stroke is not well defined. The set of behavioral tests used in the present study allowed us to detect transient sensorimotor and memory impairments in the mice subjected to cerebral
ischemia. We noted improvement in body balance and motor coordination in the treated with E2- GNPs in comparison to untreated MCAO controls, demonstrating that treatment with E2-GNPs improves functional recovery from ischemic injury by promoting tissue survival. Interpretation of the behavioral data presented here is limited due to the fact that many animals were unable to perform the tasks at 24 and 48 h post-infarct. We suggest that significant functional differences between our treatment groups may become more apparent if recovery is assessed for a longer duration. However, Farr et al. reported no recovery of sensorimotor function up to 4 weeks after permanent MCAO in ovariectomized female rats treated with slow release E2 pellets,41 so functional recovery is not guaranteed. Regardless, the strength of our infarct volume results justifies a more thorough exploration of E2-mediated functional recovery.
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Despite advances in the acute treatment of ischemic stroke, the current reperfusion-based therapies are insufficient to prevent long-term disability in stroke victims.2 Therefore, function- saving neuroprotective therapies that can be used in addition to life-saving reperfusion therapies are needed. Towards this goal, we investigated intranasal (IN) drug delivery using gelatin nanocarriers. IN drug delivery is desirable because it is non-invasive, can target therapeutics to the brain, and can reduce systemic side effects. Overall, this work has successfully demonstrated the utility of intranasal gelatin nanoparticles in the delivery of estrogen for the treatment of ischemic stroke.
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Table and Figure Captions
Table 1. In vitro nanoparticle characterization.
SAMPLE PARTICLE DIAMETER (MEAN±SD)
E2 LOADING (WT%)*
LOADING EFFICIENCY
(%)†
GNP 298.3 ± 99.4 0.3 ± 0.2% 17.4 ± 16.8%
WS-E2-GNP 301.6 ± 74.7 1.6 ± 0.3% 94.6 ± 16.9%
5% -CD-GNP 315.7 ± 96.8 1.4 ± 0.1% 85.2 ± 4.9%
10% -CD-GNP 362.3 ± 151.4 1.6 ± 0.1% 95.5 ± 8.2%
* E2 loading (w/w%) = Amount of E2
Amount of E2−GNPs× 100%
† Loading efficiency (w/w%) = Amount of E2 loaded
Amount of E2 applied× 100%
Figure 1. In vitro evaluation of -CD-GNPs and WS-E2-GNPs. Scanning electron micrograph of β-cyclodextrin conjugated gelatin nanoparticles before (A) and after (B) estrogen loading.
Release of estrogen from different nanoparticle formulations (C) as a percent of total amount released, n=4. Error bars represent standard deviation. Scale bar = 1 µm.
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Figure 2. Endogenous estrogen production in brain and gonads (n=3). Comparison of standard and estrogen free diet (A). Differences between wild type and aromatase knockouts (B). ND = none detected.
Figure 3. Estrogen content in tissues after IN E2-GNPs. Error bars represent standard error of the mean, n=4, * p<0.05, ** p<0.0001
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Figure 4. Effect of intranasal estrogen on infarct volume. E2-GNP dose response study (A), 100 ng E2-GNPs in ERKO and WT mice (B), comparison of 100 ng E2-GNPs with 100 ng WS-E2 in WT mice (C), and representative images (D). Error bars represent standard error of the mean, n=4, *p<0.05 vs MCAO
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Table 1. In vitro nanoparticle characterization.
SAMPLE PARTICLE DIAMETER (MEAN±SD)
E2 LOADING (WT%)*
LOADING EFFICIENCY
(%)†
GNP 298.3 ± 99.4 0.3 ± 0.2% 17.4 ± 16.8%
WS-E2-GNP 301.6 ± 74.7 1.6 ± 0.3% 94.6 ± 16.9%
5% -CD-GNP 315.7 ± 96.8 1.4 ± 0.1% 85.2 ± 4.9%
10% -CD-GNP 362.3 ± 151.4 1.6 ± 0.1% 95.5 ± 8.2%
* E2 loading (w/w%) = Amount of E2
Amount of E2−GNPs× 100%
† Loading efficiency (w/w%) = Amount of E2 loaded
Amount of E2 applied× 100%
Graphical Abstract_Text
Using cyclodextrin modified gelatin to make nanoparticles allows direct encapsulation of estrogen for intranasal delivery. Intranasal estrogen loaded gelatin nanoparticles were shown to not only reduce infarct volume in the post-ischemic mouse brain, but also allow for direct nose-to-brain targeting thereby reducing potential systemic side effects.
