- Email: [email protected]
Intranasal delivery of siRNA to the olfactory bulbs of mice via the olfactory nerve pathway
Neuroscience Letters 513 (2012) 193–197
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Intranasal delivery of siRNA to the olfactory bulbs of mice via the olfactory nerve pathway Dan B. Renner ∗ , William H. Frey II, Leah R. Hanson Alzheimer’s Research Center at Regions Hospital, HealthPartners Research Foundation, 640 Jackson St., St. Paul, MN 55101, USA
a r t i c l e
i n f o
Article history: Received 1 November 2011 Received in revised form 9 February 2012 Accepted 12 February 2012 Keywords: siRNA Intranasal Olfactory nerve pathway Olfactory bulbs
a b s t r a c t Adopting RNAi technology for targeted manipulation of gene expression in the central nervous system (CNS) will require delivery of RNAi constructs to the CNS followed by cellular transfection and induction of the RNAi machinery. Significant strides have been made in enhancing RNAi transfection and tailoring knockdown toward specific gene targets, however, delivery of the RNAi constructs to the CNS remains a significant challenge. One possible solution for targeting siRNA to the CNS is intranasal administration, which noninvasively delivers a variety of compounds to the CNS. The current study examined delivery of fluorescently labeled siRNA from the nasal cavity to the olfactory bulbs via the olfactory nerve pathway. siRNA was observed along the length of the olfactory nerve bundles, from the olfactory mucosa of the nasal cavity to the anterior regions of the olfactory bulbs. In the olfactory mucosa, labeled siRNA was found within the olfactory epithelium, Bowman’s glands, and associated with blood vessels and bundles of olfactory nerves. In the olfactory bulbs, siRNA was observed in the olfactory nerve, glomerular and mitral cell layers. These results demonstrate a role of the olfactory nerve pathway in targeting siRNA to the olfactory bulbs. Additional investigations will be required to assess the distribution of intranasal siRNA to additional regions of the brain and explore the capacity of the delivered siRNA to silence gene expression in the CNS. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction With the capacity to post-transcriptionally silence the expression of targeted genes, siRNA has quickly become a valuable tool for the induction of tailored cellular responses. Although routinely used in vitro, the adaptation to in vivo use has been met with a host of challenges [11]. These include the susceptibility of the short double-stranded RNAs to enzymatic degradation, low cellular uptake, non-specific targeting and rapid clearance from the blood [31]. Through the introduction of covalent modifications, significant progress has been achieved in increasing the stability of the siRNA. The adoption of various transfection strategies has greatly increased the efficiency of transfection into the cytoplasm of different cell types. Yet, the lack of options for efficient delivery has largely limited the in vivo applications of siRNA to tissues in which the siRNA can be directly administered. Clinical examples where siRNA has been shown to be effective include the treatment of macular degeneration through local injections into the eye and the treatment of respiratory syncytial virus infection by intranasal inhalation into the lungs [23]. The challenge of siRNA delivery is particularly significant for delivery to the central nervous system
∗ Corresponding author at: Regions Hospital, Mail Stop 11203A, 640 Jackson St., St. Paul, MN 55101, USA. Tel.: +1 651 254 1092; fax: +1 651 254 3661. E-mail address: [email protected] (D.B. Renner). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.037
(CNS) [21]. siRNAs are approximately 12 kDa in size and highly charged, thus the blood–brain barrier efficiently inhibits delivery to the CNS from the systemic circulation. Additional delivery methods are required to conveniently deliver siRNA to the CNS for pre-clinical and therapeutic neurological applications [33]. Intranasal delivery has been shown to increase the delivery of peptides of significant size and charge to the CNS, exceeding the efficiency of delivery and targeting observed when these compounds are administered intravenously [5,14,15]. For example, intranasal administration of the 26.5 kDa recombinant human nerve growth factor in rats resulted in CNS concentrations 10–45fold higher (different brain regions) than concentrations observed following intravenous administration [4]. Similarly, intranasal delivery of hypocretin-1 increased targeting to various brain regions by 7–13-fold [9]. Enhancement of delivery and targeting to the brain has been demonstrated with intranasal delivery of a wide range of compounds including galanin-like peptide [24], insulin-like growth factor [32], insulin [13], interferon-1b [29], transforming growth factor-1 [22] and hypocretin-1 [9]. Pathways by which these compounds are preferentially delivered to the CNS have been described [10]. Along the olfactory nerve pathway, intranasally administered compounds are delivered to the CNS by traveling within the extracellular spaces surrounding bundles of olfactory nerves that extend from the olfactory mucosa to the anterior regions of the olfactory bundles [34]. The trigeminal pathway, which has been shown to possess high concentrations
194
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197
of intranasally administered compounds, has also been proposed to mediate extracellular delivery to the CNS, including brain stem, caudal regions of the brain, and spinal cord [32]. Intranasal delivery to the CNS, in which compounds are first deposited on the olfactory epithelium followed by delivery to the CNS, is a technique distinct from previously described intranasal inhalation for delivery to the lungs [23]. Two groups have reported effects in the CNS following intranasal administration of siRNA. Bortolozzi et al. [3] targeted the serotonin (5-HT1A ) receptor through intranasal siRNA and found reduced expression of this receptor in the raphe nuclei while also inducing anti-depressant like effects in forced swim and tail suspension tests (consequences consistent with gene silencing). Kim et al. [19] detected labeled siRNA in the olfactory bulbs and gene knockdown of alpha Bcrystallin in the olfactory bulbs, amygdala and hypothalamus 12 h following intranasal administration of targeted siRNA to rats. Successful intranasal delivery of siRNA would provide researchers with an easy and non-invasive tool to investigate in vivo gene expression in the CNS. In the present study, the capacity of the olfactory nerve pathway to deliver labeled siRNA from the olfactory epithelium to the olfactory bulbs was qualitatively investigated. The distribution of fluorescently labeled siRNA containing Dharmacon’s siSTABLE modification was evaluated in the olfactory mucosa and olfactory bulbs of mice 30 min following intranasal administration. Confocal microscopy was used to capture the localization of siRNA in the olfactory epithelial and lamina propria layers of the olfactory mucosa. Images show the delivery of siRNA across the cribriform plate of the ethmoid bone as it entered the olfactory bulbs. The resulting distribution in the bulbs and the confirmation of fully intact siRNA in the bulbs is also presented.
2. Methods Labeled siRNA targeting mouse lamin A/C (Sense: 5 P-CUGGACUUCCAGAAGAACAdTdT-DY647-3 Antisense 5 PUGUUCUUCUGGAAGUCCAGdTdT) was prepared by Dharmacon RNA Technologies (Lafayette, CO). The 21 base pair siRNA with the DyLight 647 fluorescent dye (DY647) had a molecular weight of 13,832 Da and an extinction coefficient of 360,005 (l/mole cm). Dharmacon’s siSTABLE modification (proprietary) was included for enhanced stability. The siRNA was prepared for in vivo use via ion exchange high-performance liquid chromatography purification, counter ion (Na+) exchange, dialysis, sterile filtration, and endotoxin testing. Intactness was verified with matrix-assistedlaser desorption ionization/time-of-flight mass spectrometry. Lyophilized labeled siRNA was dissolved in diethyl pyrocarbonate (DEPC) treated water and stored in frozen aliquots at −80 ◦ C. Approximately 1 h prior to administration, aliquots of siRNA were brought to room temperature, diluted in DEPC treated water and mixed with an equal volume of 10% glucose to obtain a 5 g/l siRNA concentration in 5% glucose. Purity and intactness of siRNA in the delivery solution were verified by electrophoresis on a 15% urea denaturing polyacrylamide gel followed by in-gel fluorescent detection using the Maestro Imaging System (data not shown). Adult male C57BL/6J mice (20–25 g, Jackson Laboratories, Bar Harbor, ME) were housed under a 12-h light/dark cycle with food and water provided ad libitum. Mice were received at 7 weeks of age and tested within 4 weeks of arrival at our animal care facility. Animals were cared for in accordance with institutional guidelines, and all experiments were approved by HealthPartners Research Foundation Animal Care and Use Committee (IACUC Protocol # 07-008), Regions Hospital. Intranasal administration was conducted as described previously [16], with the modifications described below. To obtain a
general representation of siRNA delivery to the olfactory bulbs a total of ten mice were imaged following the intranasal administration of labeled siRNA. From previous studies with radio-labeled insulin [13] and IGF-1 [32], we estimated this number of mice would be sufficient to obtain a general representation. An additional five mice received a dosing solution without labeled drug (5% glucose) to verify background fluorescence was not detectable with set conditions. Three mice were used to assess intactness and were sufficient to show qualitative evidence of intact siRNA in the bulb. Mice were anesthetized with a cocktail consisting of 100 mg/kg ketamine, 10 mg/kg xylazine and 1.0 mg/kg atropine and placed on their backs on a heating pad set to 37 ◦ C. Head position was stabilized in a horizontal position by placing a small piece of rolled paper (4 mm diameter) under the back of the neck. A pipette (P20) was used to intranasally administer 3 l drops of the siRNA dosing solution to alternating nostrils every minute. Drops were placed at the opening of the nostril, allowing the animal to snort each drop into the nasal cavity. A total of 24 l of dose solution, containing 120 g of labeled siRNA was delivered over a course of 8 min. Tissue collection was initiated 30 min following the first nasal application. The fully anesthetized mice were transcardially perfused with 20 ml of saline (for euthanasia and removal of blood) at 10 ml/min, followed by 60 ml of 4% phosphate buffered paraformaldehyde. The brain was removed and two millimeter thick sagittal sections (for seven mice) or coronal sections (for three mice) that included the brain, olfactory region of the nasal mucosa, and/or the cribriform plate were carefully prepared. Tissue sections containing bone were decalcified in Formical-4 (formalin and formic acid, Decal Corporation, Tallman, NY) overnight at 4 ◦ C. Tissue was infiltrated with 20% buffered sucrose (24 h at 4 ◦ C), embedded in Tissue-Tek Optimal Cutting Temperature Compound (Sakura Finetek, Torrance, CA) and cryosectioned at 35–70 m on the Leica CM3050 cryostat. Tissue sections were selected at regular intervals within the 2 mm tissues for analysis. Slides were simultaneously stained with the fluorescein labeled lectin, Ulex Europeans Agglutinin I at a 1:200 dilution (Vector Laboratories, Burlingame, CA) and DAPI at 300 nM (Invitrogen, Carlsbad, CA) for 20 min in PBS. Microscopic images were obtained with the Olympus FLUOVIEW FV1000 confocal laser scanning microscopy system (Biomedical Imaging and Processing Center, University of Minnesota). Three fluorescent channels were collected; DAPI (excitation and emission wavelengths of 405 nm and 461 nm), Fluorescein (488 nm and 519 nm) and DY647 (635 and 668 nm). Fluorophores were sequentially illuminated for detection. Intensity of the individual channels was adjusted in Olympus Fluoview Ver.1.7a viewer. Images were cropped and adjusted for total brightness in Adobe Photoshop CS4 Extended. Size adjustments and image labeling was performed in Adobe Illustrator CS4. Presented images represent the general distributions observed following successful delivery via the olfactory nerve pathway. For the three mice receiving intranasal siRNA for intactness assessment, olfactory bulbs and olfactory mucosa were immediately dissected following saline perfusion and flash frozen in liquid nitrogen. Total RNA was isolated from samples using Qiagen’s RNeasy Mini Kit with modifications for purifying small RNAs (Valencia, CA). Indicated quantities of RNA were separated with urea-denaturing polyacrylamide gel electrophoresis and fluorescence was detected with the Storm 860 Phosphorimager/Fluorimager (Molecular Dynamics).
3. Results Thirty minutes following intranasal administration, fluorescently labeled siRNA was distributed throughout the olfactory epithelia and lamina propria and was delivered to the olfactory
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197
195
Fig. 2. Labeled siRNA was intranasally delivered to the olfactory bulbs via the olfactory nerve pathway. siRNA was detected along the olfactory nerve bundles (ONB), exiting the olfactory mucosa (OM), crossing the cribriform plate (CP) and entering the olfactory nerve layer (ONL) of the olfactory bulbs 30 min following administration to mice. Glomerular layer (GL). Scale bar = 30 m.
Fig. 1. Thirty minutes following the intranasal administration of fluorescently labeled siRNA (red) to mice, label was observed in the olfactory epithelium (OE) and lamina propria (LP). (A, C) Within olfactory epithelium, siRNA was associated with Bowman’s Ducts (BD), olfactory receptor neurons (ORN) and sustentacular cells (SC). (A–C) Within the lamina propria, siRNA was localized with Bowman’s glands (BG), blood vessels (BV) and olfactory nerve bundles (ONB). (B) siRNA was also occasionally observed along the basal lamina (BL). (C) siRNA was found along the mucosal surface (MS) of the epithelium and within the BD and BG. Image consists of a 63 m z-stack of 42 confocal images. (A, B) Scale bar = 30 m, (C) scale bar = 100 m. * = surface of the ONB. 120 g of labeled siRNA was administered. Red = DyLight647 (siRNA), green = fluorescein labeled Ulex agglutinin, blue = DAPI.
bulbs along the olfactory nerve pathway (Fig. 1A–C). Within the olfactory epithelium, the siRNA was detected within Bowman’s Ducts (Fig. 1A and C), along sustentacular (or supporting) cells, olfactory receptor neurons (Fig. 1A), and occasionally along the
basal lamina (Fig. 1B). Labeled siRNA was also concentrated in vesicles near the surface of the olfactory mucosa (Fig. 1C). In the lamina propria, siRNA was found within Bowman’s Glands (Fig. 1A and C) and associated with both blood vessels and olfactory nerve bundles (Fig. 1B). Delivery via the olfactory nerve pathway was observed as siRNA was detected along the length of olfactory nerves, exiting the olfactory mucosa, crossing the cribriform plate and entering the anterior regions of the olfactory bulbs (Fig. 2). Intranasal administration of labeled siRNA resulted in delivery to the anterior regions of the olfactory bulbs. The relative intensity of label detected in the bulbs and the degree of penetration into the bulbs varied between individual mice. Observed concentrations of label were either limited to the olfactory nerve layer (Fig. 2) or extended into the anterior layers of the bulbs (Fig. 3A). The relative concentrations of siRNA decreased with the distance from the surface of the bulbs, representing a general concentration gradient originating from the point of entry into the olfactory bulb. Exceptions to this gradient distribution consisted of a preference of labeled siRNA for the peri-glomerular cells (Fig. 3A) and the mitral cells (Fig. 3A and B). Label was also detected in the external plexiform layer and granule cell layer (Fig. 3A and B). The presence of fully intact labeled siRNA in the olfactory bulbs of mice was verified through the extraction of total RNA, gel electrophoresis and in-gel fluorescent detection (Fig. 3C). Identically sized fluorescent bands (of the appropriate size) were detected in the lane containing the siRNA dosing solution and the lanes containing RNA extracted from the olfactory mucosa and the olfactory bulbs. 4. Discussion Results presented here support a role of the olfactory nerve pathway in targeting labeled siRNA to the olfactory bulbs of mice 30 min following intranasal administration. Within the nasal cavity, fluorescently tagged siRNA had crossed the olfactory epithelium into the lamina propria, where it was found within Bowman’s glands (which produce nasal secretions) and associated with the blood vessels and olfactory nerve bundles. Although the intensity of label found in the Bowman’s Ducts and Glands was relatively strong, leakage from these structures was not apparent, suggesting this form of absorption by the nasal mucosa did not contribute to the siRNA delivered to the olfactory bulbs (Fig. 1A and C). The
196
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197
Fig. 3. Intranasally delivered siRNA reached multiple layers within the olfactory bulbs of mice. (A) Highest concentrations reaching the bulbs were detected in the olfactory nerve layer (ONL). siRNA was also found to penetrate the glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL) and the granule cell layer (GCL). The siRNA was localized with peri-glomerular cells and mitral cells. Scale bar 100 m. (B) Magnified image of the mitral cell layer. Scale bar 30 m. (C) The presence of intact fluorescently labeled siRNA was verified in tissue extracts collected 30 min following delivery (120 g). Arrow marks the full length labeled siRNA. Lane 1, 0.6 ng of siRNA dosing solution (DS); Lane 2, 42 g of total RNA from the nasal mucosa (NM); Lane 3, 14.7 g of total RNA from the olfactory bulbs.
labeled siRNA found associated with blood vessels in the mucosa (Fig. 1B) suggests potential delivery within either the peri-vascular spaces or the lumen of vessels that span the nasal and cranial cavities. Delivery to the cranial cavity along the olfactory nerve pathway was clearly demonstrated. siRNA was detected along the length of the olfactory nerve bundles, as it extended from the olfactory epithelium, through the foramina in the cribriform plate, to the olfactory bulbs (Fig. 2). This delivery pathway has also been imaged in rats by Jansson and Bjork [18] using a fluorescently labeled 3 kDa dextran. Within the olfactory bulb, a general concentration gradient was observed as the concentration of siRNA was found to decrease with the depth of each cell layer. A significant portion of the siRNA in the glomerular layer and mitral cell layer was co-localized with peri-glomerular cells and mitral cells, suggesting these cell populations may be particularly susceptible to intranasal targeting in the bulbs. Supporting the presence of active siRNA, fully intact labeled siRNA was isolated from the bulbs. siRNA delivery to the bulbs has potential for investigating and/or treatment of disorders observed in the olfactory system. For example, intranasal delivery may target the tau pathology and olfactory deficits observed early in the development of Alzheimer’s and Parkinson’s disease [25]. In addition to being delivered to the olfactory bulbs, intranasally administered therapeutics have been detected throughout the CNS [10]. For example, the peptides insulin-like growth factor [32], insulin [13], interferon-1b [29], transforming growth factor1 [22] and the 13-mer polynucleotide, GRN163, a telomerase inhibitor [17] and even stem cells [7,8] have all been shown to be intranasally delivered throughout the CNS of rodents and induce specific neurological responses. In humans, intranasally administered insulin has been shown to improve memory in adults and patients with Alzheimer’s disease [1,2,6,26–28]. As evidenced in two recent publications [3,19], intranasally administered siRNA is also delivered to deeper brain regions. With the potential to specifically treat a broad spectrum of disorders, RNAi technology has quickly progressed toward therapeutic use [30]. Adopting siRNA for neurological applications is currently limited by the lack of efficient methods for delivering RNAi constructs to the CNS. Several features unique to the olfactory nerves and olfactory nerve pathway may contribute to its adaption in targeting compounds to the olfactory bulbs. These include the
continuous regeneration of olfactory sensory neurons, dendrites that are directly exposed to the mucosal surface and the presence of channels that encapsulate the bundles of olfactory nerve axons that extend to the olfactory bulbs [12,20]. This investigation supports further development of the intranasal delivery method toward targeted siRNA manipulation of gene expression in the CNS.
Acknowledgments We thank Kate Faltesek for assistance with animal care and anesthesia (HealthPartners Research Foundation, Alzheimer’s Research Center at Regions Hospital, St. Paul, MN).
References [1] C. Benedict, W.H. Frey 2nd, H.B. Schioth, B. Schultes, J. Born, M. Hallschmid, Intranasal insulin as a therapeutic option in the treatment of cognitive impairments, Exp. Gerontol. 46 (2011) 112–115. [2] C. Benedict, M. Hallschmid, A. Hatke, B. Schultes, H.L. Fehm, J. Born, W. Kern, Intranasal insulin improves memory in humans, Psychoneuroendocrinology 29 (2004) 1326–1334. [3] A. Bortolozzi, A. Castane, J. Semakova, N. Santana, G. Alvarado, R. Cortes, A. Ferres-Coy, G. Fernandez, M.C. Carmona, M. Toth, J.C. Perales, A. Montefeltro, F. Artigas, Selective siRNA-mediated suppression of 5-HT(1A) autoreceptors evokes strong anti-depressant-like effects, Mol. Psychiatry (2011) (Epub ahead of print). [4] X.Q. Chen, J.R. Fawcett, Y.E. Rahman, T.A. Ala, I.W. Frey, Delivery of nerve growth factor to the brain via the olfactory pathway, J. Alzheimers Dis. 1 (1998) 35–44. [5] W.H.F.I. Christoph Reinjard, Delivery of Polynucleotide Agents to the Central Nervous System, US 2009/0264506 A1 (2009). [6] S. Craft, L.D. Baker, T.J. Montine, S. Minoshima, G.S. Watson, A. Claxton, M. Arbuckle, M. Callaghan, E. Tsai, S.R. Plymate, P.S. Green, J. Leverenz, D. Cross, B. Gerton, Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: a pilot clinical trial, Arch. Neurol. 69 (2012) 29–38. [7] L. Danielyan, R. Schafer, A. von Ameln-Mayerhofer, F. Bernhard, S. Verleysdonk, M. Buadze, A. Lourhmati, T. Klopfer, F. Schaumann, B. Schmid, C. Koehle, B. Proksch, R. Weissert, H.M. Reichardt, J. Van den Brandt, G.H. Buniatian, M. Schwab, C.H. Gleiter, W.H. Frey 2nd, Therapeutic efficacy of intranasally delivered mesenchymal stem cells in a rat model of Parkinson disease, Rejuvenation Res. 14 (2011) 3–16. [8] L. Danielyan, R. Schafer, A. von Ameln-Mayerhofer, M. Buadze, J. Geisler, T. Klopfer, U. Burkhardt, B. Proksch, S. Verleysdonk, M. Ayturan, G.H. Buniatian, C.H. Gleiter, W.H. Frey 2nd, Intranasal delivery of cells to the brain, Eur. J. Cell Biol. 88 (2009) 315–324. [9] S.V. Dhuria, L.R. Hanson, W.H. Frey 2nd, Intranasal drug targeting of hypocretin1 (orexin-A) to the central nervous system, J. Pharm. Sci. 98 (2009) 2501–2515.
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197 [10] S.V. Dhuria, L.R. Hanson, W.H. Frey 2nd, Intranasal delivery to the central nervous system: mechanisms and experimental considerations, J. Pharm. Sci. 99 (2010) 1654–1673. [11] D.M. Dykxhoorn, J. Lieberman, Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs, Annu. Rev. Biomed. Eng. 8 (2006) 377–402. [12] P. Field, Y. Li, G. Raisman, Ensheathment of the olfactory nerves in the adult rat, J. Neurocytol. 32 (2003) 317–324. [13] G.J. Francis, J.A. Martinez, W.Q. Liu, K. Xu, A. Ayer, J. Fine, U.I. Tuor, G. Glazner, L.R. Hanson, W.H. Frey 2nd, C. Toth, Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy, Brain 131 (2008) 3311–3334. [14] W.H. Frey 2nd, Method of Administering Neurologic Agents to the Brain 5,624,898 (1997). [15] L.R. Hanson, W.H. Frey 2nd, Intranasal delivery bypasses the blood–brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease, BMC Neurosci. 9 (Suppl. 3) (2008) S5. [16] L.R. Hanson, P.M. Martinez, S. Taheri, L. Kamsheh, E. Mignot, W.H.I. Frey, Intranasal administration of hypocretin 1 (Orexin A) bypasses the blood–brain barrier & targets the brain: a new strategy for the treatment of narcolepsy, Drug Deliv. Technol. 4 (2004) 81–86. [17] R. Hashizume, T. Ozawa, S.M. Gryaznov, A.W. Bollen, K.R. Lamborn, W.H. Frey 2nd, D.F. Deen, New therapeutic approach for brain tumors: Intranasal delivery of telomerase inhibitor GRN163, Neurol. Oncol. 10 (2008) 112–120. [18] B. Jansson, E. Bjork, Visualization of in vivo olfactory uptake and transfer using fluorescein dextran, J. Drug Target. 10 (2002) 379–386. [19] I. Kim, S. Kim, J. Lee, Gener knockdown in the olfactory bulb, amygdala, and hypothalamus by intranasal siRNA administration, Kor. J. Anat. 42 (2009) 285–292. [20] Y. Li, P.M. Field, G. Raisman, Olfactory ensheathing cells and olfactory nerve fibroblasts maintain continuous open channels for regrowth of olfactory nerve fibres, Glia 52 (2005) 245–251. [21] P. Lingor, M. Bahr, Targeting neurological disease with RNAi, Mol. Biosyst. 3 (2007) 773–780. [22] Y.P. Ma, M.M. Ma, S. Ge, R.B. Guo, H.J. Zhang, W.H. Frey 2nd, G.L. Xu, X.F. Liu, Intranasally delivered TGF-beta1 enters brain and regulates gene expressions of its receptors in rats, Brain Res. Bull. 74 (2007) 271–277.
197
[23] T. Nguyen, E.M. Menocal, J. Harborth, J.H. Fruehauf, RNAi therapeutics: an update on delivery, Curr. Opin. Mol. Ther. 10 (2008) 158–167. [24] N. Nonaka, S.A. Farr, H. Kageyama, S. Shioda, W.A. Banks, Delivery of galaninlike peptide to the brain: targeting with intranasal delivery and cyclodextrins, J. Pharmacol. Exp. Ther. 325 (2008) 513–519. [25] J.M. Nores, B. Biacabe, P. Bonfils, [Olfactory disorders in Alzheimer’s disease and in Parkinson’s disease. Review of the literature], Ann. Med. Intern. (Paris) 151 (2000) 97–106. [26] M.A. Reger, G.S. Watson, W.H. Frey 2nd, L.D. Baker, B. Cholerton, M.L. Keeling, D.A. Belongia, M.A. Fishel, S.R. Plymate, G.D. Schellenberg, M.M. Cherrier, S. Craft, Effects of intranasal insulin on cognition in memory-impaired older adults: modulation by APOE genotype, Neurobiol. Aging 27 (2006) 451–458. [27] M.A. Reger, G.S. Watson, P.S. Green, L.D. Baker, B. Cholerton, M.A. Fishel, S.R. Plymate, M.M. Cherrier, G.D. Schellenberg, W.H. Frey 2nd, S. Craft, Intranasal insulin administration dose-dependently modulates verbal memory and plasma amyloid-beta in memory-impaired older adults, J. Alzheimers Dis. 13 (2008) 323–331. [28] M.A. Reger, G.S. Watson, P.S. Green, C.W. Wilkinson, L.D. Baker, B. Cholerton, M.A. Fishel, S.R. Plymate, J.C. Breitner, W. DeGroodt, P. Mehta, S. Craft, Intranasal insulin improves cognition and modulates beta-amyloid in early AD, Neurology 70 (2008) 440–448. [29] T.M. Ross, P.M. Martinez, J.C. Renner, R.G. Thorne, L.R. Hanson, W.H. Frey 2nd, Intranasal administration of interferon beta bypasses the blood–brain barrier to target the central nervous system and cervical lymph nodes: a non-invasive treatment strategy for multiple sclerosis, J. Neuroimmunol. 151 (2004) 66–77. [30] E.I. Schwartz, Potential application of RNAi for understanding and therapy of neurodegenerative diseases, Front. Biosci. 14 (2009) 297–320. [31] M.S. Shim, Y.J. Kwon, Efficient and targeted delivery of siRNA in vivo, FEBS J. 277 (2010) 4814–4827. [32] R.G. Thorne, G.J. Pronk, V. Padmanabhan, W.H. Frey 2nd, Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration, Neuroscience 127 (2004) 481–496. [33] K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery, Nat. Rev. 8 (2009) 129–138. [34] H. Wu, K. Hu, X. Jiang, From nose to brain: understanding transport capacity and transport rate of drugs, Expert Opin. Drug Deliv. 5 (2008) 1159–1168.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Intranasal delivery of siRNA to the olfactory bulbs of mice via the olfactory nerve pathway Dan B. Renner ∗ , William H. Frey II, Leah R. Hanson Alzheimer’s Research Center at Regions Hospital, HealthPartners Research Foundation, 640 Jackson St., St. Paul, MN 55101, USA
a r t i c l e
i n f o
Article history: Received 1 November 2011 Received in revised form 9 February 2012 Accepted 12 February 2012 Keywords: siRNA Intranasal Olfactory nerve pathway Olfactory bulbs
a b s t r a c t Adopting RNAi technology for targeted manipulation of gene expression in the central nervous system (CNS) will require delivery of RNAi constructs to the CNS followed by cellular transfection and induction of the RNAi machinery. Significant strides have been made in enhancing RNAi transfection and tailoring knockdown toward specific gene targets, however, delivery of the RNAi constructs to the CNS remains a significant challenge. One possible solution for targeting siRNA to the CNS is intranasal administration, which noninvasively delivers a variety of compounds to the CNS. The current study examined delivery of fluorescently labeled siRNA from the nasal cavity to the olfactory bulbs via the olfactory nerve pathway. siRNA was observed along the length of the olfactory nerve bundles, from the olfactory mucosa of the nasal cavity to the anterior regions of the olfactory bulbs. In the olfactory mucosa, labeled siRNA was found within the olfactory epithelium, Bowman’s glands, and associated with blood vessels and bundles of olfactory nerves. In the olfactory bulbs, siRNA was observed in the olfactory nerve, glomerular and mitral cell layers. These results demonstrate a role of the olfactory nerve pathway in targeting siRNA to the olfactory bulbs. Additional investigations will be required to assess the distribution of intranasal siRNA to additional regions of the brain and explore the capacity of the delivered siRNA to silence gene expression in the CNS. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction With the capacity to post-transcriptionally silence the expression of targeted genes, siRNA has quickly become a valuable tool for the induction of tailored cellular responses. Although routinely used in vitro, the adaptation to in vivo use has been met with a host of challenges [11]. These include the susceptibility of the short double-stranded RNAs to enzymatic degradation, low cellular uptake, non-specific targeting and rapid clearance from the blood [31]. Through the introduction of covalent modifications, significant progress has been achieved in increasing the stability of the siRNA. The adoption of various transfection strategies has greatly increased the efficiency of transfection into the cytoplasm of different cell types. Yet, the lack of options for efficient delivery has largely limited the in vivo applications of siRNA to tissues in which the siRNA can be directly administered. Clinical examples where siRNA has been shown to be effective include the treatment of macular degeneration through local injections into the eye and the treatment of respiratory syncytial virus infection by intranasal inhalation into the lungs [23]. The challenge of siRNA delivery is particularly significant for delivery to the central nervous system
∗ Corresponding author at: Regions Hospital, Mail Stop 11203A, 640 Jackson St., St. Paul, MN 55101, USA. Tel.: +1 651 254 1092; fax: +1 651 254 3661. E-mail address: [email protected] (D.B. Renner). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.037
(CNS) [21]. siRNAs are approximately 12 kDa in size and highly charged, thus the blood–brain barrier efficiently inhibits delivery to the CNS from the systemic circulation. Additional delivery methods are required to conveniently deliver siRNA to the CNS for pre-clinical and therapeutic neurological applications [33]. Intranasal delivery has been shown to increase the delivery of peptides of significant size and charge to the CNS, exceeding the efficiency of delivery and targeting observed when these compounds are administered intravenously [5,14,15]. For example, intranasal administration of the 26.5 kDa recombinant human nerve growth factor in rats resulted in CNS concentrations 10–45fold higher (different brain regions) than concentrations observed following intravenous administration [4]. Similarly, intranasal delivery of hypocretin-1 increased targeting to various brain regions by 7–13-fold [9]. Enhancement of delivery and targeting to the brain has been demonstrated with intranasal delivery of a wide range of compounds including galanin-like peptide [24], insulin-like growth factor [32], insulin [13], interferon-1b [29], transforming growth factor-1 [22] and hypocretin-1 [9]. Pathways by which these compounds are preferentially delivered to the CNS have been described [10]. Along the olfactory nerve pathway, intranasally administered compounds are delivered to the CNS by traveling within the extracellular spaces surrounding bundles of olfactory nerves that extend from the olfactory mucosa to the anterior regions of the olfactory bundles [34]. The trigeminal pathway, which has been shown to possess high concentrations
194
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197
of intranasally administered compounds, has also been proposed to mediate extracellular delivery to the CNS, including brain stem, caudal regions of the brain, and spinal cord [32]. Intranasal delivery to the CNS, in which compounds are first deposited on the olfactory epithelium followed by delivery to the CNS, is a technique distinct from previously described intranasal inhalation for delivery to the lungs [23]. Two groups have reported effects in the CNS following intranasal administration of siRNA. Bortolozzi et al. [3] targeted the serotonin (5-HT1A ) receptor through intranasal siRNA and found reduced expression of this receptor in the raphe nuclei while also inducing anti-depressant like effects in forced swim and tail suspension tests (consequences consistent with gene silencing). Kim et al. [19] detected labeled siRNA in the olfactory bulbs and gene knockdown of alpha Bcrystallin in the olfactory bulbs, amygdala and hypothalamus 12 h following intranasal administration of targeted siRNA to rats. Successful intranasal delivery of siRNA would provide researchers with an easy and non-invasive tool to investigate in vivo gene expression in the CNS. In the present study, the capacity of the olfactory nerve pathway to deliver labeled siRNA from the olfactory epithelium to the olfactory bulbs was qualitatively investigated. The distribution of fluorescently labeled siRNA containing Dharmacon’s siSTABLE modification was evaluated in the olfactory mucosa and olfactory bulbs of mice 30 min following intranasal administration. Confocal microscopy was used to capture the localization of siRNA in the olfactory epithelial and lamina propria layers of the olfactory mucosa. Images show the delivery of siRNA across the cribriform plate of the ethmoid bone as it entered the olfactory bulbs. The resulting distribution in the bulbs and the confirmation of fully intact siRNA in the bulbs is also presented.
2. Methods Labeled siRNA targeting mouse lamin A/C (Sense: 5 P-CUGGACUUCCAGAAGAACAdTdT-DY647-3 Antisense 5 PUGUUCUUCUGGAAGUCCAGdTdT) was prepared by Dharmacon RNA Technologies (Lafayette, CO). The 21 base pair siRNA with the DyLight 647 fluorescent dye (DY647) had a molecular weight of 13,832 Da and an extinction coefficient of 360,005 (l/mole cm). Dharmacon’s siSTABLE modification (proprietary) was included for enhanced stability. The siRNA was prepared for in vivo use via ion exchange high-performance liquid chromatography purification, counter ion (Na+) exchange, dialysis, sterile filtration, and endotoxin testing. Intactness was verified with matrix-assistedlaser desorption ionization/time-of-flight mass spectrometry. Lyophilized labeled siRNA was dissolved in diethyl pyrocarbonate (DEPC) treated water and stored in frozen aliquots at −80 ◦ C. Approximately 1 h prior to administration, aliquots of siRNA were brought to room temperature, diluted in DEPC treated water and mixed with an equal volume of 10% glucose to obtain a 5 g/l siRNA concentration in 5% glucose. Purity and intactness of siRNA in the delivery solution were verified by electrophoresis on a 15% urea denaturing polyacrylamide gel followed by in-gel fluorescent detection using the Maestro Imaging System (data not shown). Adult male C57BL/6J mice (20–25 g, Jackson Laboratories, Bar Harbor, ME) were housed under a 12-h light/dark cycle with food and water provided ad libitum. Mice were received at 7 weeks of age and tested within 4 weeks of arrival at our animal care facility. Animals were cared for in accordance with institutional guidelines, and all experiments were approved by HealthPartners Research Foundation Animal Care and Use Committee (IACUC Protocol # 07-008), Regions Hospital. Intranasal administration was conducted as described previously [16], with the modifications described below. To obtain a
general representation of siRNA delivery to the olfactory bulbs a total of ten mice were imaged following the intranasal administration of labeled siRNA. From previous studies with radio-labeled insulin [13] and IGF-1 [32], we estimated this number of mice would be sufficient to obtain a general representation. An additional five mice received a dosing solution without labeled drug (5% glucose) to verify background fluorescence was not detectable with set conditions. Three mice were used to assess intactness and were sufficient to show qualitative evidence of intact siRNA in the bulb. Mice were anesthetized with a cocktail consisting of 100 mg/kg ketamine, 10 mg/kg xylazine and 1.0 mg/kg atropine and placed on their backs on a heating pad set to 37 ◦ C. Head position was stabilized in a horizontal position by placing a small piece of rolled paper (4 mm diameter) under the back of the neck. A pipette (P20) was used to intranasally administer 3 l drops of the siRNA dosing solution to alternating nostrils every minute. Drops were placed at the opening of the nostril, allowing the animal to snort each drop into the nasal cavity. A total of 24 l of dose solution, containing 120 g of labeled siRNA was delivered over a course of 8 min. Tissue collection was initiated 30 min following the first nasal application. The fully anesthetized mice were transcardially perfused with 20 ml of saline (for euthanasia and removal of blood) at 10 ml/min, followed by 60 ml of 4% phosphate buffered paraformaldehyde. The brain was removed and two millimeter thick sagittal sections (for seven mice) or coronal sections (for three mice) that included the brain, olfactory region of the nasal mucosa, and/or the cribriform plate were carefully prepared. Tissue sections containing bone were decalcified in Formical-4 (formalin and formic acid, Decal Corporation, Tallman, NY) overnight at 4 ◦ C. Tissue was infiltrated with 20% buffered sucrose (24 h at 4 ◦ C), embedded in Tissue-Tek Optimal Cutting Temperature Compound (Sakura Finetek, Torrance, CA) and cryosectioned at 35–70 m on the Leica CM3050 cryostat. Tissue sections were selected at regular intervals within the 2 mm tissues for analysis. Slides were simultaneously stained with the fluorescein labeled lectin, Ulex Europeans Agglutinin I at a 1:200 dilution (Vector Laboratories, Burlingame, CA) and DAPI at 300 nM (Invitrogen, Carlsbad, CA) for 20 min in PBS. Microscopic images were obtained with the Olympus FLUOVIEW FV1000 confocal laser scanning microscopy system (Biomedical Imaging and Processing Center, University of Minnesota). Three fluorescent channels were collected; DAPI (excitation and emission wavelengths of 405 nm and 461 nm), Fluorescein (488 nm and 519 nm) and DY647 (635 and 668 nm). Fluorophores were sequentially illuminated for detection. Intensity of the individual channels was adjusted in Olympus Fluoview Ver.1.7a viewer. Images were cropped and adjusted for total brightness in Adobe Photoshop CS4 Extended. Size adjustments and image labeling was performed in Adobe Illustrator CS4. Presented images represent the general distributions observed following successful delivery via the olfactory nerve pathway. For the three mice receiving intranasal siRNA for intactness assessment, olfactory bulbs and olfactory mucosa were immediately dissected following saline perfusion and flash frozen in liquid nitrogen. Total RNA was isolated from samples using Qiagen’s RNeasy Mini Kit with modifications for purifying small RNAs (Valencia, CA). Indicated quantities of RNA were separated with urea-denaturing polyacrylamide gel electrophoresis and fluorescence was detected with the Storm 860 Phosphorimager/Fluorimager (Molecular Dynamics).
3. Results Thirty minutes following intranasal administration, fluorescently labeled siRNA was distributed throughout the olfactory epithelia and lamina propria and was delivered to the olfactory
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197
195
Fig. 2. Labeled siRNA was intranasally delivered to the olfactory bulbs via the olfactory nerve pathway. siRNA was detected along the olfactory nerve bundles (ONB), exiting the olfactory mucosa (OM), crossing the cribriform plate (CP) and entering the olfactory nerve layer (ONL) of the olfactory bulbs 30 min following administration to mice. Glomerular layer (GL). Scale bar = 30 m.
Fig. 1. Thirty minutes following the intranasal administration of fluorescently labeled siRNA (red) to mice, label was observed in the olfactory epithelium (OE) and lamina propria (LP). (A, C) Within olfactory epithelium, siRNA was associated with Bowman’s Ducts (BD), olfactory receptor neurons (ORN) and sustentacular cells (SC). (A–C) Within the lamina propria, siRNA was localized with Bowman’s glands (BG), blood vessels (BV) and olfactory nerve bundles (ONB). (B) siRNA was also occasionally observed along the basal lamina (BL). (C) siRNA was found along the mucosal surface (MS) of the epithelium and within the BD and BG. Image consists of a 63 m z-stack of 42 confocal images. (A, B) Scale bar = 30 m, (C) scale bar = 100 m. * = surface of the ONB. 120 g of labeled siRNA was administered. Red = DyLight647 (siRNA), green = fluorescein labeled Ulex agglutinin, blue = DAPI.
bulbs along the olfactory nerve pathway (Fig. 1A–C). Within the olfactory epithelium, the siRNA was detected within Bowman’s Ducts (Fig. 1A and C), along sustentacular (or supporting) cells, olfactory receptor neurons (Fig. 1A), and occasionally along the
basal lamina (Fig. 1B). Labeled siRNA was also concentrated in vesicles near the surface of the olfactory mucosa (Fig. 1C). In the lamina propria, siRNA was found within Bowman’s Glands (Fig. 1A and C) and associated with both blood vessels and olfactory nerve bundles (Fig. 1B). Delivery via the olfactory nerve pathway was observed as siRNA was detected along the length of olfactory nerves, exiting the olfactory mucosa, crossing the cribriform plate and entering the anterior regions of the olfactory bulbs (Fig. 2). Intranasal administration of labeled siRNA resulted in delivery to the anterior regions of the olfactory bulbs. The relative intensity of label detected in the bulbs and the degree of penetration into the bulbs varied between individual mice. Observed concentrations of label were either limited to the olfactory nerve layer (Fig. 2) or extended into the anterior layers of the bulbs (Fig. 3A). The relative concentrations of siRNA decreased with the distance from the surface of the bulbs, representing a general concentration gradient originating from the point of entry into the olfactory bulb. Exceptions to this gradient distribution consisted of a preference of labeled siRNA for the peri-glomerular cells (Fig. 3A) and the mitral cells (Fig. 3A and B). Label was also detected in the external plexiform layer and granule cell layer (Fig. 3A and B). The presence of fully intact labeled siRNA in the olfactory bulbs of mice was verified through the extraction of total RNA, gel electrophoresis and in-gel fluorescent detection (Fig. 3C). Identically sized fluorescent bands (of the appropriate size) were detected in the lane containing the siRNA dosing solution and the lanes containing RNA extracted from the olfactory mucosa and the olfactory bulbs. 4. Discussion Results presented here support a role of the olfactory nerve pathway in targeting labeled siRNA to the olfactory bulbs of mice 30 min following intranasal administration. Within the nasal cavity, fluorescently tagged siRNA had crossed the olfactory epithelium into the lamina propria, where it was found within Bowman’s glands (which produce nasal secretions) and associated with the blood vessels and olfactory nerve bundles. Although the intensity of label found in the Bowman’s Ducts and Glands was relatively strong, leakage from these structures was not apparent, suggesting this form of absorption by the nasal mucosa did not contribute to the siRNA delivered to the olfactory bulbs (Fig. 1A and C). The
196
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197
Fig. 3. Intranasally delivered siRNA reached multiple layers within the olfactory bulbs of mice. (A) Highest concentrations reaching the bulbs were detected in the olfactory nerve layer (ONL). siRNA was also found to penetrate the glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL) and the granule cell layer (GCL). The siRNA was localized with peri-glomerular cells and mitral cells. Scale bar 100 m. (B) Magnified image of the mitral cell layer. Scale bar 30 m. (C) The presence of intact fluorescently labeled siRNA was verified in tissue extracts collected 30 min following delivery (120 g). Arrow marks the full length labeled siRNA. Lane 1, 0.6 ng of siRNA dosing solution (DS); Lane 2, 42 g of total RNA from the nasal mucosa (NM); Lane 3, 14.7 g of total RNA from the olfactory bulbs.
labeled siRNA found associated with blood vessels in the mucosa (Fig. 1B) suggests potential delivery within either the peri-vascular spaces or the lumen of vessels that span the nasal and cranial cavities. Delivery to the cranial cavity along the olfactory nerve pathway was clearly demonstrated. siRNA was detected along the length of the olfactory nerve bundles, as it extended from the olfactory epithelium, through the foramina in the cribriform plate, to the olfactory bulbs (Fig. 2). This delivery pathway has also been imaged in rats by Jansson and Bjork [18] using a fluorescently labeled 3 kDa dextran. Within the olfactory bulb, a general concentration gradient was observed as the concentration of siRNA was found to decrease with the depth of each cell layer. A significant portion of the siRNA in the glomerular layer and mitral cell layer was co-localized with peri-glomerular cells and mitral cells, suggesting these cell populations may be particularly susceptible to intranasal targeting in the bulbs. Supporting the presence of active siRNA, fully intact labeled siRNA was isolated from the bulbs. siRNA delivery to the bulbs has potential for investigating and/or treatment of disorders observed in the olfactory system. For example, intranasal delivery may target the tau pathology and olfactory deficits observed early in the development of Alzheimer’s and Parkinson’s disease [25]. In addition to being delivered to the olfactory bulbs, intranasally administered therapeutics have been detected throughout the CNS [10]. For example, the peptides insulin-like growth factor [32], insulin [13], interferon-1b [29], transforming growth factor1 [22] and the 13-mer polynucleotide, GRN163, a telomerase inhibitor [17] and even stem cells [7,8] have all been shown to be intranasally delivered throughout the CNS of rodents and induce specific neurological responses. In humans, intranasally administered insulin has been shown to improve memory in adults and patients with Alzheimer’s disease [1,2,6,26–28]. As evidenced in two recent publications [3,19], intranasally administered siRNA is also delivered to deeper brain regions. With the potential to specifically treat a broad spectrum of disorders, RNAi technology has quickly progressed toward therapeutic use [30]. Adopting siRNA for neurological applications is currently limited by the lack of efficient methods for delivering RNAi constructs to the CNS. Several features unique to the olfactory nerves and olfactory nerve pathway may contribute to its adaption in targeting compounds to the olfactory bulbs. These include the
continuous regeneration of olfactory sensory neurons, dendrites that are directly exposed to the mucosal surface and the presence of channels that encapsulate the bundles of olfactory nerve axons that extend to the olfactory bulbs [12,20]. This investigation supports further development of the intranasal delivery method toward targeted siRNA manipulation of gene expression in the CNS.
Acknowledgments We thank Kate Faltesek for assistance with animal care and anesthesia (HealthPartners Research Foundation, Alzheimer’s Research Center at Regions Hospital, St. Paul, MN).
References [1] C. Benedict, W.H. Frey 2nd, H.B. Schioth, B. Schultes, J. Born, M. Hallschmid, Intranasal insulin as a therapeutic option in the treatment of cognitive impairments, Exp. Gerontol. 46 (2011) 112–115. [2] C. Benedict, M. Hallschmid, A. Hatke, B. Schultes, H.L. Fehm, J. Born, W. Kern, Intranasal insulin improves memory in humans, Psychoneuroendocrinology 29 (2004) 1326–1334. [3] A. Bortolozzi, A. Castane, J. Semakova, N. Santana, G. Alvarado, R. Cortes, A. Ferres-Coy, G. Fernandez, M.C. Carmona, M. Toth, J.C. Perales, A. Montefeltro, F. Artigas, Selective siRNA-mediated suppression of 5-HT(1A) autoreceptors evokes strong anti-depressant-like effects, Mol. Psychiatry (2011) (Epub ahead of print). [4] X.Q. Chen, J.R. Fawcett, Y.E. Rahman, T.A. Ala, I.W. Frey, Delivery of nerve growth factor to the brain via the olfactory pathway, J. Alzheimers Dis. 1 (1998) 35–44. [5] W.H.F.I. Christoph Reinjard, Delivery of Polynucleotide Agents to the Central Nervous System, US 2009/0264506 A1 (2009). [6] S. Craft, L.D. Baker, T.J. Montine, S. Minoshima, G.S. Watson, A. Claxton, M. Arbuckle, M. Callaghan, E. Tsai, S.R. Plymate, P.S. Green, J. Leverenz, D. Cross, B. Gerton, Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: a pilot clinical trial, Arch. Neurol. 69 (2012) 29–38. [7] L. Danielyan, R. Schafer, A. von Ameln-Mayerhofer, F. Bernhard, S. Verleysdonk, M. Buadze, A. Lourhmati, T. Klopfer, F. Schaumann, B. Schmid, C. Koehle, B. Proksch, R. Weissert, H.M. Reichardt, J. Van den Brandt, G.H. Buniatian, M. Schwab, C.H. Gleiter, W.H. Frey 2nd, Therapeutic efficacy of intranasally delivered mesenchymal stem cells in a rat model of Parkinson disease, Rejuvenation Res. 14 (2011) 3–16. [8] L. Danielyan, R. Schafer, A. von Ameln-Mayerhofer, M. Buadze, J. Geisler, T. Klopfer, U. Burkhardt, B. Proksch, S. Verleysdonk, M. Ayturan, G.H. Buniatian, C.H. Gleiter, W.H. Frey 2nd, Intranasal delivery of cells to the brain, Eur. J. Cell Biol. 88 (2009) 315–324. [9] S.V. Dhuria, L.R. Hanson, W.H. Frey 2nd, Intranasal drug targeting of hypocretin1 (orexin-A) to the central nervous system, J. Pharm. Sci. 98 (2009) 2501–2515.
D.B. Renner et al. / Neuroscience Letters 513 (2012) 193–197 [10] S.V. Dhuria, L.R. Hanson, W.H. Frey 2nd, Intranasal delivery to the central nervous system: mechanisms and experimental considerations, J. Pharm. Sci. 99 (2010) 1654–1673. [11] D.M. Dykxhoorn, J. Lieberman, Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs, Annu. Rev. Biomed. Eng. 8 (2006) 377–402. [12] P. Field, Y. Li, G. Raisman, Ensheathment of the olfactory nerves in the adult rat, J. Neurocytol. 32 (2003) 317–324. [13] G.J. Francis, J.A. Martinez, W.Q. Liu, K. Xu, A. Ayer, J. Fine, U.I. Tuor, G. Glazner, L.R. Hanson, W.H. Frey 2nd, C. Toth, Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy, Brain 131 (2008) 3311–3334. [14] W.H. Frey 2nd, Method of Administering Neurologic Agents to the Brain 5,624,898 (1997). [15] L.R. Hanson, W.H. Frey 2nd, Intranasal delivery bypasses the blood–brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease, BMC Neurosci. 9 (Suppl. 3) (2008) S5. [16] L.R. Hanson, P.M. Martinez, S. Taheri, L. Kamsheh, E. Mignot, W.H.I. Frey, Intranasal administration of hypocretin 1 (Orexin A) bypasses the blood–brain barrier & targets the brain: a new strategy for the treatment of narcolepsy, Drug Deliv. Technol. 4 (2004) 81–86. [17] R. Hashizume, T. Ozawa, S.M. Gryaznov, A.W. Bollen, K.R. Lamborn, W.H. Frey 2nd, D.F. Deen, New therapeutic approach for brain tumors: Intranasal delivery of telomerase inhibitor GRN163, Neurol. Oncol. 10 (2008) 112–120. [18] B. Jansson, E. Bjork, Visualization of in vivo olfactory uptake and transfer using fluorescein dextran, J. Drug Target. 10 (2002) 379–386. [19] I. Kim, S. Kim, J. Lee, Gener knockdown in the olfactory bulb, amygdala, and hypothalamus by intranasal siRNA administration, Kor. J. Anat. 42 (2009) 285–292. [20] Y. Li, P.M. Field, G. Raisman, Olfactory ensheathing cells and olfactory nerve fibroblasts maintain continuous open channels for regrowth of olfactory nerve fibres, Glia 52 (2005) 245–251. [21] P. Lingor, M. Bahr, Targeting neurological disease with RNAi, Mol. Biosyst. 3 (2007) 773–780. [22] Y.P. Ma, M.M. Ma, S. Ge, R.B. Guo, H.J. Zhang, W.H. Frey 2nd, G.L. Xu, X.F. Liu, Intranasally delivered TGF-beta1 enters brain and regulates gene expressions of its receptors in rats, Brain Res. Bull. 74 (2007) 271–277.
197
[23] T. Nguyen, E.M. Menocal, J. Harborth, J.H. Fruehauf, RNAi therapeutics: an update on delivery, Curr. Opin. Mol. Ther. 10 (2008) 158–167. [24] N. Nonaka, S.A. Farr, H. Kageyama, S. Shioda, W.A. Banks, Delivery of galaninlike peptide to the brain: targeting with intranasal delivery and cyclodextrins, J. Pharmacol. Exp. Ther. 325 (2008) 513–519. [25] J.M. Nores, B. Biacabe, P. Bonfils, [Olfactory disorders in Alzheimer’s disease and in Parkinson’s disease. Review of the literature], Ann. Med. Intern. (Paris) 151 (2000) 97–106. [26] M.A. Reger, G.S. Watson, W.H. Frey 2nd, L.D. Baker, B. Cholerton, M.L. Keeling, D.A. Belongia, M.A. Fishel, S.R. Plymate, G.D. Schellenberg, M.M. Cherrier, S. Craft, Effects of intranasal insulin on cognition in memory-impaired older adults: modulation by APOE genotype, Neurobiol. Aging 27 (2006) 451–458. [27] M.A. Reger, G.S. Watson, P.S. Green, L.D. Baker, B. Cholerton, M.A. Fishel, S.R. Plymate, M.M. Cherrier, G.D. Schellenberg, W.H. Frey 2nd, S. Craft, Intranasal insulin administration dose-dependently modulates verbal memory and plasma amyloid-beta in memory-impaired older adults, J. Alzheimers Dis. 13 (2008) 323–331. [28] M.A. Reger, G.S. Watson, P.S. Green, C.W. Wilkinson, L.D. Baker, B. Cholerton, M.A. Fishel, S.R. Plymate, J.C. Breitner, W. DeGroodt, P. Mehta, S. Craft, Intranasal insulin improves cognition and modulates beta-amyloid in early AD, Neurology 70 (2008) 440–448. [29] T.M. Ross, P.M. Martinez, J.C. Renner, R.G. Thorne, L.R. Hanson, W.H. Frey 2nd, Intranasal administration of interferon beta bypasses the blood–brain barrier to target the central nervous system and cervical lymph nodes: a non-invasive treatment strategy for multiple sclerosis, J. Neuroimmunol. 151 (2004) 66–77. [30] E.I. Schwartz, Potential application of RNAi for understanding and therapy of neurodegenerative diseases, Front. Biosci. 14 (2009) 297–320. [31] M.S. Shim, Y.J. Kwon, Efficient and targeted delivery of siRNA in vivo, FEBS J. 277 (2010) 4814–4827. [32] R.G. Thorne, G.J. Pronk, V. Padmanabhan, W.H. Frey 2nd, Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration, Neuroscience 127 (2004) 481–496. [33] K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery, Nat. Rev. 8 (2009) 129–138. [34] H. Wu, K. Hu, X. Jiang, From nose to brain: understanding transport capacity and transport rate of drugs, Expert Opin. Drug Deliv. 5 (2008) 1159–1168.