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Nose-to-brain delivery: Investigation Into Transport of nanoparticles with different surface characteristics and size in excised porcine olfactory epithelium Lisbeth Illum, Alpesh Mistry, and Snjezana Stolnik Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00088 • Publication Date (Web): 21 May 2015 Downloaded from http://pubs.acs.org on May 31, 2015

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Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

P80-PS Nanoparticles 200nm

test Lumen

Porcine olfactory epithelium ACS Paragon Plus Environment


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Table of content

Abstract

Page 2

Keywords

Page 2

Introduction

Page 3

Materials and methods

Page 4

Nanoparticle test systems

Page 5

Nanoparticle physicochemical characterisation

Page 6

Nanoparticle quantification

Page 6

Dissection of nasal epithelia from the pig

Page 6

Vertical Franz Diffusion Chamber transport study

Page 7

Measurement of tissue viability

Page 8

Electrophysiological epithelial barrier integrity assessment

Page 9

Blank transport studies

Page 9

Sample preparation for histological examination

Page 9

Statistical analysis

Page 10

Results

Page 10 Nanoparticle transport study

Page 11

Evaluation of epithelial viability

Page 14

Discussion

Page 19

Conclusion

Page 24

Acknowledgements

Page 25

References

Page 25

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Nose to brain delivery: Investigation into transport of nanoparticles with different surface characteristics and size in excised porcine olfactory epithelium Alpesh Mistry1, Snjezana Stolnik1 and Lisbeth Illum2 1

Advanced Drug Delivery and Tissue Engineering Division, School of Pharmacy, University of

Nottingham, Nottingham, NG7 2RD, UK 2

IDentity, 19 Cavendish Crescent North, The Park, Nottingham NG71BA, UK

•

Corresponding author email: [email protected]

•

Telephone: +44 (0)1159481866

1 ACS Paragon Plus Environment


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Abstract The ability to deliver therapeutically relevant amounts of drugs directly from the nasal cavity to the central nervous system to treat neurological diseases is dependent on the availability of efficient drug delivery systems. Increased delivery and/or therapeutic effect has been shown by drugs encapsulated in nanoparticles, however the factors governing the transport of the drugs and/or the nanoparticles from nasal cavity to the brain are not clear. The present study evaluates the potential transport of nanoparticles across the olfactory epithelium in relation to nanoparticle characteristics. Model systems - 20, 100 and 200 nm sized fluorescent carboxylated polystyrene (PS) nanoparticles, non-modified or surface modified with polysorbate 80 (P80-PS) or chitosan (C-PS) - were assessed for transport across excised porcine olfactory epithelium mounted in a vertical Franz Diffusion Cell. Assessment of the nanoparticle content in the donor chamber of the diffusion cell, accompanied by the fluorescence microscopy of dismounted tissues, revealed loss of nanoparticle content from the donor suspension and their association with the excised tissue, depending on the surface properties and particle size. Chitosan surface modification of PS nanoparticles resulted in the highest tissue association amongst the tested systems, with the associated nanoparticles primarily located in the mucus, while the Polysorbate 80 modified nanoparticles showed some penetration into the epithelial cell layer. Assessment of the bioelectrical properties, metabolic activity and histology of the excised olfactory epithelium showed that C-PS nanoparticles applied in pH 6.0 buffer produced damaging effect on the epithelial cell layer in a size dependent manner with ‘fine’ 20 nm sized nanoparticles causing substantial tissue damage, relative to the 100 and 200 nm counterparts. Although, histology showed that the olfactory tissue was affected by the application of citrate buffer which was augmented by addition of chitosan in solution, this was not reflected in the bioelectrical parameters and the metabolic activity of the tissue. Regarding the transport across the excised olfactory tissue, none of the nanoparticle systems tested, irrespective of particle size or surface modification, was transported across the epithelium to appear in measurable amounts in the receiver chamber. Keywords Transepithelial transport, chitosan, polysorbate 80, excised tissue, olfactory epithelium, nose-tobrain delivery 2 ACS Paragon Plus Environment

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1. Introduction In recent years, a large number of studies in animal models have demonstrated the ability of small molecular weight drugs, peptides and proteins to be transported directly from the nasal cavity to the central nervous system (CNS) 1-8. However, the amount of drug transported via this route was shown to be small (well below 1%) and unless steps are taken by means of drug delivery technology to enhance the transport across the olfactory epithelium and/or via the olfactory and trigeminal nerves, this route is only therapeutically viable for very potent drugs 9. Some early studies showed that nanoparticles in the form of elemental particular materials such as iron (II) oxide 1, manganese oxide

10

, carbon

11,12

and gold

13

applied to the nasal cavity can

reach the brain, albeit in small quantities. The mechanism of nose-to-brain transport of these nanoparticles was not completely elucidated in these early published works. However, it has later been suggested that nanoparticles after nasal application can reach the brain using three main routes of transport, a) transport through the nasal respiratory epithelium into the systemic circulation followed by transport across the blood brain barrier (BBB) (‘systemic pathway’), b) direct transport paracellularly or transcellularly via the olfactory neuron (‘olfatory neural pathway’) or the olfactory epithelium (‘olfactory epithelial pathway’) or c) by transport via the trigeminal nerves situated in in respiratory and olfactory epithelia (‘trigeminal pathway’)14. The particles are expected to be taken up by the cells by processes of endocytosis ie micropinocytosis or phagocytosis. Fluorescent or radiolabeled nanoparticles, produced from materials such as polystyrene, PLA, PLA-PEG and chitosan, have also shown indication of accumulation in the brain especially when functionalized with specific lectins chosen for the ability to attach to receptors in the olfactory region

15-18

. However, it should be noted that in these papers the

nanoparticles were identified and localized on the basis of fluorescent signals or radioactivity measures. Furthermore, an increasing number of publications have described an improvement of drug concentration in the brain after nasal application of a drug encapsulated in nanoparticles as compared to a simple drug solution, e.g. nimodipine in methoxy-PEG-PLGA nanoparticles19, coumarin in lectin conjugated PEG-PLA nanoparticles15,20, rhodamine in chitosan nanoparticles20 and leucine-enkephalin in trimethyl chitosan nanoparticles22. However, very few studies have attempted to elucidate whether the drug was transported to the brain encapsulated in the 3 ACS Paragon Plus Environment


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nanoparticles by the neural or transepithelial pathways or whether the drug was released on the surface of the nasal epithelium or inside the tissue and then transported across the membrane. The site of release of the drug is important since it determines the approach for optimization of the nanoparticles, either by increasing the adherence to the mucus or promoting transport into the tissue. Encapsulated drug may also be protected against the degradative effects of extracellular enzymes and avoid membrane efflux pumps23, especially if the nanoparticles containing the drug are able to be transported into/across the mucosal epithelial cell layer. This could also explain the increased transport of drug to the CNS. Little information is available about factors such as surface characteristics and size that may affect the transport of nanoparticles from the nasal cavity to the CNS. Due to the size of the tight junctions in the nasal cavity being in the order of 3.9 – 8.4 Å, the transport of molecules across the tight junctions larger that 15Å being negligible24 and the suggestion that absorption enhancers opens the tight junctions only 10 - 15 times25, it is likely that only nanoparticles with a diameter less than 20 nm can achieve extracellular transport from the nasal cavity to the brain. Furthermore, it is to be expected that only nanoparticles with a diameter less than that of the olfactory axons can be intracellularly transported to the brain via the olfactory neural pathway. De Lorenzo13 showed in electron microscopy studies the average diameter of olfactory axons in 2 month old rabbits to be about 200 nm with many axons less than 100 nm in diameter, whereas the olfactory axons of humans are between 100 and 700 nm26. In general, cellular internalization of nanoparticles is suggested to be dependent on concentration, particle size, surface charge and other surface characteristics such as lipophilicy27-29. The aim of the present work was to investigate the mechanism involved in the transport of nanoparticles into, and potentially across, the olfactory nasal epithelium in an in vitro porcine animal model. Porcine nasal tissue was selected as suitable for use in in-vitro diffusion chamber transport studies since (i) the olfactory epithelium is sufficiently large to obtain workable tissue pieces and (ii) the gross morphology of the epithelium and some compositional aspects of the mucus are similar between humans and pigs30-32. The excised olfactory epithelium was mounted in an in vitro vertical Franz diffusion cell model, which allowed a detailed investigation of the time-related association and movement of nanoparticles in relation to the tissue, permitting strict control of physiological conditions such as pH, temperature and oxygenation. 4 ACS Paragon Plus Environment

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A number of colloidal formulations with a range of nanoparticle diameters and surface characteristics was evaluated in this model. The model nanoparticles selected for the study were in the 20-200 nm size range, appropriate for internalization and transport by epithelial cells and/or axons. Non-modified polystyrene latex (PS) provided nanoparticles with a highly negatively charged and hydrophobic surface, chitosan coating created positively charged (C-PS) nanoparticles with a higher surface hydrophilicity, while polysorbate 80 coated nanoparticles (P80-PS) were hydrophilic with an almost neutral charge. These different nanoparticle systems were selected based on previous studies in rats and mice models33,34. After exposure to the test formulations,

the

viability

of

the

olfactory

epithelia

was

also

evaluated

using

electrophysiological and Alamar Blue® viability methods. Materials and Methods Carboxylate-modified fluorescent polystyrene nanoparticles (FluoSpheres) with diameters of 20 nm, 100 nm and 200 nm containing either a yellow-green dye or red fluorescent dye were purchased from Molecular Probes (Paisley, UK). Tween 80 (polysorbate 80, 1.31 kDa) was purchased from Sigma (Gillingham, UK). Chitosan (143 kDa, 89% deacetylation) was donated by Bioneer (Hørsholm, Denmark). Krebs Ringer Bicarbonate buffer (Sigma, Gillingham, UK) contained 1.5 mM NaH2PO4, 0.83 mM NaHPO4, 1.67 mM Mg2Cl2, 4.56 mMKCl, 119.78 mMNaCl and 10 mM D-glucose. NaHCO3, CaCl2, citric acid and all other chemicals were obtained from Sigma (Gillingham, UK). Materials for brightfield light microscopy sample preparation (glutaraldehyde, sodium cacodylate, propylene oxide, Araldite CY212, Agar 100 resin, DDSA and Dibutyl phthalate DMP 30) were all purchased from Agar Scientific (Stanstead, UK). Glycine was purchased from Fisher Scientific, Loughborough, UK and IMS and osmium tetroxide from Sigma-Aldrich, Gillingham, UK. Tissue samples were mounted onto microscope slide using Prolong Gold DAPI (Invitrogen A/S, Taastrup, Denmark). Simulated nasal solution (SNS buffer) was prepared by modifying Krebs Ringer Bicarbonate buffer according to Östh et al.35, with the addition of 1.5 mM NaHCO3 and 1.2 mM CaCl2. The osmolarity was adjusted to 300 ± 5 mol/kg by the addition of NaCl and the pH was adjusted to 7.4 with 1M NaOH. The buffer was oxygenated using a mixture of O2 (95%) and CO2 (5%). For cell viability studies 300 mM sodium azide was added to the buffer. Citrate buffers 0.1 M (pH 5 ACS Paragon Plus Environment


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4.5 or pH 6.0) were prepared from 0.1 M citric acid monohydrate and 0.1M trisodiumcitrate dihydrate. Isotonicity of the citrate buffer was achieved by the addition of NaCl as required, and pH adjusted with 1M NaOH. Nanoparticle test systems 20 nm, 100 nm or 200 nm PS nanoparticles (FluoSpheres), surface modified with either chitosan (C-PS) (in citrate buffer pH 4.5 or 6.0) or polysorbate 80 (P80-PS) (in SNS buffer pH7.4) were used in the transport studies. The surface modified nanoparticle suspensions were prepared as described previously34. Carboxylated PS latex (as purchased) suspended in SNS buffer was used for comparison purposes; referred to as non-modified nanoparticles. According to the manufacturer’s batch specifications, the differences between size and surface charge of the red and yellow-green fluorescent labelled nanoparticles were considered negligible. Nanoparticle physicochemical characterisation The Dynamic Light Scattering (Model 802, Viscotek, Worcestershire, UK) instrument was used for particle size measurements. Particle size is reported as average hydrodynamic diameter of intensity distribution from ten measurements in three independent experiments. Zeta potential was measured using a ZetaSizer 2000 (Malvern, Malvern, UK). An average of five measurements was made for each sample. Zeta potential is reported as the mean ± standard deviation. The reported zeta potential values represent a mean of three independent experiments. All particle size and zeta potential measurements were performed in the appropriate buffer that was used for transport studies. Nanoparticle quantification The 200 µL samples, taken during the transport study from the receiver and/or donor chambers were analysed for nanoparticle content by fluorescence spectroscopy. Yellow-green or red fluorescent nanoparticles were detected at 485/530 (FL600FA, Labtech Int. Ltd., Ringmer, UK) or 530/590 (FluoroSkan Ascent, Thermo, Stone, UK), respectively. The concentration of nanoparticles (number of particles/ml) in each aliquot was calculated by using a standard curve.


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Dissection of Nasal Epithelia from the pig Porcine nasal mucosa was isolated from six month old domesticated sows slaughtered in the slaughterhouse at Sutton Bonnington Campus, University of Nottingham, UK. The animals were electrically stunted and then killed by exsanguination in compliance with current UK legislation on animal experimentation (Animals Scientific Procedures Act 1986) and within the requirements of Commission Directive 86/609/EEC concerning the protection of animals used for experimental and other scientific purposes. The head was removed and the nasal cavity exposed. To gain access to the olfactory region, a ventral incision (up to and including the hard palette) was made ~5 cm rostrally in front of the eyes (Figure 1, Incision 1) and ~6cm further rostrally (Figure 1, Incision 2) to yield Section A.

Incision 2

Incision 1

Section A

Figure 1. Schematics illustrating sectioning of the pig head for retrieval of porcine nasal mucosa right side of sagittal section of mature swine skull. Adapted from from Getty 36.

Section A was then sawed horizontally along the middle of the nasal cavity and the olfactory epithelium exposed (dorsal surface of the nasal cavity) and peeled away in the rostrocaudal direction from the underlying bone or cartilage with forceps and a haemostat. The dissection was performed within 15 minutes post-mortem. 7 ACS Paragon Plus Environment


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The olfactory epithelium (retrieved from Section A (Figure 1)) was easily distinguishable from the surrounding respiratory tissue type by its yellowish appearance (due to the presence of olfactory pigment) compared to the pinkish colour of the respiratory mucosa. Within 20 minutes of slaughter, two pieces (~2 cm diameter) of olfactory epithelium (one from each side of the nasal cavity) were cut out with a cork borer. Acquired tissue samples were transported to the laboratory on ice in vials containing either citrate or SNS pre-oxygenated (O2/CO2 95:5 mix). Selection of the buffer depended on the nanoparticles systems used in the transport study. Vertical Franz Diffusion Chamber transport study The excised tissue was loaded into the vertical Franz diffusion chamber (Crown Glass Company, CA), comprising two symmetrical side-by-side diffusion chambers. The epithelium was mounted vertically between the chambers (mucosal side facing the donor chamber), using two cork-ring braces, within ~1 hour of slaughter. The braces reduced the physical tension on the tissue during the experiment and the likelihood of tissue tear. The inner chambers were filled with cold preoxygenated buffer solution to the collar of the sample port. The tissue immersion buffer was allowed to warm slowly to and was maintained at 29±1ºC during the transport experiment to mimic the temperature in the nasal cavity37. The buffer was pre-sparged with O2/CO2 and stirred by a magnetic stirrer rather than stirring the buffer by bubbling of gas38. The initial electrophysiological (TEER, PD and Isc) measurements were taken every 5 minutes in the appropriate buffer during the equilibration period (~30 minutes). For the transport study, a suitable volume of the test nanoparticles suspension was placed into the donor chamber following the equilibration period to create a final suspension containing 4.55x1010 particles/ml. Thereafter, 200 µl aliquots (~6% of the total chamber volume) were withdrawn from the receiver chamber every 10 minutes for 90 minutes and placed into a 96 well microtitre plate for nanoparticle content analysis by fluorescent spectroscopy. The buffer volume in the receiver chamber was replaced with the appropriate pre-warmed and pre-oxygenated buffer. During the transport experiment, electrophysiological measurements were taken at the 40, 60, 80 and 100 minute time points and every 5 minutes for the last 30 minutes of the study. The


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transport study was terminated after a total of ~3½ hours post-mortem, the excised tissue was carefully dismounted and evaluated for cell viability (Alamar Blue® assay) and examined by fluorescence and optical microscopy. Measurement of tissue viability Alamar Blue® Assay (AbDSerotec Ltd) was carried out as an indicator of tissue viability. The analysis was performed on (i) excess of dissected olfactory epithelium (positive control prior to the transport experiment) (ii) excess excised tissue exposed to appropriate buffer containing sodium azide (300 mM) (negative control) and (iii) tissue from dismounted olfactory epithelium following the transport experiment. For the assays, three tissue sections of ~3 mm in diameter were cut using a cork borer and placed in wells of a 96 well-plate containing 200 µl of a 10% v/v solution of Alamar Blue® stock solution and the test performed as per manufacturer’s instructions. Electrophysiological epithelial barrier integrity assessment The electrophysiological measurements to assess barrier integrity of the excised tissue were taken prior, during and following the transport study, as indicated above. An EVOM (World Precision Instruments, Stevenage, UK) epithelial voltohmmeter, combined with STX2 electrode system, was employed to gain data on transmucosal electrical resistance (TEER), potential difference (PD) and short-circuit current (Isc), as previously described by Wadell et al

39

and

35

Östh et al. . ‘Blank’ transport studies

The transport study was also performed in the absence of nanoparticles to assess potential damage to the mounted tissues arising from the experimental procedure (e.g. tissue trauma due to dissection, translocation and mounting onto in vitro apparatus) and from exposure to buffers and chitosan and Polysorbate 80 materials used for surface modification of the nanoparticles. Hence, control samples of (i) SNS, (ii) citrate buffer (pH 6.0), (iii) chitosan solution (in citrate buffer, pH 6.0) and (iv) polysorbate 80 solution (in SNS buffer) were tested. 0.87 µM and 105



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µM chitosan and polysorbate 80 solutions, respectively were applied. These concentrations exceeded the highest concentration of the materials used in the surface modification of the nanoparticles and that could be present in the diffusion chamber during the transport study 34. Sample preparation for histological examination Two small pieces (approx. 4 mm2) of olfactory epithelium were cut from the tissue dismounted from the Franz Diffusion Cell after termination of the experiment using a scalpel knife. The first sample was immediately fixed with glutaraldehyde, rinsed with water and alcohol and embedded in Araldite CY212 resin/Agar 100 resin/DDSA using procedures previously described by Bancroft and Stevens

40

. The sample was placed in a mould and heated at 60oC for 48 hours

before producing slices of 500 nm thickness for brightfield microscopy using an ultra-microtome with a glass knife. The second sample was frozen by ‘snap freezing’ (submerging the sample into isopentane, supercooled using liquid nitrogen40). The sample was then cryomounted using Bright Cryo-M-Bed cryomountant (Bright Instrument Co. Ltd. Huntingdon, UK) onto a cork disc and stored at -80°C until required for cryosectioning. Cryosectioning was performed on a B3889 (Bright Instrument Co. Ltd. Huntingdon, UK) cryostat. The tissue was sliced into 10 mm sections and subsequently mounted onto standard glass histological slides using glycerol jelly and coverslips. The jelly was prepared by dissolving 10g gelatine (Sigma, Gillingham, UK) in 60ml water and 70ml glycerol (Sigma, Gillingham, UK) and stored at 2-8°C. Imaging of histological sections was performed using a Labophot 2 fluorescent microscope (Nikon UK Ltd, Kingston Upon Thames, UK) mounted with a high-resolution DXM1200 camera (Nikon UK Ltd, Kingston Upon Thames, UK) and bandpass filter cubes for DAPI, FITC and TRITC (Nikon UK Ltd, Kingston Upon Thames, UK). Statistical analysis Results are presented as means±standard error and a one-way analysis of variance (ANOVA) with a post hoc test was applied. A Bonferroni post-hoc test was performed on groups with equal


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variances and a Games-Howell post-hoc test was performed on groups with unequal variances. A value of p0.05 ANOVA).


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The percentage losses from the donor chamber were not affected by particle size for nonmodified PS nanoparticles (p>0.05 ANOVA), and in general for the chitosan coated and polysorbate 80 coated nanoparticles, except for the 20 nm C-PS which showed a significant higher loss that than 100 nm and 200 nm C-PS nanoparticles and the 200 nm P80-PS which showed a higher loss than the 20 nm and 100 nm P80-PS nanoparticles. Table 1. Percentage loss of particles from donor chamber during transport study with PS and PS surface modified nanoparticles

Nanoparticle system

% number loss (mean ± standard error)

Non-modified polystyrene latex (PS) suspended in SNS 20 nm

0.0±0.0*

100 nm

7.9 ± 9.0

200 nm

6.1 ± 1.8

Polysorbate 80 modified polystyrene nanoparticles (P80-PS) suspended in SNS 20 nm

0.0±0.0*

100 nm

4.2 ± 4.9

200 nm

22.2 ± 6.4a

Chitosan modified nanoparticles (C-PS) suspended in citrate buffer pH 6.0 20 nm

16.4 ± 7.6a

100 nm

5.7 ± 5.0

200 nm

8.5 ± 2.7

Chitosan modified nanoparticles (C-PS) suspended in citrate buffer pH 4.5 100 nm

39.9 ± 4.6a,b

a

Statistically significant compared to uncoated particles (ANOVA with Bonfferoni post hoc test p

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