KUOPION YLIOPISTON JULKAISUJA D. LÄÄKETIEDE 414
KUOPIO UNIVERSITY PUBLICATIONS D. MEDICAL SCIENCES 414
LEENA PITKÄNEN
Retinal Pigment Epithelium as a Barrier
in Drug Permeation and as a Target
of Non-Viral Gene Delivery
Doctoral dissertation
To be presented by permission of the Faculty of Medicine of the University of Kuopio
for public examination in Auditorium, Tietoteknia building, University of Kuopio,
on Friday 31 st August 2007, at 12 noon
Department of Pharmaceutics and
Department of Ophthalmology
University of Kuopio and Kuopio University Hospital
JOKA
KUOPIO 2007
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Series Editors: Professor Esko Alhava, M.D., Ph.D.
Institute of Clinical Medicine, Department of Surgery
Author´s address: Department of Ophthalmology
University of Kuopio
P.O. Box 1627
FI-70211 KUOPIO
FINLAND
Tel. +358 17 172 476
Fax +358 17 172 486
Supervisors: Professor Arto Urtti, Ph.D.
Drug Delivery and Development Technology Center
University of Helsinki
Lecturer Veli-Pekka Ranta, Ph.D.
Department of Pharmaceutics
University of Kuopio
Docent Markku Leino, M.D. Ph.D.
Department of Ophthalmology
University of Kuopio
Reviewers: Professor Stefaan De Smedt, Ph.D.
Laboratory of General Biochemistry & Physical Pharmacy
Ghent University
Belgium
Professor Ilkka Immonen, M.D., Ph.D.
Department of Ophthalmology
University of Helsinki
Opponent: Professor emerita Lotta Salminen, M.D., Ph.D.
Department of Ophthalmology
University of Tampere
Professor Raimo Sulkava, M.D., Ph.D.
School of Public Health and Clinical Nutrition
Professor Markku Tammi, M.D., Ph.D.
Institute of Biomedicine, Department of Anatomy
ISBN 978-951-27-0674-7
ISBN 978-951-27-0751-5 (PDF)
ISSN 1235-0303
Kopijyvä
Kuopio 2007
Finland
Pitkänen, Leena. Retinal pigment epithelium as a barrier in drug permeation and as a target of nonviral gene delivery. Kuopio University Publications D. Medical Sciences 414. .2007. 75 p.
ISBN 978-951-27-0674-7
ISBN 978-951-27-0751-5 (PDF)
ISSN 1235-0303
ABSTRACT
Retinal pigment epithelium (RPE) is a unique monolayer of cells which lie between the neural retina
and the choroid. It plays an essential role in maintaining visual acuity and metabolic integrity in the
retina. As a part of the blood-retina barrier, RPE restricts the molecules from blood flow and outer
ocular layers from gaining access to the neural retina. Many retinal diseases may be relieved by
delivery of neuroprotective or antiangiogenic factors to the retina. RPE is an interesting target for
transfer of genes to induce a production of therapeutic proteins. Viral gene transfer vectors are most
often used in gene therapy, but non-viral polymeric and liposomal vectors are more biocompatible and
easier to produce. However, their gene transfer efficacy does not match that of viral vectors. For
example, intravitreally given DNA must permeate the vitreous and the neural retina before reaching
the RPE.
In the present study, the limiting barriers after intravitreal non-viral gene delivery were identified.
In vitro experiments with bovine vitreous and retina demonstrated that both vitreous and neural retina
restrict the uptake of cationic gene complexes (DOTAP, PEI and PLL complexes) into the RPE. The
large size and especially the positive charge of the complex are the reasons for a limited access into
the RPE. Though, the exact mechanisms remain unclear. Oligonucleotides in solution were efficiently
taken up by RPE cells, but the neural retina limits their permeation. The uptake of naked plasmid into
RPE cells was very low even without the presence of vitreous or neural retina.
Prolonged drug delivery to the posterior segment of the eye is a challenge in ophthalmology. After
subconjunctival administration, the drug or gene product (resulting from cell or gene therapy) has to
permeate across the sclera, choroid and RPE to reach the neural retina. The influence of lipophilicity
(beta-blockers) and size (FITC-dextrans) of the permeants were assessed using isolated bovine
choroid-RPE. For hydrophilic compounds, the choroid-RPE was 10-100 times less permeable than the
sclera, whereas for lipophilic compounds the RPE and sclera were equal barriers emphasizing the
important role of the RPE in permeation. The permeability of the RPE is a key factor also in the drug
delivery from the systemic blood circulation into the retina.
Choroid and RPE contain melanin that binds many drugs. Synthetic melanin is often used for
binding studies. In the present study it was demonstrated that the melanin isolated from bovine ocular
tissue and synthetic melanin differ in terms of size, surface area, shape, aggregation properties, and
drug binding. Based on the data supplemented with further calculations it was estimated that the
choroid-RPE contains 3-19 times more melanin bound betaxolol and metoprolol compared to the free
drug. In contrast, phosphodiesterase oligonucleotides and carboxyfluorescein did not bind to melanin.
In conclusion, the vitreous and neural retina are barriers to the non-viral gene transfer to the RPE.
In transscleral delivery, the RPE-choroid is the rate-limiting barrier for large and hydrophilic
molecules but not necessarily for lipophilic drugs. Furthermore, melanin binding modifies the
pharmacokinetics of betaxolol and metoprolol at the cellular level in the posterior eye segment.
National Library of Medicine Classification: QU 110, QV 132, WB 340, WW 103, WW 245, WW
250, WW 270
Medical Subject Headings: Adrenergic beta-Antagonists/ pharmacokinetics; Choroid/metabolism;
Dextrans/pharmacokinetics; Drug Delivery Systems; Gene Transfer Techniques; Liposomes;
Melanins; Permeability; Pigment Epithelium of Eye; Polymers; Retina; Vitreous Body
To My Family
ACKNOWLEDGEMENTS
The series of studies was carried out in the department of Pharmaceutics in University of
Kuopio in 1997-2006.
I express my deepest gratitude to my principal supervisor, Professor Arto Urtti for his
professional guidance. His expertise, optimism and patience were essential for this work. I
thank my supervisor, Veli-Pekka Ranta, Ph.D., for actively using his admirable knowledge to
advance this work. I thank my supervisor, Docent Markku Leino for his valuable comments
and encouragement.
I wish to thank Professor Jukka Mönkkönen, the Dean of the Faculty of Pharmacy,
Professor Jukka Gynther, the former Dean of the Faculty of Pharmacy and Professor Kristiina
Järvinen, Head of the Department of Pharmaceutics for providing excellent facilities and
working environment.
I express my gratitude to Professor Stefaan De Smedt and Professor Ilkka Immonen for
reviewing the manuscript and for valuable comments.
I warmly thank my co-authors Marika Ruponen, Ph.D., Hanna Moilanen, M.Sc., Professor
Jukka Pelkonen, Seppo Rönkkö, Ph.D. and Jenni Nieminen M.Sc. for pleasant collaboration.
I thank Acting Professor Iiris Sorri for her advice and support, and Professor Hannu
Uusitalo and Professor emerita Maija Mäntyjärvi for their help concerning my research. I am
grateful to Docent Markku Teräsvirta and Docent Tuomo Puustjärvi for their co-operation. I
thank Docent Kaija Tuppurainen for flexible arrangements of my clinical training during this
work. Furthermore, I want to thank all dear colleagues and personnel of the Department of
Ophthalmology of Kuopio University Hospital and Kuopion Näkökeskus for their support.
I thank the whole personnel of Department of Pharmaceutics for pleasant atmosphere and
helpfulness. I am especially grateful to Professor Paavo Honkakoski, Mika Reinisalo, M.Sc.
and Ilpo Jääskeläinen, Ph.D. for promoting this work with their ideas and valuable advice. I
thank laboratory assistants Hanna Eskelinen and Kaarina Pitkänen for their skillful practical
help, especially in the beginning of this work when I was really lost in the lab. I also want to
specially thank Johanna Jyrkkärinne M.Sc., Antti Valjakka, Ph.D., Elisa Toropainen, Ph.D.
and Zanna Hyvönen, Ph.D. for their friendly support and my most long-term room mates
pharmacist Päivi Tiihonen, Marjukka Suhonen, Ph.D., and Hannu Mönkkönen, Ph.D. for nice
company.
I own my warmest thanks to my husband Veijo for his love and support and to our sons
Eemeli, Arttu and Santeri for the joy that they have brought to my life. I thank my parents
Katri and Vesa Siira for their support. I am grateful to Heikki and Hanne Siira, Lea and Veijo
Saarelainen, Helena and Heikki Pitkänen, Virpi Lindi and all other friends and relatives for
giving me joy and strength.
This work has been financially supported by the Academy of Finland, the Special
Government Funding from Kuopio University Hospital, the National Agency of Technology
(TEKES), the Finnish Cultural Foundation of Northern Savo, the Eye- and Tissuebank
Foundation, the Friends of the Blinds, the Finnish Medical Foundation, the Research and
Science Foundation of Farmos and Retina Finland which are gratefully acknowledged.
Kuopio, June 2007
Leena Pitkänen
ABBREVIATIONS
AAV
adeno associated virus
AMD
age related macular degeneration
ARPE
adult retinal pigment epithelium
BET
Brunauer, Emmet, Teller-method
BRB
blood-retina-barrier
CF
carboxyfluorescein
DNA
deoxyribonucleic acid
DMEM
Dulbecco`s Modified Eagles Medium
DOPE
dioleylphosphatidylethanolamine
DOTAP
1,2-dioleoyl-3-trimethylammonium-propane
EDTA
ethylenediaminetetraacetic acid
EMA
ethidium monoazide
FACS
fluorescence activated cell sorter
FITC
fluorescein isothiocyanate using
FRAP
fluorescence recovery after photobleaching
GAG
glycosaminoglycan
GFP
green fluorescent protein
HPLC
high performance liquid chromatography
HEPES
N-2-hydroxyethylpiperazine-N-´2-ethane sulphonic acid
HSV
herpes simplex virus
HSV-tk
herpes simplex virus-thymidine kinase
IgG
immunoglobulin
ILM
inner limiting membrane
kb
kilobase
kDa
kilodalton
MRI
magnetic resonance imaging
mRNA
messenger ribonucleic acid
mw
molecular weight
PEG
polyethyleneglycol
PEI
polyethyleneimine
PLL
poly-L-lysine
PVR
proliferative vitreoretinopathy
pGFP
plasmid encoding green fluorescent protein
rAAV
recombinant adeno associated virus
PBS
phosphate buffered saline
RISC
RNA-induced silencing complex
RNA
ribonucleic acid
RP
retinitis pigmentosa
RPE
retinal pigment epithelium
SDS
sodium dodecyl sulphate
siRNA
small interfering ribonucleic acid
TAE
tris acetate ethylenediaminetetraacetic acid
TEER
transepithelial electrical resistance
TEP
transepithelial potential difference
VEGF
vascular endothelial growth factor
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications, referred to the text by Roman
numerals I-IV.
I
Pitkänen L, Ruponen M, Nieminen J, Urtti A: Vitreous is a barrier in nonviral gene
transfer by cationic lipids and polymers. Pharmaceutical Research 20:576-83, 2003
II
Pitkänen L, Pelkonen J, Ruponen M, Rönkkö S, Urtti A: Neural retina limits the
nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model.
The AAPS Journal 6:e25, 2004
III
Pitkänen L, Ranta VP, Moilanen H, Urtti A: Permeability of retinal pigment
epithelium: effects of permeant molecular weight and lipophilicity. Investigative
Ophthalmology and Visual Science 46:641-6, 2005
IV
Pitkänen L, Ranta VP, Moilanen H, Urtti A: Binding of betaxolol, metoprolol and
oligonucleotides to synthetic and bovine ocular melanin, and prediction of drug
binding to melanin in human choroid-retinal pigment epithelium. Pharmaceutical
Research (in press)
CONTENTS
1 INTRODUCTION
16
2 REVIEW OF LITERATURE
18
2.1 General aspects of drug delivery to the retina
18
2.2 Tissue barriers in the eye
20
2.2.1 Cornea
20
2.2.2 Conjunctiva
20
2.2.3 Sclera
21
2.2.4 Choroid
22
2.2.5 Blood-retina barrier
23
2.2.6 Binding of drugs to melanin
24
2.2.7 Vitreous humor
25
2.3 Gene delivery systems for retinal transfection
26
2.3.1 Viral vectors
26
2.3.2 Non viral methods
27
2.3.3 Ex vivo gene therapy
30
2.3.4 Oligonucleotides and ribozymes
30
2.4 Retinal gene therapy
31
3 AIMS OF THE STUDY
34
4 MATERIALS AND METHODS
35
4.1 Labeled macromolecules
35
4.2 Carriers
35
4.3 Plasmids
36
4.4 Oligonucleotides
36
4.5 Beta-blockers and carboxyfluorescein
37
4.6 Cell culture and transfection
37
4.7 Tissue preparation and permeation experiments
38
4.8 Melanin binding experiments
41
4.9 High performance liquid chromatography
43
4.10 Flow cytometric analysis
43
4.11 Fluorometry, BET and laser diffractometry
44
4.12 Microscopic analysis
45
4.13 Statistical analysis
46
5 RESULTS
5.1 Vitreous as a barrier for gene transfer by cationic lipids and polymers
47
47
5.1.1 DNA-complexes
47
5.1.2 FITC-dextrans and FITC-PLL
48
5.2 Neural retina as a barrier for gene transfer by cationic lipids and polymers 48
5.2.1 DNA-complexes
48
5.2.2 FITC-oligonucleotides and uncomplexed plasmid DNA
49
5.2.3 FITC-dextrans and FITC-PLL
49
5.3 Permeability of RPE
50
5.3.1 Tissue viability in diffusion chambers
50
5.3.2 Permeability of carboxyfluorescein and FITC-dextrans
50
5.3.3 Permeability of beta-blockers
51
5.4 Binding to melanin
51
5.4.1 Characterization of synthetic and isolated melanin
51
5.4.2 Binding of beta-blockers, oligonucleotides and 6-CF to melanin
51
6 DISCUSSION
53
6.1 The vitreous and neural retina as a barrier
53
6.2 RPE as a barrier
56
7 CONCLUSIONS
61
8 REFERENCES
62
9 ORIGINAL PUBLICATIONS
75
16
1 INTRODUCTION
The transfer of therapeutic genes to the retina represents a promising method for the treatment
of many severe eye diseases which are today without effective treatment. Potential targets of
gene therapy include age related macular degeneration, proliferative vitreoretinopathy, retinal
and choroideal neovascularization and retinitis pigmentosa. Retinal degenerations cause
impaired function of the photoreceptors and consequently lead to a gradual loss of vision. Age
related macular degeneration (AMD) is the most common retinal degeneration and the most
common cause of severe visual impairment in the industrial world. Multiple genes and
environmental factors are considered to play a role in the pathogenesis of AMD (Chamberlain
et al. 2006). Many degenerative diseases can be traced to genetic factors, e.g., retinitis
pigmentosa, which is a common term for the many mutations leading to widespread
degeneration of photoreceptors and the RPE. Proliferative vitreoretinopathy complicates
about 5 % of retinal detachments. It is characterized by vitreal, epiretinal and subretinal
membranes and traction retinal detachment. Diabetic retinopathy, retinal venous occlusion,
retinopathy of prematurity and age related macular degeneration are examples of diseases
which may be complicated by neovascularization. Laser treatment or other available therapies
are not always sufficient to control the vision threatening disease.
Drug delivery into the posterior part of the eye is a challenge. Topical delivery by
eyedrops, ointments, gels or inserts is the most common way to deliver drugs to the ocular
tissues. However, rapid drainage of the eyedrop, anterior membrane barriers (cornea,
conjunctiva, and sclera), systemic absorption via conjunctival vessels, and aqueous humour
outflow mean that the topical drug delivery into the posterior segment is rather inefficient
(Figure 1; Olsen et al. 1995; Geroski and Edelhauser 2000).
Intravenous and oral medications are used in the treatment of systemic diseases with
ocular involvement and in ocular malignancies. Photosensitization therapy, cytotoxic agents,
steroids and antibiotics are examples of drugs that are used systemically to treat ocular
diseases (Jumbe and Miller 2003). Systemic drug treatment with oral or intravenous drugs is
limited by the blood-retinal barrier which is composed of retinal capillaries and the retinal
pigment epithelium (RPE) (Maurice and Mishima 1984; Cunha-Vaz 2004). Intravitreal
injection is the most direct approach to deliver drugs into the vitreous and retina. However,
serious side effects such as endophthalmitis, cataract, hemorrhage and retinal detachment may
occur, particularly if multiple injections are needed. The same side effects are potential
complications also after insertion of intravitreally implanted sustained release devices
17
(Maurice and Mishima 1984; Geroski and Edelhauser 2000). Despite the side effects,
intravitreal injections continue to be the method of choice for the treatment of some
intraocular diseases (Chastain 2003). With a subconjunctival injection, sub Tenon`s injection
(parabulbar injection) or a drug depot, it is possible to deliver a drug locally and to avoid the
invasiveness of intravitreal injections. Lee and Robinson (2001) concluded that direct
penetration is the dominant pathway for a subconjunctivally injected compound entering the
vitreous chamber, instead of entering into the vitreous via the aqueous chamber or via the
general circulation. Furthermore, a peribulbar injection (behind the orbital septum, outside the
muscle conus), a retrobulbar injection (to the intraconal space behind the bulbus of the eye)
and a posterior juxtascleral injection are options to deliver drug into the posterior segment of
the eye (Raghava et al. 2004).
This work examines the properties of the retinal pigment epithelium as a barrier for
intravitreal, transcleral and intravenous drug delivery into the posterior part of the eye. The
behaviour of intravitreally delivered non-viral gene complexes and probe molecules of
different sizes and charges were characterized in in vitro permeation studies with bovine
vitreous and neural retina. The in vitro permeability of bovine RPE to molecules of different
size and lipophilicity was assessed, and binding properties of oligonucleotides and two betablockers (betaxolol and metoprolol) to melanin were characterized.
18
2 REVIEW OF LITERATURE
2.1 General aspects of drug delivery to the retina
Figure 1. Drug delivery routes to the retina (modified from a figure by Geroski and
Edelhauser 2000).
Several barriers limit the drug delivery to the posterior part of the eye. After topical
application, typically less than 5 % of a drug dose reaches the aqueous humour via the cornea;
the vast majority of the eye drop is absorbed systemically through blood vessels in the
conjunctiva and nasal mucosa (Geroski and Edelhauser 2000). The aqueous humour flows
down the pressure gradient from the ciliary processes into the posterior chamber and through
the pupilla into the anterior chamber, and further forward through the trabecular meshwork
into the canal of Schlemm, or through the ciliary body to uveal vessels or periocular tissues.
The small amount of the dose that permeates the cornea may be eliminated from the
intraocular tissues to the episcleral space via the canal of Schlemm or ciliary body (Macha et
al. 2003). The transcorneally permeated drug has to diffuse against the flow of the aqueous
humour to reach the tissues behind the posterior chamber. Lens epithelium, densely packed
lens fibers and nucleus form a barrier between the posterior chamber and vitreous, even
19
though the lens capsule is permeable to small molecules and proteins with a mw of up to 70
kDa and the epithelium does not greatly restrict the movement of the molecules to the fiber
mass (Maurice and Mishima 1984; Boulton and Saxby 2004). Furthermore, to reach the
retina, the drug also needs to permeate across the vitreous. Thus, transcorneal drug delivery to
the posterior part of the eye is very difficult.
The non-corneal route, i.e., delivery through the conjunctiva and sclera, has been suggested to
be a more viable route for posterior segment drug delivery, especially in the case of peptides
and oligonucleotides. These tissues have a higher permeability than the cornea (Hämäläinen
et al. 1997). It has been also shown in vivo that the transscleral route may represent a feasible
way to deliver macromolecules to the retina (Ahmed and Patton 1985; Kim et al 2002;
Ambati et al 2000). However, the conjunctival blood flow may flush away a part of the drug
dose before it enters the retina. Furthermore, if subconjunctival and sub Tenon´s injection or
retrobulbar delivery is used, then the choroidal blood flow may become an important barrier.
The RPE and retinal vessels form a blood-retinal barrier that efficiently restricts the
permeation from the uveal tract or systemic blood flow to the retina and intraocular space.
Kim et al. (2004) followed the permeation of a hydrophilic drug surrogate gadolinium-DTPA
by magnetic resonance imaging (MRI) after placing an episcleral implant at the equator. Li et
al. (2004) assessed the permeation and clearance of model ionic permeants after
subconjunctival injection. No significant in vivo permeation to the vitreous was seen in either
of these experiments. Pars plana has been suggested as being the most permeable pathway for
passive transscleral transport of the polar permeants (Li et al. 2004). Ocular membranes and
tissues express several transporters and efflux pumps that may influence drug permeation
(Aukunuru et al. 2001; Sunkara and Kompella 2003). With respect to the drugs that reach the
intravitreal space or are delivered intravitreally, drug diffusivity in the vitreous and retinal
permeability are the key factors that affect elimination from the vitreous. Lipophilic
compounds tend to exit mainly via the retina, and hydrophilic substances and compounds with
poor retinal permeability diffuse through the hyaloid membrane into the posterior chamber.
Vitreous liquefaction may change the vitreal permeability conditions and the site of injection
can affect the drug distribution in the vitreous (Friedrich et al. 1997; Araie and Maurice 1991;
Chastain 2003).
20
2.2 Tissue barriers in the eye
2.2.1 Cornea
The cornea is the main route of drug absorption from the tear fluid into the eye, and the
corneal permeation of drugs has been studied extensively. Corneal epithelium is a lipophilic
cellular tissue with tight intercellular tight junctions. It is considered to be the main
permeation barrier for very hydrophilic drugs due to the restricted paracellular permeation
(Huang et al. 1983; Chien et al. 1988). If a molecule is lipophilic enough to cross the
epithelium, then the hydrophilic stroma may become the rate-limiting layer for lipophilic
compounds (Huang et al.1983; Prausnitz and Noonan 1998). The corneal permeability is
dependent on the size, shape, lipophilicity, pKa and ionization of the permeant. For a
hydrophilic compound like atenolol, the Papp is approx. 1 x 10-6 cm/s and for the lipophilic
betaxolol of the same size, Papp is approx. 30-50 x 10-6 cm/s (Prausnitz and Noonan 1998).
The optimum logPC for corneal permeation is between 1 to 3 (Schoenwald and Ward 1978,
Schoenwald and Huang 1983). For hydrophilic polyethylene glycols, the cut-off level for the
corneal permeation was determined at a molecular weight of 400-600 by Liaw and Robinson
(1992) but Hämäläinen et al. (1997) reported that the permeability of PEGs decreased
gradually with increasing molecular weight. The unionized drugs usually permeate the
epithelium easier than the ionized form of the drug. At physiological pH, cationic molecules
permeate between the cells more easily than anions (Liaw et al. 1992).
2.2.2 Conjunctiva
The outer epithelium of the conjunctiva has tight junctions and may represent a permeability
barrier to drugs. In general, the conjunctiva is more permeable to drugs than the cornea,
especially for polar hydrophilic molecules (Ahmed et al. 1987). The surface area of the
human conjunctiva is about 18 cm2, 17 times greater than that of the cornea (Watsky et
al.1988). The estimated paracellular pore diameters of rabbit palpebral and bulbar conjunctiva
are about 5 nm and 3 nm, respectively (Hämäläinen et al. 1997), whereas the pore diameter of
apical corneal epithelium is 2.0 nm.
Molecules that permeate through the palpebral
conjunctiva end up in the systemic blood flow, while the permeation of the bulbar conjunctiva
may open a route also to ocular absorption. The pores are large enough to allow permeation of
small peptides and oligonucleotides with molecular weights 5000-10 000. In addition to the
paracellular pore size, also the pore density is greater in the conjunctiva than in the corneal
epithelium (Hämäläinen et al. 1997). The conjunctival permeability of hydrophilic
compounds is usually 10-100 times higher than the corneal permeability (Ahmed et al. 1987;
21
Huang et al. 1989; Sasaki et al. 1995; Sasaki et al. 1997; Hämäläinen et al. 1997). An increase
in lipophilicity correlates positively with increased conjunctival permeation and the greatest
effect of increasing lipophilicity on the conjunctival permeation of beta-blockers is at logPC
values of 1-3 (Wang et al 1991). The outer epithelium of the conjunctiva lies on a highly
vascularized layer, the substantia propria, and thus, transconjunctival permeation results also
in the systemic loss of the drug. Furthermore, transporter and efflux pump mechanisms, pH
and enzymatic lability of drugs may modify the conjunctival permeation of drugs (Horibe et
al. 1997; Wang et al. 1991; Ashton et al.1991; Chien et al. 1991).
2.2.3 Sclera
The sclera is a microporous tissue consisting of water (70 %), proteoglycans and closely
packed collagen fibrils. The thickness of the sclera varies in different parts of the globe from
0.3 to 1.0 mm and it is perforated by numerous arteries and veins. Scleral permeability is
comparable to that of corneal stroma (Geroski and Edelhauser 2000). Scleral permeability
decreases with increasing size of the molecule, but nonetheless the sclera does permit the
permeation of compounds of even high molecular weights. For example, human sclera is
permeable to 70 kDa dextrans, and rabbit sclera to dextrans up to 150 kDa and IgG of same
molecular weight (Olsen et al. 1995; Ambati et al. 2000). Lipophilicity seems to play a less
important role in scleral permeability (Ahmed et al. 1987; Prausnitz and Noonan 1998). When
delivered by a peribulbar or retrobulbar injection, the drug has to permeate also Tenon`s
capsule to reach the retina. Conjunctiva with Tenon`s capsule is reported to be about two
times less permeable to mannitol than plain conjunctiva (Huang et al. 1989). Thus, a subTenon injection could decrease the permeation barrier, even though larger volumes of fluid
are likely to spread and escape into the subconjunctival space (Maurice and Mishima 1984).
Elevated intraocular pressure was reported to decrease the scleral permeability of low
molecular weight molecules (Rudnick et al 1999), but Cruysberg et al 2005 concluded that the
transscleral delivery of macromolecules was relatively unaffected by the pressure gradient.
The estimated permeability rates of the rabbit noncorneal route (conjunctiva and sclera)
for polyethylene glycols of molecular weights 238 and 942 were six and nine times higher
compared to the cornea, respectively. Thus, the non-corneal route has been proposed to
represent a better option also for ocular peptide and oligonucleotide delivery to the posterior
segment (Hämäläinen et al. 1997). Compared to corneal permeation, the permeation in the
conjunctiva and sclera are less sensitive to the changes in permeant lipophilicity (Wang et al.
22
1991; Ahmed et al. 1987). There is also less peptidase activity in the conjunctiva and sclera
than present in the cornea (Hämalainen et al. 2000).
2.2.4 Choroid
After systemic delivery, choroidal and retinal capillaries transfer the drug to the posterior part
of the eye where it comes into contact with the blood retinal barrier. The choroid is a highly
vascularized tissue between the RPE and sclera. It consists of three layers: the vessel layer, a
choriocapillary layer with a network of fenestrated capillaries, and Bruch`s membrane (Macha
and Mitra 2003). The outermost layer of Bruch`s membrane forms the basement membrane of
the choriocapillaris and the innermost layer serves as the basement membrane for the RPE.
Between these layers are two collagenous zones and in the middle is the central elastinbearing layer (Hewitt 1986). Any drug that has crossed the conjunctiva and sclera comes into
contact with the heavily vascularized choroid and may be partly washed into the systemic
blood flow (Maurice and Mishima 1984, Torczynski 1995). The extent of this elimination is
not known. For systemically delivered drugs, the choroidal vasculature is the route to ocular
tissues. The blood flow in the human choroid is about 800-1200 ml/100 g tissue/minute, one
of the highest blood flow rates of all human tissues (Torczynski 1995). The abundant
fenestrations, which have diameters of 60-80 nm, are distributed on the inner wall of the
capillaries. The fenestrated capillaries are very permeable to small molecular weight
substances, and the choroid is permeable also to macromolecules, like IgG and albumin
(Törnquist et al 1990; Torczynski 1995). The choroid-Bruch’s membrane was recently shown
to restrict the permeation of lipophilic and cationic molecules (Cheruvu and Kompella 2006).
However, drug diffusion further inwards is restricted by the RPE.
23
2.2.5 Blood-retina barrier
Figure 2. Retinal layers (modified from http://137.222.110.150/calnet/visual2/page2.htm)
The retina is a delicate multilayered tissue, where large vessels are present in the optic nerve
fiber layer, and retinal capillaries are present between the inner nuclear layer and outer
plexiform layer (Figure 2). Solute transport from the systemic blood flow and choroid to the
retina is restricted by the blood-retina barrier (BRB). The RPE forms the outer part of BRB
and the endothelial membranes of the retinal blood vessels are the inner BRB (Sunkara and
Kompella 2003). The RPE is a monolayer of highly specialized cuboidal cells that are located
between the neural retina and choroid. It carries out essential biochemical functions in
maintaining the visual system, such as the phagocytosis of photoreceptor outer segments,
transport between photoreceptors and the choriocapillaris, and uptake and conversion of the
retinoids that are needed in the visual cycle. The tight junctions of the RPE restrict efficiently
the intercellular permeation of molecules. In monkeys, the permeation of horseradish
peroxidase (mw 44 000) stops at the tight junctions of the RPE (Peyman and Bok 1972, Toris
and Pederson 1984). In addition, binding of drugs to tissue proteins or melanin pigment,
active transport processes and metabolism may affect the permeability of drugs in the RPE.
Proteins that transport ions, glucose, water, amino acids and peptides, and transporter systems
affecting the pH have been characterized in the RPE (Maurice and Mishima 1984; Hughes et
al. 1998; Hamann et al. 2003). Furthermore, P-glycoprotein, a transport protein involved in
the efflux of many hydrophobic compounds, has been detected in both the apical and the
24
basolateral surfaces of human RPE cells (Kennedy and Mangini 2002). The basolateral Pglycoprotein may clear unwanted substances from subretinal space, but apical P-glycoprotein
has suggested having other functions like ATP efflux, lipid translocation, volume specific
chloride current modulation and retinoid and steroid transport (Kennedy and Mangini 2002).
In addition to restricting drug access from blood to the retina, the RPE also affects the
elimination of drugs from vitreous through the retina. RPE cells are polarized and their apical
surfaces facing the photoreceptors are phagocytosing. Furthermore, there are also active
transporters in the apical surfaces of the RPE. For example, fluorescein and penicillin are
actively transported from the retina to the choroid by the RPE (Marmor 1998).
2.2.6 Binding of drugs to melanin
Melanins are biological pigments that are acidic and polyanionic polymeric compounds (Ito
1986; Sarna 1992). In addition to being present in ocular tissues (uvea and RPE), melanin is
found in the inner ear, skin, hair follicles and brain (substantia nigra and locus coeruleus)
(Ings 1984). In the synthesis of melanin, tyrosine is converted via intermediates to melanin,
with the enzyme tyrosinase being essential in this process (Boulton 1998). There are two
basic types of melanins; eumelanin is brown or black and pheomelanin is red or yellow. The
ocular melanin is mainly eumelanin. Melanin is packed in melanosomes that are covered by a
thin membrane. The melanin inside the granule is bound to protein (Boulton 1998). In ocular
tissues, melanin is found in the pigmented melanocytes of uvea and in melanosomes of RPE
cells. Ocular melanin absorbs visible light and protects the retina from overexposure by
preventing light scatter in the eye. It may also protect the tissues against free radicals and may
also inhibit the ocular toxicity of some compounds (Sarna 1992; Leblanc et al.1998).
Various groups of drugs, for example beta-blockers, antibiotics, chloroquine and some
antipsychotic drugs, are known to bind to melanin. Drugs may bind to melanin reversibly or
irreversibly, but it has been suggested that the most basic compounds bind to melanin
reversibly as a result of an electrostatic interaction (Ings 1984). Electrostatic forces play an
important role in binding, but in addition, van der Waals forces or charge transfer may
contribute to the binding (Larsson and Tjälve 1979). The affinity for melanin correlates
positively with the lipophilicity and the alkalinity of the drug (Zane et al.1990). It has been
estimated that about 40 % of clinically used drugs are both basic and lipophilic. On the basis
of literature, Leblanc et al.1998 supported the concept that all basic, lipophilic drugs can
reasonably be expected to bind to melanin to some extent. Binding of harmful substances may
25
protect the pigmented cells and adjacent tissues but, on the other hand, it has been suggested
that high levels of noxious chemicals stored in melanin may cause degeneration of the
melanin containing cells (Larsson 1993).
Beta-blockers timolol and betaxolol are commonly used as anti-glaucoma agents. Several
studies have shown that timolol binds reversibly to ocular melanin and at least two binding
sites have been detected (Araie et al. 1982; Salminen and Urtti 1984; Abrahamsson et al.
1988; Aula et al. 1988). The decreasing effect of timolol on intraocular pressure occurs more
rapidly in blue-eyed than brown-eyed patients (Salminen et al. 1985). On the other hand,
pharmacological effects may be prolonged by pigment binding (Urtti et al 1985). Even though
melanin binding is a commonly encountered phenomenon, its role in the pharmacokinetics of
the uvea and the RPE is poorly known.
2.2.7 Vitreous humour
Vitreous contains water (98 %), collagen (40–120 µg/ ml) and hyaluronic acid (100–400
µg/ml). The rest of the solid material consists of ions and low-molecular weight solutes. A
number of non-collagenous proteins have also been isolated (Berman 1991). Vitreous is a
complicated
three-dimensional
network
of
collagens,
glycosaminoglycans
and
noncollagenous structural proteins and it functions as a molecular sieve that may particularly
restrict the diffusion of large molecules (Bishop 2000). Lipophilic (e.g. dexamethasone) and
actively transported compounds (e.g. penicillin) tend to exit mainly through the retinal
pigment epithelium, whereas hydrophilic substances and compounds with poor retinal
permeability (e.g. FITC-dextran and fluorescein glucuronide with molecular weights 66 000
and 508, respectively), diffuse through the anterior hyaloid membrane into the posterior
chamber (Araie and Maurice 1991; Chastain 2003). In general, vitreal half-lives of
compounds that are eliminated through the retina tend to be shorter than half-lives of
compounds that are eliminated via the anterior route (Chastain 2003). Injection volume and
site can influence the distribution and elimination of a drug, and vitreous liquefaction may
lead to the formation of liquefied spaces where convective transport of a drug may occur
(Friedrich et
al.
2003).
Hyaluronan
is
the
most
common negatively charged
glycosaminoglycan in the human vitreous, but the vitreous contains also chondroitin sulphate
and possibly also heparan sulphate (Bishop 2000). These glycosaminoglycans are known to
interact with polymeric and liposomal DNA complexes and to interfere with gene transfer by
the complexes (Ruponen et al. 1999).
26
2.3 Gene delivery systems for retinal transfection
RPE cells are phagocytosing cells that have been successfully transfected with numerous gene
transfer methods. RPE is the outermost layer of retina and it is located between the choroid
and neural retina. Thus, the transfected RPE cells could serve as platform for secretion of
therapeutical proteins, like neurotrophic or angiostatic factors, that are needed in the therapy
of many diseases of the retina or retinal and choroidal vasculature.
2.3.1 Viral vectors
Viruses have naturally a capacity to infect cells and to copy their genome in the host cells.
This property of viruses has been utilized in viral gene transfer by modifying non-replicative
forms of infecting viruses and by packing therapeutic genes in them. Many viral vectors have
proven to be effective in producing proteins in transfected cells. Also in ophthalmology, gene
therapy research has mainly utilized viral vectors. The main problems with the use of viral
vectors are their possible immunological, infective and oncological properties. The only gene
therapy product on the market is Gendicine®, a recombinant human adenovirus-p53 product
that was aproved for the treatment of head and neck cancer in China in 2003 (Peng 2005).
Adenoviruses - Adenoviruses are non-enveloped, double-stranded DNA viruses that
infect both dividing and non-dividing cells. The virus is not incorporated into the host genome
and, thus, the adenoviral vector gene expression is typically limited to a few weeks.
Replication-defective adenoviruses have been developed for gene transfer of up to 8 kilobases
of foreign DNA (Chaum and Hatton 2002). They have been successfully used for transfection
of several types of retinal cells in animals (Bennett et al. 1994, Jomary et al. 1994, Li 1994).
Adenoviral transduction may cause tissue inflammation and an immune response to the viral
proteins in the host, and it may also evoke potentially toxic effects on the retina (Chaum and
Hatton 2002; Sakamoto et al.1998; Tripathy et al 1996).
Adeno associated viruses (AAV) - Adeno associated viruses (AAV) are non-enveloped,
single-stranded DNA parvoviruses that can infect dividing and non-dividing cells. AAVs
insert into the host genome at a specific locus, thus increasing the likelihood of a stable
transgene expression. AAVs are not associated with any known human infectious disease and
they cause less tissue inflammation than adenoviruses. The cloning capacity of AAV is
limited to 5 kilobases (Chaum and Hatton 2002). Like adenoviruses, AAVs have been shown
27
to transduce several cell types of the retina (Bennett et al. 1999; Grant et al. 1997). Stable
restoration of rod and cone photoreceptor function in dogs affected with a disease caused by
RPE65 deficiency has been achieved with rAAV-mediated gene therapy (Acland GM et al.
2001).
Herpes simplex virus (HSV) - Replication- defective vectors, amplicon vectors (deleted
for all essential HSV genes), and attenuated replication competent herpes simpex viruses can
be used for gene transfer of dividing and non-dividing cells. HSV is a double stranded DNA
virus with the capacity to carry more than 30 kilobases of foreign DNA (Jolly 1994). Several
ocular cell types have been successfully transfected with HSV, including RPE cells (Fraefel et
al. 2005; Liu et al. 1999; Spencer et al, 2000).
HSV thymidine kinase gene can be transfected by using other viruses. Viral thymidine
kinase has a higher affinity than the cellular thymidine kinase for the prodrug ganciclovir, and
thus the delivered ganciclovir is converted to a cytotoxic metabolite. This kind of “suicide
gene therapy” might be useful in gene therapy of retinoblastoma or proliferative
vitreoretinopathy (Hurwitz et al. 1999; Sakamoto et al 1995).
Retroviruses - Retroviruses are RNA viruses that can integrate their DNA into the
genome of the host cell after reverse-transcription and therefore they exhibit long-term and
stable gene expression. They can carry about 8 kilobases of foreign DNA and require cell
division to infect the host cell (Jolly 1994). Lentiviruses are a subclass of retroviruses that can
infect both dividing and non dividing cells. Pseudotyped lentiviruses have been used also for
transfection of retinal cells (Miyoshi et al. 1997).
Baculovirus -Baculovirus is a double-stranded DNA-virus that does not replicate in
vertebrate cells. Its safety and its capacity to carry large fragments of recombinant DNA are
two advantages of baculovirus vector. RPE and other retinal cells have been transfected with
baculovirus vector (Haeseleer et al. 2001).
2.3.2 Non-viral methods
In non-viral gene therapy, the gene that codes the therapeutic protein is subcloned into a
plasmid DNA, which contains also the structural elements for modulating the duration and
expression level of the protein. After local administration, transgene expression has been
achieved with naked plasmid DNA in some tissues in animal models (Wolff et al. 1990;
Hickman et al. 1994; Nomura et al. 1997). However, naked DNA is vulnerable to degradation
28
in the tissues and therefore several kinds of physical and chemical gene carrier systems have
been developed to increase the transfer efficiency of plasmid DNA into target cells or tissues.
Chemical methods - Liposomes are vesicles that are comprised of phospholipid bilayers.
They can be used as gene carriers because they encapsulate DNA (Nicolau and Cudd, 1989).
In general, liposomes with a neutral and negative charge cannot efficiently interact with DNA,
but cationic liposomes and DNA form charged complexes that can transfer DNA into cells
(Felgner et al. 1987). Colipids like dioleylphosphatidylethanolamine (DOPE) or cholesterol
have been used to increase the transfection efficacy of liposomal vectors. Poly-L-lysine (PLL)
and polyethyleneimine (PEI) are examples of polymer based vectors. Modifications, like
pegylation and incorporation of several kinds of ligands, have been used to stabilize these
vectors and to increase their efficacy (Petersen et al. 2002; Wagner et al. 1991).
Cationic lipids and polymers condense DNA by neutralizing its negative charges, and they
form complexes with DNA. The size and morphology of the complexes depend on several
factors like the carrier, solution, concentration, charge ratio and technical factors during the
complex preparation (Hyvönen et al. 2000; Hirota et al. 1999). The positively charged
lipid/DNA complexes bind to the negatively charged cell surface and are most likely
internalized by endocytosis (Figure 3) (Haensler and Szoka 1993; Wrobel and Collins 1995;
Friend et al. 1996). Most of the endocytosed complexes are trapped in endosomes and are
exposed to lysosomal degradation. Plasmid DNA can be protected from lysosomal
degradation by complexation with carriers, and the cationic lipids destabilize membranes
allowing the DNA to escape from the endosome (Wattiaux et al. 1997; Wattiaux et al. 2000).
If it is to be expressed, the plasmid DNA has to diffuse through the cytoplasm and to enter the
nucleus. The dismantling of the nuclear envelope during cell division may make the nuclear
entrance of plasmid DNA easier and this may explain the better efficacy of gene transfer with
cationic complexes in dividing cells compared to non-dividing cells (Fasbender et al. 1997).
Numerous commercial liposome products are available for transfections in the laboratory, but
not for clinical gene therapy. Primary and secondary dividing and also differentiated RPE
cells have been successfully transfected with liposomes in vitro (Urtti et al 2000; Jääskeläinen
et al. 2000; Mannermaa et al 2005). There are also some reports of in vivo transfections of
retinal cells with liposomal vectors (Masuda et al. 1996; Hangai et al. 1996).
Peptide based vectors have been designed to mimic the properties of viruses. Thus, in some
cases they help the plasmid to overcome the barriers it faces on its way to the nucleus
(Wagner et al. 1992).
29
Calcium phosphate precipitation is a chemical transfection method in which DNA is
precipitated with calcium phosphate before its delivery to cells (Chen and Okayama 1987).
Unfortunately, cytotoxicity and low transfection efficacy limit the use of this method.
In general, non-viral methods are considered to be safe and the efficiency of non viral vectors
may be improved by modifications, such as selective targeting moieties, nuclear-localizing
sequences or by inhibiting lysosome digestion.
Figure 3. Scheme of non-viral gene transfer.
Physical methods - In electroporation, short electric pulses create temporary aqueous
pores in the cell membrane. This method has been used to transfect several cell types in vitro
and in vivo and also retinal ganglion cells and RPE cells have been successfully transfected
by electroporation (Mir et al 1999; Zhang et al. 2002; Mo et al. 2002; Chalberg et al 2005). It
has been claimed that electrotransfection of the ciliary muscle could be used for the cure of
diseases of both anterior and posterior parts of the eye (Bloquel et al. 2006). Ultrasound has
been found to increase the transfection efficiency of ultrasound reflective liposomes (Huang
et al. 2001). In magnetofection, targeted gene delivery of vectors with superparamagnetic
nanoparticles is achieved via the application of a magnetic field (Scherer et al. 2002).
30
2.3.3 Ex vivo gene therapy
Implantation of ex-vivo modified cells overcomes many problems of in vivo gene transfer.
Selection and cloning of the targeted cells is easier and in vivo targeting of gene transfer and
extracellular barriers of efficient gene transfer can be avoided. The assessment of safety
concerns and efficacy is easier with modified cells than with in vivo methods (Chaum and
Hatton 2002). Gene transfection of the donor organ during organ preservation is an attractive
method for prevention of rejection of transplanted allographs (Isobe et al. 2004). Encapsulated
genetically engineered cells that produce growth factor have been shown to protect
photoreceptors after these cells were implanted in the intravitreal space of dogs with retinal
degeneration (Tao et al. 2002).
2.3.4 Oligonucleotides and ribozymes
Oligonucleotides are single-stranded DNA or RNA chains, usually consisting of 7-25
nucleotides. The antisense oligonucleotides bind to mRNA and inhibit protein synthesis.
Oligonucleotides may also bind to DNA to form a triplex and thus prevent transcription
(antigene oligonucleotides) or to proteins thereby inhibiting their function (aptamers).
Oligonucleotides consist of natural phosphodiester compounds, but the poor stability of these
oligonucleotides against nucleases has limited their therapeutic use (Stein 1996).
In phosphorothioate oligonucleotides a single oxygen at a non-bridging position of the
phosphate bridge is replaced by sulphur. They are highly resistant to nuclease activity, form
reasonably stable duplexes with specific target mRNAs and have high water-solubility. They
can catalyze mRNA cleavage by eliciting the activity of RNAse H, a ubiquitous enzyme that
cleaves the mRNA strand of the RNA-DNA duplex. The phosphorothioate oligonucleotides
bind more avidly to proteins than the phosphodiester oligonucleotides (Stein 1996).
Ribozymes are catalytic RNA molecules that can cleave specific message RNA
sequences. Mutation-specific cleavage of the transcript prevents the synthesis of an abnormal
protein. In mutation-independent ribozyme therapy, both the mutant and wild type transcripts
are cleaved, but a modified transcript coding for the normal protein is introduced and this
modified transcript is not cleaved by the ribozyme (O`Neill et al 2000).
Small interfering RNAs (siRNA) are new very promising tools which can silence genes.
The long double stranded RNAs are cleaved in the cells into 21-22 nucleotide siRNAs. In
conjunction with multiple enzyme complexes (RISC), siRNAs locate to a specific site on the
31
mRNA and degrade it. Silencing of gene promotors by siRNAs can also reduce protein
production (Wadhwa et al 2004). This is a part of an endogenous gene silencing system.
The
first
oligonucleotide-based
drug
marketed was fomivirsen,
an antisense
oligonucleotide which specifically inhibits replication of human cytomegalovirus by binding
to complementary sequences on the mRNA transcribed from the major immediate-early
transcriptional unit of the virus (Vitravene®, Perry and Balfour 1999). Pegaptanib is a
pegylated modified oligonucleotide, which adopts a three-dimensional conformation that
enables it to bind to extracellular vascular endothelial growth factor (VEGF). It is used in the
treatment of neovascular age-related macular degeneration (Macugen ®, Gragoudas et al.
2004). Both of these oligonucleotide drugs are administered as intravitreal injections.
2.4 Retinal gene therapy
Gene therapy is a promising way to treat many currently untreatable retinal diseases. Retinitis
pigmentosa, genetic and acquired retinal dystrophies, age-related macular degeneration,
proliferative vitreoretinopathy, retinoblastoma and neovascular diseases, including diabetic
retinopathy, have all been considered as targets for gene therapy research (Chaum and Hatton
2002). Retinitis pigmentosa (RP) is a genetically and clinically heterogenous group of retinal
degenerations. RP occurs in autosomal dominant, autosomal recessive and X-linked recessive
forms (Phelan and Bok 2000). At least 45 known genes/loci have been identified in nonsyndromic RP, including 15 for autosomal dominant RP, 24 for autosomal recessive RP , five
for X-linked- inheritance, and one, which has been found mutated only in the rare digenic
form of RP. It has been estimated that the cloned genes account for about 50 % of dominant
RP, 40 % of recessive RP and approximately 80 % of X-linked RP (Hamel 2006). The
existence of several animal models for RP has contributed substantially to the research of
gene therapy in this group of diseases.
When the degeneration is known to be a consequence of the production of an abnormal
form of protein, as in autosomal dominant forms of RP, it could be possible to inhibit the
translation of the mutant protein from its transcript by ribozymes or by the use of antisense
oligonucleotides. Also growth factor and antiapoptotic therapies have been shown to slow the
disease process in animal models (Lewin et al 1998, Chaum and Hatton 2002). Recessive
degenerations are characterized by an inability to produce a normal gene product. The aim of
gene therapy in these degenerations is to transfect the retina with a wild-type gene copy that
produces the lacking functional protein, or use genes that produce growth factors or
32
antiapoptotic factors to improve photoreceptor survival (Chaum and Hatton 2002). Retinal
degenerations in animal models of Sly syndrome (mucopolysaccharidosis type VII) and
Leber´s congenital amarosis have been successfully treated by delivering a functioning copy
of the deficiently functioning gene (Li and Davidson 1995; Acland et al. 2001).
Retinoblastoma is a primary intraocular malignancy of childhood. Mutations and/or allelic
loss of the retinoblastoma gene are essential for the tumorigenesis of a retinoblastoma. The
inactivation of the retinoblastoma gene can be inherited through the germ line (hereditary
form) or somatically acquired (nonhereditary form) (Huang et al 2003). Cytotoxic HSV
ribonuclease reductase mutants and ganciclovir after HSV thymidine kinase transfection have
demonstrated efficacy against the tumor cell lineY79 in animal models (Kogishi et al 1999;
Hurwitz et al. 1999). Unfortunately, attempts to induce a wild-type retinoblastoma gene into
cell lines have been less successful (Xu et al.1991; Muncaster et al. 1992).
Proliferative vitreoretinopathy (PVR) is characterized by the formation of vitreal,
epiretinal or subretinal membranes after retinal reattachment surgery or ocular trauma. RPE
cells that undergo metaplastic changes, glial cells and several other cell types have been
identified in these tissues (Charteris et al. 2007). In some cases, the membranes cause traction
and distortion of the retina, and PVR is the most common cause of failed retinal detachment
surgery (Andrews et al. 1999). Destruction of the proliferating cells of PVR achieved by
suicide gene therapy by transferring HSV-tk into the cells has been demonstrated in vitro and
in vivo (Kimura et al. 1996a). Multiple growth factors and signalling enzymes may be
involved in PVR, though the platelet-derived growth-factor is considered as being one of the
essential contributors. Gene therapy with a retrovirus used to express a dominant negative
alpha platelet-derived growth-factor receptor has attenuated PVR in a rabbit model of the
disease (Ikuno and Kazlauskas 2002). Modulation of growth factors, receptors or structural
gene expression by antisense gene therapy may have a role in the gene therapy of PVR
(Capeans et al. 1998; Roy et al.1999).
In age related macular degeneration (AMD), the degenerative changes of the RPE and
Bruch`s membrane result in degeneration of the neural retina. Although ageing and
environmental and genetic factors are known to be related to the degeneration, the
pathophysiology of AMD is not completely known. Recently, gene polymorphisms in
complement system regulator proteins (complement factor H, complement component 2complement factor B) and in some other genes (LOC387715, promoter region of HtrA serine
peptidase 1) with less defined functions have been identified as major risk factors for AMD
(Despriet et al. 2006; Dewan et al.2007; Scholl et al 2007).
33
Several growth factors stimulate DNA synthesis and RPE proliferation in vitro and have
demonstrated neuroprotective effects in animal models of retinal degeneration and
detachment (Chaum and Hutton 2002). Basic fibroblast growth factor has been shown to
stimulate phagocytic activity in the Royal College of Surgeons-rat model (McLaren and Inana
1997). Thus, modulation of RPE cell growth factor gene expression could be a possibility to
gene therapy of AMD. It has been claimed that antiapoptotic gene therapy might slow the
degeneration progress of AMD as has been achieved in RP models (Chaum and Hatton 2002).
Furthermore, gene therapies for producing anti-angiogenetic factors, like endostatin, may
provide a new therapeutic approach to treat ocular neovascularization of AMD. A recent
phase I clinical study with adenoviral vector-delivered natural anti-angiogenetic factor,
pigment epithelial growth factor, provided promising results in patients with advanced
neovascular AMD (Campochiaro et al 2006).
34
3 AIMS OF THE STUDY
The general purpose of this study was to evaluate the retinal pigment epithelium as a barrier
to drug permeation and as a target of non-viral gene delivery.
The specific aims were:
1.
To evaluate the roles of vitreous and neural retina in the delivery of genes and other
macromolecules into retinal pigment epithelium
2.
To determine the barrier properties of RPE, particularly the effects of permeant size and
lipophilicity on the permeability in RPE
3.
To characterize the melanin binding of beta-blockers and oligonucleotides. To compare
the size, shape, specific surface area and binding of betaxolol to synthetic and isolated
bovine melanin. To predict the binding of lipophilic beta-blockers to melanin in human
choroid-RPE.
35
4 MATERIALS AND METHODS
4.1 Labeled macromolecules
FITC-dextrans - The FITC-dextrans with mean molecular weights of 4400 and 20 000 were
purchased from Sigma and the FITC-dextrans with mean molecular weights 70 000, 500 000
and 2 000 000 were from Molecular Probes. FITC-dextrans were dissolved in DMEM at 1.5
mg/ml (I) and in 5 % glucose at 3.0 mg/ml (II). The molecular sizes of the FITC-dextrans
4400, 70 000, 500 000 and 2 000 000 were measured with a NICOMP 380 submicron particle
sizer at 3 mg/ml, and the mean diameter of the molecules was assessed on the basis of
NICOMP number-weighted analysis (I). The mean diameter of the largest FITC-dextran (mw
2 000 000) was 30 nm and that of other FITC-dextrans was below 10 nm. FITC-dextrans
(Sigma-Aldrich) of molecular weights 4400, 9300, 21200, 38200, 77000 at concentrations of
4 mg/ml, 6 mg/ml, 6 mg/ml, 8 mg/ml and 8 mg/ml, respectively, in BSS PLUS ™ with
HEPES 10 mM, pH 7.4, were used as donor solutions in the RPE permeation studies (III).
FITC-labeled poly-L-lysines - FITC-labeled poly-L-lysine (PLL) of mean molecular
weight 20 000 and unlabeled poly-L-lysines of mean molecular weights of 20 000 and 200
000 were from Sigma. PLL of mw 200 000 was labeled with FITC (I). PLL and FITC-label
were separately diluted in 0.05 M sodium bicarbonate. The solutions were combined and
stirred, and free label was separated by gel filtration. Labeled PLL was precipitated with
ethanol, centrifuged, and dissolved in sterile water. Dialysis was performed overnight,
precipitation was repeated as described above, and the yield was determined. For vitreal
permeation experiments, the FITC-labeled poly-L-lysines were used at a concentration of
6.25 µg/ml, and 744 µg/ml of unlabeled PLL was added. PLL-solutions were diluted in
DMEM before testing (I). For neural retinal permeation studies, FITC-PLL of molecular
weight 20 000 was used at a concentration 0.1 mg/ml and 0.65 mg/ml of unlabeled PLL was
added. In these experiments, the PLLs were diluted in 5 % glucose (II).
4.2 Carriers
Polyethyleneimine (PEI) with a mean molecular weight of 25 000 was from Aldrich (St.
Louis, MO, USA) and was used as 10 mM aqueous stock solution (I, II). Poly-L-lysine (PLL)
with a mean molecular weight of 200 000 was from Sigma and it was diluted with water to 3
mg/ml (I, II). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from
Avanti Polar Lipids (Pelham, AL, USA). Cationic liposomes composed of DOTAP were
36
prepared by evaporating a chloroform solution of lipids, resuspending the lipid in water at a
concentration of 3.2 mM and sonication under argon (I-II).
4.3 Plasmids
The green fluorescent protein GFP S65T mutant was excised from pTR5-DC/GFP plasmid (a
gift from Dr. Mosser, Montreal, Canada; Mosser et al. 1997) as a BamHI fragment. It was
inserted into the BamHI-site of a cytomegalovirus-driven pCR3-plasmid to yield the plasmid
(pGFP) that was used in the complexes (I, II). The pGFP was labeled with ethidium
monoazide which forms covalent bonds with DNA bases during photoactivation. The
procedure of Zabner et al. (1995) was used with minor modifications. EMA in water was
added to the GFP expressing plasmid in water. After incubation, the solution was exposed to
UV-light at a wavelength of 312 nm. Gel filtration was used to purify labeled DNA from free
EMA. To remove intercalated but not covalently bound EMA, cesium chloride was added,
and the plasmid was extracted with CsCl-saturated isopropanol. CsCl was removed by
dialysis and the labeled EMA-DNA plasmid was recovered by ethanol precipitation (I-II).
The reporter gene plasmid that encodes beta-galactosidase under the control of
cytomegalovirus promoter (pCMV ) was a gift from Dr F.C. Szoka Jr. (University of
California San Francisco, CA, USA). Rhodamine-labeled beta-galactosidase encoding
plasmid (p-Gene-Grip™ , Rhodamine/ß-galactosidase Vector, San Diego, CA) was used for
the complexation in histological analysis (II).
4.4 Oligonucleotides
FITC-oligonucleotide for the neural retinal permeation study (phosphorothioate, 5´-FITCTGG CGT CTT CCA TTT 3´) was purchased from A.I.Virtanen Institute (Kuopio, Finland)
and diluted into 5 % glucose at a concentration of 50 µg/ml for delivering to RPE or retina
(II).
For melanin binding studies, FITC-labelled 21-mer and 10-mer phosphodiesterase
oligonucleotides (5`-Fluorescein-GCC TCG GCT TGT CAC ATC TGC-3` and 5´-fluorescein
-TCA CAT CTG C-3`; Oligomer, Finland) were mixed in PBS. Polystyrene tubes were used
in every phase to avoid adsorption of oligonucleotides to the tube walls.
37
4.5 Beta-blockers and carboxyfluorescein
For permeation studies, 6-carboxyfluorescein (Sigma, St. Louis, MO, USA) was diluted with
glucose glutathione bicarbonate solution (BSS PLUS ™; Alcon, For Worth, Tx, USA) with
HEPES 10 mM, pH 7.4, at a concentration of 0.0376 mg/ml (III). Atenolol, nadolol, pindolol,
metoprolol, betaxolol (Sigma) and timolol (donated by Merck, Sharp & Dohme Research
Laboratory, Rahway, NJ, USA) were diluted with BSS PLUS ™ with HEPES 10 mM, pH 7.4
(III). In the melanin binding experiments, 6-CF, metoprolol and betaxolol were dissolved in
HEPES- BSS® (IV).
4.6 Cell culture and transfection
The D407 cell line (human retinal pigment epithelial cells) was a gift from the laboratory of
Dr Richard Hunt (University of South Carolina, Medical School, Columbia, SC, USA), and
the cells were cultured in DMEM medium supplemented with 1 % penicillin-streptomycin, 5
% fetal bovine serum and 2 mM L-glutamine at + 37° C in 7 % CO2 (I).
One day before cellular uptake or transfection experiments, the cells were divided into
wells (I). Plasmid DNA and the cationic polymers or cationic liposomes were both diluted
first to 5 %-glucose solution. Solutions of DNA and carrier were mixed before transfection at
charge ratios of 2:1 or 4:1 (positive charges of carrier over negative charges of DNA). DNAcarrier complexes were prepared at room temperature. Before transfection, the solutions were
allowed to remain standing for at least 20 minutes (I-II).
At the beginning of the experiments, the DMEM culture medium was aspirated, the cells
were washed with PBS, and 1 ml (about one mm thick layer) of vitreous, hyaluronan solution
(0.3 mg/ml or 1.0 mg/ml) or DMEM was added onto the cells (I). Then, DNA-complexes,
FITC-dextran or FITC-PLL were carefully pipetted onto the surface of the wells. Three kinds
of treatments were used at + 37° C: incubation of 5 or 48 hours without stirring and 5 hours
with stirring. For cellular uptake measurements, the cells were washed twice with PBS,
detached from the bottom of the well with trypsin-EDTA (Gibco) and fixed with 1 %
paraformaldehyde. For GFP-transfections, the cells were washed twice with PBS after
incubation and the culture medium was added for 24 hours. Thereafter, the cells were treated
as described above.
The sizes of the complexes were assessed with a NICOMP 380 submicron particle sizer,
which determines size based on the extent of light scattering (NICOMP Particle Sizing
38
Systems Inc., Santa Barbara, CA, USA). For the measurements, PEI, PLL and DOTAP were
complexed with plasmid DNA (pGFP). The complexes were prepared in 5% glucose and the
concentrations of DNA were 20 µg/ml (I) and 50 µg/ ml (II). Size distributions were
determined on the basis of NICOMP volume-weighted analysis (I) or NICOMP numberweighted (II) analysis.
4.7 Tissue preparation and permeation experiments
The bovine eyes were obtained from a local abattoir. Fresh bovine eyes were kept at + 9°C.
They were cleaned of extraocular material and dipped in 0.9 % NaCl (III) or 1 % penicillinstreptomycin (Gibco BRL, Grand Island NY, USA) in 0.9 % NaCl (II). The eyes were opened
circumferentially about 8 mm behind the limbus and the anterior tissues and the vitreous were
separated gently from the neural retina.
Vitreal permeation studies –In the vitreal permeation studies, vitreous was pushed with
a syringe through a nylon mesh to make it easier for handling and administration into the cell
culture plates (I). The viscosity of vitreous was measured with a rotation viscometer
(Brookfield, Middleboro, MA, USA). The viscosity of vitreous was 7-24 mPa depending on
the shear rate (6-100 rpm) at + 21° C. The viscosities of 5 % glucose, DMEM, and hyaluronan
0.3 mg/ml and 1.0 mg/ml were measured with capillary viscometer, and the viscosities were
1.1418 mPa, 1.0380 mPa, 1.211 mPa and 1.7646 mPa, respectively (I). For determination of
nucleases, a plasmid that encodes beta-galactosidase under the control of cytomegalovirus
promoter was mixed with DMEM, hyaluronan (1 mg/ml in DMEM) or vitreous (ad 200
l/sample). The solutions were incubated at + 37° C. The reactions were stopped with 50 l of
5x bromophenol blue (30 % glycerol, 0.25 % SDS, 50 mM Tris-HCl, 50 mM NaCl, 20 mM
EDTA, 0.1 % bromophenol blue) at time points 0 h; 2.5 h; 5 h; 24 h and 48 h, and stored at –
20 C until gel electrophoresis. Then, 18 µl samples were pipetted in the wells of gel (1 %
agarose in 1 % TAE), and the gel electrophoresis was performed in 1 % TAE buffer with
ethidium bromide by EPS 600 (Pharmacia Biotech) (75 V, 1 hour). For the detection of the
effect of nucleases on complexed DNA, a beta-galactosidase coding plasmid was complexed
with PEI, PLL and DOTAP at charge ratios +/- 2 and +/- 4 as described above. Then, 125 µ l
of the complex solution was mixed with 75 µl of DMEM, 1 mg/ml hyaluronan or vitreous.
Samples were taken at time points 0 min, 24 h and 48 h, and gel electrophoresis was
performed as described earlier.
39
Neural retinal permeation studies - To study the permeability of the neural retina (II), it
was either left in its place or gently peeled and collected at the optic disc and cut with scissors
near to the optic nerve head. To avoid blood cell contamination from cut vessels, the optic
nerve head was gently wiped with a piece of paper tissue. The eyecups were set in the 6-well
plates. DNA-carrier complexes, FITC-dextrans, FITC-oligonucleotides, EMA-labeled
plasmid or 5 % glucose were pipetted onto the RPE or neural retina in the eye cup. Incubation
of two hours at + 37°C was started within less than 3 hours after the death of the animal.
Then, the sample solution was removed and the exposed surface of the RPE or retina was
gently rinsed with PBS. If the retina was still present, it was gently removed. The optic nerve
head was wiped with a piece of paper tissue, and the underlying RPE was rinsed with 1 ml of
PBS. The eye cup was kept steady in the well when the neural retina was separated and the
eyecup was rinsed.
A previously described method by Feeney-Burns and Berman (1982) with some
modifications was used for the isolation of the RPE cells. Trypsin-EDTA was pipetted onto
the surface of the RPE and incubated for 1-2 minutes. After this incubation, the cells were
gently loosened from Bruch´s membrane with a small brush. Only the cells that were under
the surface of trypsin-EDTA solution were brushed. The loosened cells were collected and
fixed with 1 % paraformaldehyde and centrifuged. To remove any contaminants, the cells
were suspended in buffered sucrose and centrifuged. The supernatant was removed and the
centrifugation in buffered sucrose was repeated twice. In microscopic evaluation after the
three sucrose centrifugations, some samples contained only RPE cells, but occasionally some
outer segment fragments, erythrocytes and pigment granules were present. After the last
sucrose centrifugation, the pellet was washed with 1 % paraformaldehyde, centrifuged and
diluted with 1 % paraformaldehyde for flow cytometric analysis.
An additional experiment to study the permeation of oligonucleotides was done by
applying FITC-oligonucleotide to the retina and RPE as described above. Samples were taken
from the solution after 15 and 30 minutes or 60 and 120 minutes after the start of the
incubation. The sample was centrifuged and the supernatant was stored at – 20°C for HPLC
analysis.
RPE-permeation studies - For RPE-permeation studies in the diffusion chamber, a
circular piece with a diameter of 20 mm was punched from the pigmented part of the eyecup
(III). The sclera was detached from the RPE with forceps and the retina-choroid block was
moved to BSS PLUS ™-solution. The neural retina was separated gently and the RPEchoroid-block was transferred carefully onto a piece of nylon mesh (holes 1x1mm). The
40
choroid part was set to face the nylon mesh. The tissue block with the nylon mesh was moved
to a vertical diffusion chamber (Costar, Cambridge, MA). The chamber was equipped with
silicone adapters with a circular aperture of 9.5 mm (exposed area of 0.709 cm2) and vacuum
grease was used to seal the margins of the tissue to the adapters. The experiments with
fluorescent probes were preceded by equilibration for five minutes with pre-warmed diffusion
medium on both sides of the tissue (BSS PLUS ™). To start the experiment, one milliliter of
the diffusion medium was removed from either the choroidal or retinal side of the tissue and
replaced with an equal volume of the fluorescent probe-containing medium. The initial
concentration of 6-carboxyfluorescein (Sigma, St. Louis, MO) in the donor chamber was
0.0376 mg/ml (100 µM). The initial donor concentrations of FITC-dextrans with mean
molecular weights of 4.4, 9.3, 21.2, 38.2, and 77.0 kDa (Sigma) were 4, 6, 6, 8, and 8 mg/ml
(909, 645, 283, 209, and 104 µM), respectively. In beta-blocker studies, the experiment was
started by pipetting 5 ml of diffusion medium into the receptor side and an equal volume of
test solution to the donor side. N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)
(HEPES) (Sigma) was dissolved in BSS Plus at a concentration of 10 mM and the pH of the
solution was adjusted to pH 7.4. The test solution was a mixture of atenolol, nadolol, pindolol,
metoprolol, betaxolol and timolol in BSS Plus with 10 mM HEPES (pH adjusted to 7.4). The
concentration of metoprolol and betaxolol in the test solution was 100 µM and the
concentration of the other beta-blockers was 400 µM. The whole diffusion apparatus was
covered with aluminium foil to protect the solutions from light. The temperature of the
chamber was maintained at 37° C by using a heating block (Costar) and a circulating water
bath (M3, Lauda, Köningshofen, Germany). Gentle bubbling with a stream of low oxygen gas
(5 % CO2, 10 % O2, 85 % N2) provided oxygen to the tissue and mixed the donor and receptor
solutions during the experiment, and the pH was maintained at 7.4-7.5 by CO2.
Samples were taken from the receptor solution at 15, 30, 45, 60, 75, 90, 105, 120, 150,
180, 210 and 240 minutes and replaced with pure diffusion medium. A sample was taken
from the donor solution at 10 minutes in the case of fluorescent probes, and at 240 minutes
from all donor solutions. The FITC-dextrans were analyzed immediately, but the 6-CF and
beta-blocker samples were stored at - 20° C before analysis.
The permeability of betaxolol through pigmented and non-pigmented bovine choroid-RPE
was studied in a diffusion chamber as described above. In this experiment, betaxolol was
alone in the donor solution. The non-pigmented and pigmented tissue samples were taken
from the same eye and the experiments were performed in triplicate.
41
The apparent permeability coefficient (P app) characterizes the diffusion rate of solute transfer
across RPE.
Papp = flux / (SRPEC0)
Flux is the slope of the linear portion of the permeability curve for each of the probe
molecules, SRPE is the exposed surface area of the choroid-RPE tissue (0.709 cm2) and C0 is
the initial concentration of the probe molecule in the donor solution.
The permeation lag time was determined by extrapolating the linear portion of the
permeability curve and determining its intercept on the time axis.
4.8. Melanin binding experiments
Isolation of melanin. The bovine eyes were dissected as described before and the neural
retina was gently removed. The RPE-choroid was separated from the sclera and frozen in
potassium phosphate buffer, pH 8.
The melanin granules were isolated by protease digestion (a method by Sauer and
Anderson, 1994) and sucrose gradient centrifugation (Seiji et al.1961; Boulton and Marshall,
1985). Subtlisin Carlsberg protease type VIII (Sigma Aldrich, St. Louis, MO, USA) solution
in PB8 was added into the collected RPE-choroid tissue. The tissue in the protease solution
was incubated at 56° C for one hour with stirring and heated up to 99° C for 15 minutes. After
centrifuging, the supernatant was removed, the pellet was washed with PB8, and centrifuged
again. Melanin was isolated from the sediment by centrifuging melanin containing sucrose
gradients of eight concentrations from 1 M to 2 M. The melanin sediment was washed four
times with phosphate buffer and frozen at -70 for one hour before being lyophilized for 18
hours (ModulyoD230, Thermo Savant, Holbrook, NY, USA). Isolated melanin was stored at 18° C.
Binding experiments. For assessing the binding of beta-blockers and carboxyfluorescein
to melanin, isolated bovine melanin or synthetic (Sigma) melanin was mixed at concentration
of 2 mg/ml to 50 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES;
Sigma, St. Louis, USA) BSS® (Alcon Laboratories Inc., Fort Worth, Texas, USA), pH was
adjusted to 7.4 and the solution was sonicated for 15 minutes (Bandelin Sonorex, Super RK
102 H, Bandelin electronic, Germany). For the experiments with oligonucleotides, PBS
(Gibco BRL, Grand Island NY, USA) was used as the solvent. An amount of 750 l of
42
melanin solution and the same amount of test solution were mixed and incubated with stirring
at 37° C (Swip KS-10 Edmund Bühler, Germany). Polystyrene tubes were used for
oligonucleotides to minimize the attachment of the molecules to the tube walls.
Polypropylene tubes were used for other compounds.
Concentrations of 100 nM and 1 µM at timepoints 1, 3, 6 and 24 hours were used to
determine the kinetics of binding of betaxolol to isolated and synthetic melanin. The
maximum binding capacity and dissociation constant were determined for betaxolol and
metoprolol (Sigma-Aldrich) in the concentration range 0.05 -100 µM after 4 hours incubation.
The binding of FITC-labelled 21-mer and 10-mer phosphodiesterase oligonucleotides (5`Fluorescein-GCC TCG GCT TGT CAC ATC TGC-3` and 5´-Fluorescein -TCA CAT CTG
C-3`; Oligomer, Finland) to isolated bovine melanin was examined over a range of
concentrations from 3 to 28 nM with a 2 hours` incubation. Binding of 20 nM 6carboxyfluorescein (Sigma-Aldrich) was determined at incubation times of 0.5, 1, 5 and 24
hours. Controls were prepared by incubating each test compound in pure incubation medium
at all the concentrations used in the binding studies, and by incubating melanin in incubation
medium without any test compounds. The experiments consisted of 3-4 replicate samples and
controls.
After incubation, the suspensions from the beta-blocker and 6-CF experiments were
centrifuged (13 000 rpm, 15 minutes; Centra-M2, International Equipment Company, USA) at
37° C.
The suspensions from the oligonucleotide experiments were centrifuged at 5000 rpm for
15 minutes (FP-510 Centrifuge, Labsystems Oy, Finland). A sample of 400-700 µl of the
supernatant was taken for analysis.
Calculation of the binding parameters. The calculation of binding parameters was
based on Langmuir binding isotherm:
Bmax[L]
B = ----------Kd +[L]
where B is the observed binding of the ligand to melanin (nmol/mg), when the measured free
concentration of the ligand is equal to bound ligand [L]( M), Bmax is the maximum binding
of the ligand to melanin (nmol/mg) and Kd is the equilibrium dissociation constant for the
binding ( M). Scatchard plots were used for determining the number of classes of binding
sites by visual inspection. Thereafter, nonlinear fitting based on the Langmuir binding
isotherm with two binding sites was performed using Prism software (version 4; GraphPad
43
Software, San Diego, CA, USA) with 1/Y weighting to determine separate Bmax and Kd values
for high affinity and low affinity binding sites.
4.9 High performance liquid chromatography
II: The HPLC system was a Beckham System Gold High Performance Liquid
Cromatography equipped with a System Gold 168 Detector, 126 Solvent Mixing Module,
507e Autosampler, a Rheodyne Model 7725 injector (Cotati, CA, USA) fitted with 100 µl
sample loop. The software was 32 Karat™ from Beckman.
The analytical HPLC column was DNA Pac ™ PA-100 column 4 x 250 (Dionex). Buffer
A was 20% acetonitrile in 0.1 M acetate buffer (pH 8.0) and buffer B was 20 % acetonitrile
and 0.4 M NaClO4 in 0.1 M acetate buffer (pH 8.0). The gradient consisted of buffer A with
increasing amounts of buffer B according to the following scheme: increase from 10 % to 35
% over 5 min and increase to 55 % over 25 min. The flow rate was 1 ml / min. Column
equilibration took 15 min. Oligonucleotides eluting from the column were detected by
ultraviolet absorption at a wavelength of 260 nm. Suitably diluted standards and samples were
loaded onto the system through an autosampler.
Gradient HPLC with combined ultraviolet and fluorescence detection was used to assay
the beta-blockers (III, IV).
III: The HPLC system consisted of a Beckman System Gold Programmable Solvent
Module 168 with a diode array UV detector and a model 507e autosampler (Beckman
Instruments, Fullerton, CA) with a 50 µl sample loop. A Meta Therm column temperature
controller (MetaChem Technologies, Torrance, CA) was used with the HPLC. A HewlettPackard 1046A fluorescence detector (Waldbronn, Germany) was used together with UV
detection, and signals from the two detectors were collected by the 32 Karat software package
version 3.0. The chromatographic conditions and calibration procedures have been described
earlier (Ranta et al. 2002).
4.10 Flow cytometry analysis
Cellular uptake and transgene expression were measured with a fluorescence activated cell
sorter (FACS-scan flow cytometry, Becton Dickinson, San Jose, CA, USA) with an argon ion
laser (488 nm) as the excitation source (I-II). Fluorescence of GFP was collected at 525 nm
(FL 1) and fluorescence of EMA was collected at 670 nm (FL3). The cells were visualized on
44
a FSC (Forward Angle Light Scatter) versus SSC (90 degrees light scatter) display. For each
sample, a maximum of 10 000 events were collected. The RPE cells which were alive before
fixation were selected for analysis by gating. The dead RPE cells and possible erythrocytes,
photoreceptor outer segments and pigment granules were excluded on the basis of their size
and other scattering properties. The result was discarded, if less than 1000 living RPE cells
could be analysed from a sample.
EMA-DNA was used as a marker for intracellular delivery of DNA. The gate of positive
events for each carrier was adjusted according to the negative control. The controls were
unlabeled beta-galactosidase DNA/carrier complexes for EMA-DNA complexes (I-II) and
unlabeled pGFP for naked plasmid DNA (II).
GFP positive cells were separated from the autofluorescence by setting a gate (I). For
FITC positive cells after application of FITC-dextran, FITC- oligonucleotide and FITC-PLL
(II), 5 % glucose was used as a control. The percentage of the positive cells was calculated as
the number of positive events divided by the total number of events in the gate of living cells.
4.11 Fluorometry, BET and laser diffractometry
Carboxyfluorescein and FITC-dextrans were determined using a 96-well fluorescence plate
reader (FL 500, Bio-Tek Instruments, Burlington, VT) with 485 nm excitation and 530 nm
emission filters (III,IV). The fluorescence of oligonucleotide samples was measured with
Victor2 1420-012, Software version 2.0, Perkin Elmer - Wallac, Turku, Finland) (IV).
Standard curves of fluorescence versus concentration were obtained by serial dilution of
fluorescent compounds in diffusion medium. Concentrations were determined by linear
regression analysis within the linear portion of the standard curve.
The specific surface area (SSA) of melanin samples was determined by the single-point
BET method (Micromeritics Flowsorb II 2300, Norgross, GA). Samples of synthetic and
isolated melanin were first dried under vacuum at +37° C for 20 hours. The measuring gas
was a nitrogen/helium (70 %/30 %) gas mixture. A mean of five measurements was used for
calculation of the SSA (IV).
The particle size of synthetic and isolated melanin was examined at a concentration of 2
mg/ml in water with a laser diffractometer (Malvern Instruments Ltd, Malvern, UK). The
measurements were performed after 15 minutes sonication, after 4 hours of stirring at 37° C
and after being kept for 48 hours at room temperature (IV).
45
4.12 Microscopic analysis
To determine the location of fluorescent permeants in bovine retina, solutions of PEI, PLL
and DOTAP- DNA complexes, FITC-labeled oligonucleotide, FITC-dextran of molecular
weight of 20 000, FITC-PLL of molecular weight of 20 000 and 5 % glucose were pipetted
onto neural retinas of eye cups (II). After the incubation, the eye cups were rinsed with PBS,
fixed with 4 % paraformaldehyde for 30 minutes at room temperature and rinsed again. Pieces
of 8 mm in diameter, containing all posterior ocular layers, were cut from the area that was
exposed to the incubating solution. The samples were frozen in the OCT-compound (TissueTek®) in isopentane that was kept cold with pieces of dry ice in ethanol. Cryostat cuts of 1014 µm were embedded in glycerol and they were evaluated with a fluorescence microscope
(Nikon Eclipse/UltraVIEW Confocal Imaging System Perkin Elmer Life Sciences,
Cambridge, United Kingdom, connected to Nikon Eclipse TE 300 inverted microscope,
Nikon Corporation, Tokyo, Japan).
The role of diffusion in the vitreal barrier was clarified by confocal microscopy (I).
Divided cover glasses were covered by FITC-poly-L-lysine (I). A layer of vitreous about 1
mm in thickness was pipetted on the wells and DOTAP, PLL and PEI complexed with
rhodamine labeled DNA (Rhodamine/beta-Gal, pGeneGrip, Gene Therapy Systems, Inc., San
Diego, CA, USA) at charge ratios of +/- 2 and +/- 4 were added on the vitreous. After 2 hours
of incubation at + 37 C, the sample was examined with confocal microscopy on a
UltraVIEW confocal imaging system (Perkin Elmer Life Sciences, Boston, MA, USA) with
an Eclipse TE 300 microscope (Nikon, Melville, N.Y., USA) using a 100 x oil immersion
objective. The bottom of the glass was detected by the FITC-poly-L-lysine dots that were
imaged by using the 488 nm excitation line of krypton/argon laser, and green fluorescence
was detected at 515-545 nm. Rhodamine-labeled DNA was detected at 590-610 nm after
excitation at 568 nm. Confocal images were collected with a cooled digital charge-coupled
device camera (Perkin Elmer Life Sciences). Ten serial images with fluorescein and
rhodamine fluorescence at about 25 µm Z intervals were recorded and then co-localized.
Images were processed and analyzed by using the confocal assistant software program
(UltraVIEW). The rhodamine fluorescent dots were counted from the two lowest and two
topmost images of each sample to obtain a semi-quantitative view of the diffusion of the
complexes. For controls, the same amount of each complex solution was mechanically mixed
evenly to vitreous and, thereafter, handled and detected as described above.
46
For investigating the shape of the melanin granules, they were coated with gold (Polarin
Sputter Coater 11-E5100, Polaron Equipment Ltd, Watford, UK) and examined with scanning
electron microscopy (XL 30 ESEM TMP, FEI Company, Brno, Czech Republic) (IV).
4.13 Statistical analysis
The Mann-Whitney´s U-test was used for statistical analysis of permeation studies of vitreous
and neural retina (I-II). The results of experiments with FITC-dextrans of molecular weights
500 000 and 2 000 0000 were also analysed with a t-test (I).
Kruskal-Wallis analysis was used to compare multiple experimental groups of the RPEpermeation study (III). When the difference was significant (P < 0.05) multiple comparisons
versus a control group were performed with the Dunn test. The control group was
carboxyfluorescein in the comparison of the fluorescescent probes, and atenolol for betablockers. The Mann-Whitney test was used to test for a difference between the inward and
outward permeability of each probe (III).
47
5 RESULTS
5.1 Vitreous as a barrier for gene transfer by cationic lipids and polymers
5.1.1 DNA-complexes
According to the FACS-analysis, cellular uptake of PEI/DNA complexes ranged from 35 to
42 % during 5 h in DMEM. The respective levels of cellular uptake of DOTAP/DNA and
PLL complexes were about 20 % and 6–15 %. In the presence of vitreous the cellular uptake
of all carrier/DNA complexes decreased to less than 2 % and also the presence hyaluronan
decreased the uptake of DNA significantly (I: Fig.1). The only exception was PLL +/- 4 in 0.3
mg/ml hyaluronan. Decreased cellular uptake of the complexes was also noted when stirring
was used during the incubation of 5 hours and when the incubation time was increased up to
48 hours (I: Fig. 2).
Less than 1 % of the cells displayed expression of transfected GFP after incubation for 5
hours with the PEI, PLL and DOTAP complexes in DMEM. The transfection efficacy of PLL
+/-4 and PEI +/-2 complexes was practically zero also in DMEM. Vitreous and hyaluronan
decreased the expression levels of GFP to practically zero (I: Fig.3). Incubation of 48 h with
the GFP-carrier complexes decreased the fraction of GFP expressing cells from 0.2 - 0.4 % in
DMEM to less than 0.02 % in the vitreous. PEI +/- 2 and PEI +/- 4 complexes caused GFP
expression in 4.5 +/- 1.6 % and 9.7 +/- 1.3 % of the cells, respectively, when stirring was used
during incubation. The other complexes mediated transfection levels of less than 1 %. The
presence of vitreous decreased the GFP-transfection to less than 0.01 % in all cases.
In confocal microscopy, rhodamine fluorescent particles were seen in the bottom of the
vitreous in all samples. Not remarkably more particles were seen in the upper part of the
vitreous (about 250 µm upwards from the bottom of the well) in any of the specimens.
Compared to the mixed control samples, there was no evidence for reduced rhodamine
fluorescence in the bottom of the unmixed vitreous samples.
No nuclease activity was seen in DMEM or hyaluronan solutions, but the uncomplexed
plasmid had degraded after 24 and 48 hours in the vitreous. There were possibly partial
changes from a supercoiled DNA to a circular plasmid already after 2.5 and 5 hours of
incubation in the vitreous (I). In the case of DOTAP at charge ratios +/- 2 or +/- 4, DNA was
separated from complexes by the electric current and/or SDS (0.05%) present in the samples.
The separated DNA was detectable in electrophoresis, but the released DNA appeared to be
48
intact even after 48 hours of incubation in DMEM, hyaluronan or vitreous. When DNA was
complexed with PLL and PEI, no release or degradation of DNA was seen (I).
The mean diameters of the complexes at the concentration of 20 µg/ml were: 180 nm (PEI
+/-2), 110 nm (PEI +/- 4), 200 nm (DOTAP +/- 2), 90 nm (DOTAP +/-4), 110 nm (PLL +/-2)
and 170 nm PLL +/-4). In particular the sizes of PEI complexes varied extensively. In number
weighted analysis there were mostly very small complexes (< 10 nm), therefore, a fraction of
uncomplexed PEI cannot be excluded (I). The respective mean diameters of the PEI, PLL and
DOTAP complexes at charge ratio +/- 4 at the 50 µg/ ml concentration were 114 nm, 105 nm
and 110 nm, respectively (II).
5.1.2. FITC-dextrans and FITC-PLL
The cellular uptake (i.e. the percentage of the FITC-positive cells) of FITC-dextrans of
molecular weights of 4400 and 70 000 was decreased by the presence of vitreous from almost
100 % to 65 %. At molecular weights of 500 000 and 2 000 000, the vitreous decreased the
cellular uptake from 60–70 % to about 35 % (I: Fig.4). Hyaluronan 0.3 and 1.0 mg/ml caused
only a modest and in most cases not significant change in the cellular uptake of FITCdextrans.
The cellular uptake of positively charged FITC-labeled PLL 20 000 and 200 000 were
99% and 76 % in DMEM, respectively. The vitreous decreased the cellular uptake of FITCPLL probes significantly and more than the uptake of any of the FITC-dextrans. The cellular
uptake of FITC-labeled PLL 20 000 was significantly decreased by hyaluronan although to a
lesser extent than was the case with the vitreous, but with the PLL 200 000, the presence of
hyaluronan did not cause any significant decrease in the cellular uptake (I: Fig.5).
5.2 Neural retina as a barrier for gene transfer by cationic lipids and polymers
5.2.1 DNA-complexes
When the complexes were pipetted directly onto the RPE cells resting in the eyecup of the
bovine eyes, according to the FACS analysis 7 % of the cells took up PEI complexes. The
uptake of PEI complexes into the RPE was decreased to 1 %, when the complexes were
delivered onto the neural retina (II: Figure 2). Without the neural retina, the uptake of
DOTAP and PLL complexes in the RPE was 10 % and 15 %, respectively. The neural retina
decreased the cellular uptake of DOTAP and PLL complexes practically to zero (II: Figure
49
2). The mean fluorescence intensity did not increase in the presence of the neural retina and
thus the total fluorescence of positive cells decreased significantly in all cases. In fluorescence
microscopy, the fluorescence of rhodamine labeled DNA-carrier complexes was seen only
superficially at the level of the inner limiting membrane (I: Figure 4 A, B, C).
5.2.2 FITC-oligonucleotides and uncomplexed plasmid DNA
The cellular uptake of FITC oligonucleotides to bovine RPE was 67 % (i.e., the percentage of
fluorescent cells by FACS), but neural retina decreased the uptake to 2 % (II: Figure 3 C). A
decrease was also seen in the mean fluorescence intensity and total fluorescence of the
positive cells. When pipetted onto the neural retina, the concentration of FITColigonucleotide in the donor solution decreased already at 15 minutes by 44 ± 11 %, but no
further decrease took place in two hours. When the FITC-oligonucleotides were pipetted
directly onto the RPE, the FITC-oligonucleotide level in the solution decreased by 24 ± 14 %
during an incubation period of 15 minutes. According to this data, FITC-oligonucleotides
penetrate and bind into the neural retina, but do not easily gain access to the underlying RPE.
The uptake of EMA-labelled plasmid into the RPE was 1 % and the presence of the neural
retina did not significantly change the extent of uptake (II: Figure 3 C). Two fluorescent
bands were seen in fluorescence microscopy of the retinas after incubation with FITColigonucleotides: at the level of the inner limiting membrane and ganglion cells and at the
level of the inner part of the inner nuclear layer (II: Figure 4 F).
5.2.3 FITC-dextrans and FITC-PLL
The cellular uptake levels of FITC-dextrans with mean molecular weights of 20 000, 500 000
and 2 000 000 to the RPE cells were 96 %, 36 % and 34%, respectively, when the neural
retina had been removed. When the dextrans were pipetted onto the neural retina, the cellular
uptake levels in the RPE were 87 %, 26 % and 10 %, respectively (II: Figure 3 A). The
uptake decreased significantly in the case of FITC-dextran 20 000 and 2 000 000, but in the
case of FITC-dextran 500 000, no significant decrease of uptake was seen in the presence of
neural retina due to the variation in the cellular uptake. In histological analysis after 2 hours
of incubation with FITC-dextran of molecular weight of 20 000, the RPE showed a bright
fluorescence and diffuse fluorescence was seen at the level of photoreceptors (II: Figure 4 G).
50
The mean fluorescence intensities of FITC-dextrans did not show any clear trend and no
molecular weight dependence was seen in total fluorescence.
The uptake of FITC-poly-L-lysine (mean mw 20 000) into the RPE was 98 % without
neural retina, but decreased to 3 % when the neural retina was present (II: Figure 3). Also the
mean fluorescence intensity and total fluorescence of the positive cells decreased
significantly.
In fluorescence microscopy, FITC-PLL was seen superficially at the level of ILM.
5.3 Permeability of RPE
5.3.1 Tissue viability in diffusion chambers
In the experiments with fluorescent probes, TEER was 115 ± 23 and 124 ± 29
x cm2 at 20
and 210-240 min, respectively. In beta-blocker studies, TEER was 125 ± 35 and 123 ± 32
x
cm2 at 30 and 210-240 min, respectively (III: Table 1 and 2). In the experiments with
fluorescent probes, TEP was 6.9 ± 2.3 and 5.5 ± 2.5 mV at 20 and 210-240 min, respectively.
In beta-blocker studies, TEP was 6.4 ± 2.9 and 3.4 ± 1.5 mV at 30 and 210-240 min,
respectively. No significant differences between initial and final TEER and TEP values in
different experiments were detected (III).
The transepithelial electrical resistances (TEER) of the non-pigmented RPE specimens
(83-189
* cm2) were rather similar to the pigmented samples (unpublished data).
5.3.2 Permeability of carboxyfluorescein and FITC-dextrans
The inward permeability of fluorescent probes decreased with the increasing size of the
molecule, and there was a 35-fold difference in the permeability between carboxyfluorescein
and FITC-dextran of 80 kDa (III Table 1 and Fig. 1 and 2). The choroid-to-retina
permeability of carboxyfluorescein was 2.4 times lower than measured in the opposite
direction but there was no directionality in the permeation of FITC-dextran 10 kDa. The
permeation lag time increased clearly with the molecular size, with values from 30 to 100 min
(III Table 1 and Fig. 1).
51
5.3.3 Permeability of beta-blockers
The inward permeability coefficients of the most lipophilic beta-blockers, metoprolol,
timolol, and betaxolol, were 7-8 times higher than that of atenolol (III Table 2 and Fig. 3).
However, even the most hydrophilic beta-blockers, atenolol and nadolol, permeated two times
faster than carboxyfluorescein (III Tables 1 and 2). There was no directionality in the case of
atenolol and nadolol, but the more lipophilic beta-blockers permeated faster in the inward
direction than outwards. The lag times of atenolol, nadolol and carboxyfluorescein were about
40 min, while the lag times of the more lipophilic beta-blockers were generally about 100 min
(III Table 2). The lag times of beta-blockers were similar in both directions, except for
betaxolol (III Table 2). The permeability of betaxolol through the unpigmented part of the
RPE-choroid (including tapetum fibrosum) was clearly lower than its permeation through the
pigmented part (Papp 1.1 ± 0.9 x 10-6 cm/s and 13.0 ± 6.8 x 10-6 cm/s, respectively).
5.4 Binding to melanin
5.4.1 Characterization of synthetic and isolated bovine melanin
In SEM, the shape of the isolated melanin granules was round or oval with a diameter of
about 1µm, while the size of the synthetic melanin granules was smaller and the shape of the
particles was irregular (IV: Figure 1). In laser diffractometry carried out in water immediately
after sonication, the mean diameters of the isolated melanin granules varied from 0.2 to 10.0
m, and the diameter of the synthetic melanin ranged from 0.3 to 50 m (modes 1.9 m and
1.1 m, respectively). After 4 hours, there was practically no change in the size of isolated
melanin granules, but at that time, a large part of the synthetic melanin had formed aggregated
granules with a diameter of 10-300 nm with a mode of 46 m (IV: Figure 2). After two days,
also the isolated melanin had partly aggregated (data not shown). The specific surface areas of
the synthetic and isolated melanins were 11.75 m2/g and 5.94 m2/g, respectively.
5.4.2 Binding of beta-blockers, oligonucleotides and 6-CF to melanin
Binding of betaxolol and metoprolol to melanin was examined at 13 concentrations from 25
nM to 100 M. Saturation was not reached even at the highest concentration (IV: Figure 3).
Betaxolol was bound to melanin more than metoprolol, and comparing melanin binding per
mass unit, more binding was always seen to synthetic melanin than to isolated melanin (IV:
52
Figure 3). When binding of beta-blockers was compared per surface area, there was less of a
difference between binding to isolated and synthetic melanin (IV: Figure 4). The binding of
betaxolol to synthetic or isolated melanin was not increased at long incubation times. In
synthetic and isolated melanin, at least two binding sites for melanin were found in the
Scatchard plots (IV: Figure 5). For betaxolol, the maximum binding capacity and dissociation
constant for the high affinity site were much lower in isolated melanin than in synthetic
melanin. The dissociation constant for the low affinity site was much higher in isolated
melanin than in synthetic melanin. The capacity of the low affinity site in isolated melanin
was about 2100 times higher than that of the high affinity site, whereas the difference with
synthetic melanin was about 240 fold. For metoprolol the binding parameters for the high
affinity site were in same range for both melanin types, but the dissociation constant for the
low affinity site was greater for isolated melanin. Neither 6-carboxyfluorescein nor
oligonucleotides showed any tendency to bind to melanin.
53
6 DISCUSSION
6.1. The vitreous and neural retina as a barrier
The network structure of the vitreous which is composed of collagen fibers, hyaluronan and
water, restricts the passage of large molecules (Bishop 2000). The density of the vitreous
varies, being highest in the marginal parts of the vitreous compartment. The basal layer of the
vitreous allows passage of molecules of mean molecular sizes of 15-20 nm and smaller
(Balazs and Denlinger 1984). In our experiments, the FITC-dextran of the highest molecular
weight permeated the vitreous (30 nm; mw 2 000 000). The vitreous was treated by pushing it
through a sieve and, hence its structure was partly disrupted. This may have diminished the
barrier function of the vitreous. On the other hand, the structure of the vitreous varies also in
vivo, because it has a tendency to liquify with age as well as in several pathological processes
(Bishop 1999 et al.). Peeters et al. (2005) recently used FRAP- techniques (fluorescence
recovery after photobleaching) to reveal that FITC-dextrans at least up to a mw of 2 000 000
were able to diffuse through the vitreous as quickly as through water. On the basis of studies
with coated nanospheres, they concluded that the maximum particle size to diffuse through
vitreous was between 220 and 575 nm. However, also in their study the vitreous was collected
from bovine eyes and, thus, its structure was not intact (Peeters et al. 2005).
The effect of a positive charge of the molecule on the permeation in the vitreous has not
been previously examined. Since it is a polyanionic gel, the vitreous might bind the positively
charged molecules and DNA complexes, reorganize their structure or release the DNA. In
addition to hyaluronan, vitreous contains other glycosaminoglycans such as chondroitin
sulphate and possibly also heparan sulphate, and the positive gene-carrier complexes have
been previously shown to interact with glycosaminoglycans (Bishop 2000; Ruponen et al.
1999). At a 3-fold excess of negative charges, hyaluronan partly or totally inhibited
transfections of rabbit aortic media smooth muscle cells with PEI, but not those mediated by
DOTAP or PLL. Chondroitin sulphates and heparan sulphate inhibited the transfection with
PEI, PLL and DOTAP (Ruponen et al.1999). Coating of complexes with hyaluronan or
heparan sulphate decreased the cellular uptake of the PEI-complexes, but did not decrease the
cellular uptake of DOTAP and PLL complexes (Ruponen et al. 2001). In those experiments,
the concentration of glycosaminoglycans was low and there was no diffusional barrier
between the cells and complexes, as in this study. In our experiment, vitreous and hyaluronan
significantly decreased the cellular uptake of all complexes, with one single exception
54
(cellular uptake of PLL complexes at 0.3 mg/ml hyaluronan was not changed). The sizes of
the complexes were mostly bigger (110-200 nm) than the largest FITC-dextran (30 nm), but
the DOTAP +/- 4 complexes and the PEI complexes were in the same or smaller range.
Nonetheless, their cellular uptake was decreased. Interestingly, confocal microscopy showed
that the complexes did not remain on the surface of the vitreous and thus, a simple diffusional
barrier does not explain the decrease in the cellular uptake. The vitreous decreased more
efficiently the cellular uptake of FITC-PLL with mw of 20 000 than the cellular uptake of the
FITC dextrans with mw´s of 500 000 or 2 000 000, which also indicates the negative
importance of the positive charges of the molecules on vitreal permeation.
Peeters et al. noted that nonpegylated cationic lipoplexes aggregate in the vitreous and
they suggested that the negatively charged GAGs might neutralize the zeta-potential of the
complexes leading to aggregation of the complexes (Peeters et al. 2005). Furthermore,
making the surfaces of the complexes hydrophilic by attaching PEG-chains on their surfaces
diminished the aggregation of the complexes and binding to the fibrillar stuctures of the
vitreous (Peeters et al. 2005). In our experiments, aggregation and binding to the fibrillar
structures may also have decreased the cellular uptake rate of complexes in the presence of
the vitreous and hyaluronan.
Convection and movements of vitreous gel may modify the permeation in vivo, but
despite stirring, the vitreous still remains as a barrier for the complexes (Xu et al. 2000;
Michaelsson 1980). The structural components of human and bovine vitreous are virtually
identical and thus it is presumable that the conclusions of this study would be the same if
performed with human vitreous.
Even though the vitreous was removed by vitrectomy, the intravitreally administered
molecules must permeate through neural retina to reach the RPE. The tight junctions of the
RPE and retinal capillaries form a blood-retina barrier which restricts the passage of large
molecules into the retina. In animal models, after an intravitreal injection, numerous growth
factors, for example, basic fibroblast growth factor and brain-derived neurotrophic factor
(molecular weights 20 000-30 000), adequately permeate through neural retina to counteract
photoreceptor degeneration (Faktorovich et al.1990; La Vail et al. 1998). In addition,
rhodamine B isothiocyanate labeled dextran with a molecular weight of 20 000 penetrated
through the rabbit neural retina (Kamei et al. 1999). On the other hand, the neural retina has
been previously suggested to restrict the passage of larger molecules. For example, thorium
dioxide particles of 10 nm in diameter diffused from the vitreous to the outer limiting
membrane of the cat retina but not further (Smelser GK et al. 1965). Likewise, a tissue
55
plasminogen activator (mw 70 000) did not permeate through the rabbit neural retina (Kamei
et al. 1999). In our experiments with bovine eyes, even the FITC-dextran with a molecular
weight of 2 000 000 permeated to some extent through the neural retina (II: Figure 3). The
comparison of different sets of data in the literature is difficult due to non-standardized
experimental conditions and different methods of detection. Furthermore, chemical
interactions of test molecules with the retinal tissues may differ.
Intact RPE took up DNA complexes effectively if they were administered directly on the
RPE. This indicates that if DNA complexes can reach the cells in an active form, then they
can be delivered effectively into non-dividing and differentiated RPE (II: Figure 2). This is in
line with the report of Mannermaa et al. (2005), who concluded on the basis of a study with
polarized ARPE-19 cells, that differentiated RPE cells can be transfected to express the
transgene for prolonged periods with selected lipoplexes. With our method only the cellular
uptake of RPE was measurable, because measuring of the transfection efficacy would require
a prolonged culturing of RPE cells. When the neural retina was present, the cellular uptake of
positively charged molecules decreased remarkably (II: Figures 2 and 3 B). The poor
permeation of DNA carrier complexes and FITC-PLL was also seen in the fluorescence
microscopy data (II: Figure 4 A, B, C and E). According to the cellular uptake measurements
by FACS, even the largest neutral FITC-dextran (mw 2 000 000) permeated the neural retina
better than the positively charged PLL with molecular weight of 20 000. Several retinal
layers, for example the inner limiting membrane (ILM) and interphotoreceptor matrix, contain
negatively charged GAGs which may interact with positively charged molecules (Chai and
Morris 1994). In the fluorescence microscopy, the DNA-carrier complexes and FITC-PLL
were seen on the level of ILM. The GAGs of ILM and interphotoreceptor matrix may undergo
similar interactions with complexes in the neural retina as discussed previously in conjunction
with the vitreous.
When the neural retina was absent, the naked plasmid DNA was poorly taken into RPE
cells (cellular uptake < 1%), but the FITC-oligonucleotide was taken up by 67 % of the cells.
The presence of neural retina decreased the cellular uptake of the oligonucleotides, even
though they were negatively charged. In confocal microscopy, the FITC-oligonucleotide was
not found in the level of the RPE but in the levels of ganglion cells and inner nuclear layer. In
the experiments conducted by Rakoczy et al. (1996), intravitreally injected fluorescein labeled
cathepsin S antisense oligonucleotide was found in ganglion cells after two hours from
injection. We used this same incubation time. In the experiment of Rakozky et al. (1996)
fluorescein labeled cathepsin S antisense oligonucleotide was found in all retinal layers after 3
56
days, and FITC fluorescence was seen in the RPE even after 56 days. Accumulation of
oligonucleotides into the retina has been detected also in kinetic studies with intravitreally
delivered phosphorothioate oligonucleotide, fomivirsen (Vitravene®, Perry and Balfour,
1999). Phosphorothioate oligonucleotides are generally recognized as undergoing extensive
protein binding, which might explain the retinal accumulation and slow release of
oligonucleotides from the retina (Bennett 1998). The oligonucleotides showed no binding to
melanin in our study (IV), thus, binding to melanin does not explain the accumulation to RPE.
The neural retina is a soft and fragile multilayered tissue and it is technically demanding
to detach it intactly from the eye. We used a fresh post mortem bovine eyecup to study the
permeation properties of the neural retina. In this study, the retina remained untouched in its
natural environment, including the interphotoreceptor matrix. Nevertheless, possible
metabolic differences compared to an in vivo situation cannot be excluded, and the incubation
time that can be used with this model is limited.
FACS does not distinguish the fluorescence inside the RPE cells from fluorescence
attached to the cell surface. It is unlikely that the neutral or negatively charged molecules like
dextrans, oligonucleotides or plasmid DNA would become attached firmly on the cell surface.
However, in the case of positively charged molecules this might happen. Trypsination of the
RPE cells during the detaching procedure disrupts the structure of the proteoglycans on the
cell surfaces and consequently should separate the attached molecules from the cell surface.
In the permeation experiments, the molecules must, however, permeate the neural retina
before they can reach the surface of the RPE cell. Therefore, the above mentioned factors do
not affect our conclusions on the role of the neural retina as a barrier.
6.2 RPE as barrier
The RPE restricts drug permeation through the transscleral route to the neural retina and
vitreous after local administration by subconjunctival, retrobulbar or sub-Tenon´s injection.
After systemic delivery, the molecules which permeate from the choroidal circulation to
neural retina have to cross the RPE. After intravitreal injection drug elimination from the
vitreous may take place through the neural retina and RPE. As a part of the blood-retinal
barrier, the tight junctions of the RPE are known to restrict permeation of big and hydrophilic
molecules (Maurice and Mishima 1984; Cunha-Vaz 2004). Drugs may also bind to melanin,
which may modify their permeation and half time in pigmented tissues like the RPE.
Permeation data of drugs and other molecules in the RPE is sparse and no systematic data on
57
the permeation properties of molecules as a function of size and lipophilicity has been
published (Kimura et al. 1996b; Steuer et al. 2004). On the other hand, large proteins, like
PEDF and ovalbumin, are known to permeate through ocular tissues into the retina after an in
vivo subconjunctival injection (Amaral et al 2005; Gehlbach et al. 2003).
Carboxyfluorescein and the most hydrophilic beta-blockers, atenolol and nadolol are
expected to permeate mainly through the tight junctions of the RPE (paracellular route) and
the more lipophilic beta-blockers permeate mostly across the cell membranes of the RPE
(transcellular route). For the most hydrophilic beta-blockers, atenolol and nadolol, the
permeability of the choroid-RPE was close to carboxyfluorescein but the permeability of the
more lipophilic beta-blockers was significantly higher in both directions (III, Tables 1 and 2).
The permeability of the lipophilic beta-blockers was slightly higher inwards than outwards.
The asymmetry in the permeability of lipophilic drugs in different directions may point to a
contribution of active transporters or efflux pumps, like the P-glycoprotein efflux pump.
Based on bioelectrical measurements, the choroid-RPE tissues remained viable and in good
condition during the
permeation experiments. In addition,
the
permeability of
carboxyfluorescein in the outward direction was higher than in the opposite direction (III,
Table 1) suggesting an active transport similar to that observed in isolated rabbit and dog
RPE-choroid tissue (Kimura et al. 1996b; Tsuboi and Pederson 1986).
According to the literature, the permeation rates for hydrophilic molecules in bovine RPEchoroid are about 1/10 - 1/100 as compared to the human and rabbit sclera (III: Figure 5).
The thickness of the human sclera varies between 0.4 - 1.0 mm depending on the anatomical
location and may be as thin as 0.1 - 0.25 mm at the equator in a significant number of eyes.
The thickness of the human sclera was not reported in the study of human scleral permeation
which was used for comparison (Olsen et al.1995). However, even if the thickness were to be
5 times higher, RPE-choroid remains as the major barrier. According to our permeation
studies, the RPE-choroid is the rate limiting permeation barrier in the transscleral route of
retinal drug delivery of hydrophilic molecules and macromolecules, such as proteins and
oligonucleotides. In the case of small lipophilic molecules, both the sclera and RPE-choroid
are important barriers. Scleral permeability was not sensitive to solute lipophilicity: scleral
permeabilities of hydrophilic and lipophilic low-molecular-weight drugs were similar
(Prausnitz and Noonan 1998). They were in the same range with the RPE-choroid
permeability of the lipophilic beta-blockers in our study.
The choroidal blood flow may flush a major proportion of transsclerally delivered
molecules into the systemic blood flow. Currently little is known about the importance of
58
choroidal blood flow in drug delivery. It has been previously shown that choroidal electrical
resistance, probably including Bruch's membrane, is less than 10 % of the total resistance of
the isolated bovine RPE-choroid (Miller and Edelman 1990). Recently, Cheruvu and
Kompella (2006) demonstrated that choroid-Bruch`s layer is a more significant barrier to drug
transport than sclera and discriminates especially lipophilic and cationic molecules.
The RPE-choroid taken from non-pigmented part of the bovine eye appeared to be less
permeable to betaxolol than the RPE-choroid from a pigmented part. This may be due to the
dense fibrotic layer of choroid of the non-pigmented part of the bovine eye, called the tapetum
lucidum. The experiments indicated that in studies with bovine eyes, and probably with other
animal eyes which have a tapetum lucidum, it is important to be aware that the tapetum
lucidum may affect permeability. However, bovine sclera-choroid (with a tapetum lucidum)
and a porcine model of sclera-choroid (without a tapetum lucidum) behaved similarly with
respect to transport of molecules of different lipophilicities in the study of Cheruvu and
Kompella (2006).
The permeability of hydrophilic fluorescent probes, carboxyfluorescein and FITCdextrans, in the bovine RPE-choroid declined roughly exponentially with respect to increasing
molecular radius (III: Figure 2). FITC-dextrans can be taken into some cells by fluid-phase
endocytosis (Ruponen et al. 2004). Active transcytosis would result in asymmetry in the
permeability rates, but the permeability of FITC-dextran 10 kDa was similar in both
directions. Thus, our study indicates that endocytosis does not result in dextran transport
across the tight epithelium (III, Table 1).
The lag time of permeation in the choroid-RPE increased with the size of the molecule,
which was to be expected, because the diffusion coefficient of the molecule within the
membrane usually decreases with increasing molecular weight. Generally, the lag time is
inversely proportional to the diffusion coefficient (Crank and Park 1968; III, Table 1). The
lag time of the fluorescent probes varied from 30 to 100 minutes whereas in human and rabbit
sclera, a steady-state permeability was achieved within 15-30 min even for the largest FITCdextrans (Olsen et al.1995; Ambati et al. 2000). This would be expected also based on the
higher diffusivity within sclera.
The lag times of the lipophilic beta-blockers were longer than those of the hydrophilic
beta-blockers (III, Table 2). Timolol has been previously shown to bind reversibly to melanin
and more than one class of binding sites have been found (Araie et al. 1982; Salminen and
Urtti 1984; Abrahamsson et al. 1988; Aula et al 1988). At least two binding sites were seen
also in our study for betaxolol and metoprolol. In the permeation experiments, melanin-
59
binding increases the lag time, because the steady-state permeation begins only after
equilibrium between the free and melanin-bound drug has been achieved. Lengthening of the
incubation time did not increase binding of betaxolol to melanin. Also the binding of timolol
to melanin has been reported to rapidly reach a plateau (within 30 minutes) (Kiuschi et al
2004).
Synthetic melanin, which is produced by oxidation of tyrosine with hydrogen peroxide,
and solubilized, partly solubilized and intact melanin granules have been used for studies on
melanin binding. Synthetic melanin lacks the protein and membrane structures of
melanosomes. Larsson and Tjälve (1979) reported that the protein-moiety is not important in
the melanin binding process. Natural melanin is a copolymer that may exhibit variations of
complex and random polymers, and contains water that is thought to maintain the hydrated
state of melanin (Koeberle et al 2003). Ito et al. (1986) reported that the enzymatically
prepared dopa melanin contains a lower percentage of 5,6-dihydroxyindole-2-carboxylic acid
(DHIC)-derived monomers than natural eumelanin. The natural eumelanin from sepia
officinalis has a structural order with subunits that have a lateral dimension of
15 nm while
the synthetic eumelanin appears to be amorphous solids (Nofsinger et al 2000). In our study,
the shape of synthetic melanin appeared to be irregular and the specific surface area of
synthetic melanin was about double compared to that of isolated melanin. Furthermore,
synthetic melanin aggregated more readily than the isolated melanin. Synthetic melanin
bound more beta-blockers than isolated melanin and there were also differences in the binding
capacity and dissociation constant values of high- and low affinity binding sites. Synthetic
melanin is thus useful in testing whether or not a drug binds to melanin whereas isolated
melanin should be used to analyze binding properties in detail.
Melanin binding may lead to the accumulation of drug in the choroid-RPE, increase the
lag time in drug permeation and prolong the action of the drug. The binding of betaxolol and
metoprolol to melanin in the human choroid-RPE was predicted by using the binding
parameters obtained with bovine ocular melanin. Based on our predictions, melanin in the
human choroid-RPE may bind as much as hundreds of nanograms of betaxolol and
metoprolol at therapeutic drug concentrations. The amount of melanin bound drug was 3 to19
times higher than the amount of unbound drug. Recently, Hollo et al. (2006) reported high
concentrations of betaxolol in the pigmented tissues of human eye after 1 month of topical
ocular administration. It is important to further clarify the role of melanin binding in the
accumulation and ocular pharmacokinetics of betaxolol in the RPE. The morphology of
human and bovine melanin granules is rather similar in electron microscopy (Boulton et al
60
1990) and eumelanin is the major component in both human and bovine melanin (Boulton
1998; Liu et al 2005). However, differences in their binding properties cannot be excluded
and more information of the binding properties on human melanin is needed.
61
7 CONCLUSIONS
1.
The vitreous limits gene transfer with polymeric and liposomal gene complexes into the
RPE cells. The large size and especially the positive charge of these molecules or
complexes, decrease the extent of cellular uptake when molecules or complexes are
delivered onto a vitreous layer above the RPE cells. The mechanisms remain unclear,
but the presence of a diffusional barrier and electrostatic forces between the negatively
charged vitreous and positively charged complexes does not completely explain the
results.
2.
The neural retina is another barrier to gene transfer into the RPE with non-viral gene
complexes. As in the case of the vitreous, the cellular uptake of large and positively
charged molecules and complexes is restricted by the neural retina. Thus, vitrectomy
cannot solve the problem of poor intravitreal injection efficacy of non-viral gene
delivery systems. Oligonucleotides accumulate in the retina, but do not bind to melanin.
3.
The permeability of hydrophilic molecules through the choroid-RPE decreases with the
increasing size of the molecule and it is much lower than the permeability through the
sclera. The permeability of lipophilic beta-blockers through the choroid-RPE is much
higher than that of the hydrophilic compounds and is in the same range as the scleral
permeability. For hydrophilic drugs and macromolecules, the RPE is a tighter barrier
than the sclera for transcleral delivery to retina. The RPE seems to be a more important
barrier than the sclera for transscleral drug delivery into the posterior segment but the
role of the choroid remains still unclear.
4.
Oligonucleotides and 6-carboxyfluorescein do not bind to melanin while betaxolol and
metoprolol bind rapidly in a concentration dependent manner. The lipophilic betablockers are predicted to bind significantly to melanin in human choroid-RPE. The
physicochemical and binding properties of synthetic melanin differ markedly from
biological melanin which should be taken into consideration when designing in vitro
binding experiments and making pharmacokinetic predictions.
62
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9 ORIGINAL PUBLICATIONS I-IV
Kuopio University Publications D. Medical Sciences
D 392. Pesonen, Tuula. Trends in Suicidality in Eastern Finland, 1988–1997.
2006. 119 p. Acad. Diss.
D 393. Tuhkanen, Hanna. DNA copy number changes in the stromal and epithelial cells of
ovarian and breast tumours.
2006. 112 p. Acad. Diss.
D 394. Koskelo, Reijo. Säädettävien kalusteiden vaikutukset tuki- ja liikuntaelimistön terveyteen
lukiolaisilla.
2006. 96 p. Acad. Diss.
D 395. Elo, Mika. Stress-Related Protein Synthesis in Mammalian Cells Exposed to Hydrostatic
Pressure.
2006. 74 p. Acad. Diss.
D 396. Remes-Pakarinen, Terhi. Influences of genetic factors and regular exercise on bone
in middle-aged men.
2006. 95 p. Acad. Diss.
D 397. Saarela, Tanja. Susceptibility genes of diabetes and endothelial dysfunction in preeclampsia.
2006. 103 p. Acad. Diss.
D 398. Piippo-Savolainen, Eija. Wheezy babies - wheezy adults? Adulthood asthma, bronchial
reactivity and lung function after hospitalization for bronchiolitis in early life.
2006. 91 p. Acad. Diss.
D 399. Kauppinen, Anu. Lipocalin Allergen-Induced T Cell Response: Prospects for Peptide-Based
Immunotherapy.
2006. 81 p. Acad. Diss.
D 400. Vasara, Anna. Autologous chondrocyte transplantation: Properties of the repair tissue in
humans and in animal models.
2007. 92 p. Acad. Diss.
D 401. Andrulionyte, Laura. Transcription factors as candidate genes for type 2 diabetes: studies
on peroxisome proliferator-activated receptors, hepatic nuclear factor 4α and PPARγ coactivator Iα.
2007. 112 p. Acad. Diss.
D 402. Raatikainen, Kaisa. Health behavioural and social risks in obstetrics: effect on pregnancy
outcome.
2007. 100 p. Acad. Diss.
D 403. Kinnunen, Tuure. The role of T cell recognition in the immune response against lipocalin
allergens: prospects for immunotherapy.
2007. 76 p. Acad. Diss.
D 404. Gratz, Silvia. Aflatoxin binding by probiotics : experimental studies on intestinal aflatoxin
transport, metabolism and toxicity.
2007. 85 p. Acad. Diss.
D 405. Ming, Zhiyong. Upper limb musculoskeletal disorders with special reference to sympathetic
nerve functions and tactile sensation.
2007. 91 p. Acad. Diss.
D 406. Timonen, Leena. Group-based exercise training in mobility impaired older women.
2007. 91 p. Acad. Diss.