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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5915_Библиотеки_им_академика_М_И_Перельмана
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Chapter 10
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Microparticles as Drug Delivery Systems
for the Back of the Eye
Rocío Herrero-Vanrell
Abstract Treatment of vitreoretinal disorders often include repeated intraocular
injections to achieve effective levels of the active substance in the target site.
Intraocular drug delivery systems (IDDS) are considered an alternative to multiple
injections as they release the encapsulated drug over long periods of time. Among
them, biodegradable microparticles are very useful for intraocular administration
because they can be injected as a conventional suspension without surgical procedures, to release the active substance over weeks or months. Microparticles can be
loaded with different drugs useful to treat different pathologies affecting the back of
the eye such as proliferative vitreoretinopathy, age-related macular degeneration,
cytomegalovirus retinitis, diabetic retinopathy, endophthalmitis, glaucoma, herpes
infection, macular edema, retinal vein occlusion, retinitis pigmentosa, and uveitis.
Administration of microparticles can be performed by periocular, intravitreal, subretinal, or other intraocular routes to treat vitreoretinal disorders. Generally, microparticles are loaded with one active substance. Recently, biodegradable microparticles
loaded with more than one drug (“combo microparticles”) are being developed.
Moreover, biodegradable microspheres are potential tools for retinal repair in combination with retinal progenitor cells.
Abbreviations
PLA Poly(lactic) acid
PGA Poly(glycolic) acid
PLGA Poly(lactic-co-glycolic) acid
GPC Gel permeation chromatography
R. Herrero-Vanrell (*)
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy,
Avda Complutense s/n, Complutense University, 28040 Madrid, Spain
e-mail: rociohv@farm.ucm.es
U.B. Kompella and H.F. Edelhauser (eds.), Drug Product Development for the Back of the Eye,
AAPS Advances in the Pharmaceutical Sciences Series 2, DOI 10.1007/978-1-4419-9920-7_10,
© American Association of Pharmaceutical Scientists, 2011
231
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R. Herrero-Vanrell
e
K
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Mw Weight-average molecular weight
Mn Number-average molecular weight
PEG Polyethylene glycol
kGy Kilo Gray
Tg Glass transition temperature
Tm Crystalline melting points
Css Steady state concentration
K0 Zero-order constant
Vd Volume of the vitreous
Elimination rate constant
G Gauge
PBS Phosphate buffer solution
BSS Buffer solution
HA Hyaluronic acid
HPMC Hydroxypropylmethyl cellulose
AUC Area under the curve
5-FU 5-fluorouracil
VEGF Vascular Endothelial Growth Factor
AMD Age macular degeneration (AMD)
TA Triamcinolone acetonide
PVR Proliferative vitreoretinopathy
RPE Retinal pigment epithelium
RD Retinal detachment
RA Retinoic acid
LPS Lipopolysaccharide
TRD Tractional retinal detachment
CyS Cyclosporine
CNV Choroidal neovascularization
ARN Acute retinal necrosis
HSV Herpes simplex virus
Da Daltons
CMV Cytomegalovirus
HCMV Human cytomegalovirus
RGC Retinal ganglion cells (RGC)
ECM Extracellular matrix
MMP2 Matrix metalloproteinase
RPCs Retinal progenitor cells (RPCs)
10.1 Introduction
Successful ophthalmic therapy requires effective concentrations of the drug in the
target site. Therapeutic concentrations of the active substance in cornea and conjunctiva are mandatory for the treatment of ocular surface diseases such as dry eye
syndrome, surface inflammation, or infection. However, if the drug has to reach the
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aqueous humor as it is the case of hypotensive agents for glaucoma management,
the active substance must be present at high concentrations at the site of administration to cross through the cornea and/or conjunctiva to achieve therapeutic concentrations in the anterior segment (only 5% of the administered dose penetrates the
cornea) (Maurice and Mishima 1984). Furthermore, the drug must have specific
physical and chemical properties to cross the ocular surface barriers. While lipophilic drugs cross the epithelium well, hydrophilic substances are able to cross the
stroma. In any case, the molecular weight of the substance must be small enough to
use the transcellular or paracellular route to reach intraocular structures.
In the management of vitreoretinal disorders, the drug must reach the back of the
eye. In these cases, periocular, intravitreous, or other intraocular injections are
required. If successive administrations are needed, special care has to be taken to
avoid fibrosis and inflammation at the site of injection. Moreover, it is well known
that repeated intravitreal injections are poorly tolerated and the risk of adverse
effects (e.g., cataracts, intravitreal hemorrhages, and retinal detachment) increases
with the number of administrations (Herrero-Vanrell and Refojo 2001).
Controlled drug delivery systems can maintain concentrations of the active
substance at the target site for long periods of time. Among them, implants (>1 mm),
microparticles (1–1,000 mm), and nanoparticles (1–1,000 nm) have been developed
for the treatment of posterior segment pathologies (Herrero-Vanrell and Refojo
2001; Urtti 2006) (Fig. 10.1).
Fig. 10.1 Strategies to avoid frequent intraocular injections. “Depot” systems – Poor aqueous
soluble drugs. Once injected, the active substance is slowly dissolved in the vitreous. Drug delivery
systems (DDS): Implants, microparticles, and nanoparticles. Biodegradable DDSs disappear from
the site of administration after delivering the drug
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Fig. 10.2 Administration routes of microparticles: Intravitreal, subretinal, and periocular
Microparticles are adequate for the intraocular route, bypassing the blood– ocular
barrier. One of their advantages is that microparticles can release the drug over the
time with one single administration, having the same effect than multiple injections
(Fig. 10.2). Furthermore, injection of microparticles is carried out as a conventional
suspension.
Microparticles are usually prepared with a polymer or mixture of polymers and
one or several active substances. Depending on the nature of the polymer (erodible
or biodegradable and nonerodible or nonbiodegradable) microparticles remain or
disappear from the site of injection after delivering the drug. In the case of posterior
segment diseases, biodegradable microparticles are preferred.
Microparticles are capable to provide sustained and controlled release of the
bioactive agent, while the remaining drug still present inside the particle is protected
from degradation and physiological clearance.
By physical structure, microparticles are classified in microcapsules and microspheres. Microcapsules are constituted by a drug core, which is surrounded by a
polymer layer (reservoir structure). Conversely, in the microspheres the drug is dispersed through the polymeric network (matrix structure) (Fig. 10.3).
Among the biodegradable polymers employed to prepare microparticles are
gelatin, albumin, polyorthoesters, polyanhydrides, and polyesters (Colthrust et al.
2000; Herrero-Vanrell and Refojo 2001). Since several years ago, the most employed
polymers to prepare biodegradable microspheres are the poly(lactic) acid (PLA),
poly(glycolic) acid (PGA), and their copolymers poly(lactic-co-glycolic) acid
(PLGA). PLA and PGA have crystalline structure whereas PLGA is amorphous.
Experience has shown that the PLGA 50:50 (50% lactide and 50% glycolide)
degrades relatively fast to metabolic lactic and glycolic acid that are readily eliminated from the body after suffering metabolism to carbon dioxide and water mediated by Krebs cycle (Zimmer and Kreuter 1995). Regarding to molecular weight,
polymers with small chains degrade faster than high molecular weight polymers.
For the back of the eye, PLA and PLGA polymers have been employed to prepare
different devices: implants, scleral plugs, pellets, discs, films, and rods (Yasukawa
et al. 2004; Mansoor et al. 2009).
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Fig. 10.3 Structure of microparticles. Microcapsules (reservoir system) and microspheres (matrix
structure)
10.2 Manufacturing of Microparticles
Manufacturing of microparticles are mainly based on four basic techniques: aggregation by pH adjustment or heat, coacervation (phase separation), spray drying, and
solvent extraction/evaporation (Freitas et
of solvents and coacervating agents that can remain in the microparticles once prepared and low micrometer size is difficult to obtain. The use of supercritical gases
as phase separating agents has been introduced to avoid potentially harmful residues
in the microspheres. Spray drying is relatively simple but not useful for highly temperature-sensitive drugs. Microspheres prepared according to this technique are
highly porous. Microspheres loaded with triamcinolone acetonide (TA) and ciprofloxacin have been prepared by the spray drying technique for intraocular injection
(Paganelli et al. 2009).
The most commonly reported technique for microspheres formation is the
solvent extraction/evaporation method (evaporation of a solvent from an emulsion)
(Herrero-Vanrell et al. 2000; Amrite et al. 2006) (Fig. 10.4). It requires a dissolution
or dispersion of the active substance in a first solvent containing the matrix forming
polymer (inner phase). After that, an emulsification of the polymer organic solution
in a second continuous phase immiscible with the inner phase is carried out. Then,
an extraction of the organic solvent from the formed emulsion by evaporation is
al. 2005). Coacervation required the use
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R. Herrero-Vanrell
O/W emulsion
O/W emulsion
Dissolved
drug
Solid
drug
Solid
drug
S/O/W emulsion
Preparation of drug dispersion
Homogeneous
drug solid
dispersion
Sonication
↓T
a
(ice)
↓t(30")
↓power
S/O/W emulsion
Drug
PLGA solution
in CH
2CI2
PLGA solution
in CH
2CI2
O-phase:
Drug (dissolved)
+PLGA solution
in CH
2CI2
O-phase:
drug (solid) +
PLGA solution
in CH
2CI2
Addition of
aqueous-phase
(PVA 2% in H
2
O)
Addition of
aqueous-phase
(PVA 2% in H
2
O)
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Fig. 10.4 Schematic procedure for microspheres preparation according to solvent/extraction/
evaporation technique (O/W emulsion and S/O/W emulsion) O/W emulsion – The drug is dissolved in the inner phase of the emulsion S/O/W emulsion – The drug is suspended as solid in the
inner phase of the emulsion
performed at room temperature or under vacuum. Finally, the immature microspheres
are harvested and dried before freeze-drying or desiccation. Lyophilization is
preferred because the stability of the product increased. Depending on the solubility
of the drug, oil in water (O/W) or oil in oil (O/O) emulsions are formed. In the case
of aqueous soluble drugs an (O/O) emulsion achieves higher encapsulation
efficiencies. By the contrary, for poor soluble drugs the O/W emulsion technique
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results more adequate. In the case of polypeptides, proteins, or biotechnological
products, a double emulsion (W/O/W) or solid oil in water emulsion (S/O/W) is
employed.
10.3 Characterization of Microparticles
10.3.1
Microsphere samples can be observed by light microscopy and scanning electron
microscopy (SEM). SEM allows observation of surface morphology of microspheres
once prepared and at different stages of the in vitro release studies. For this technique,
samples have to be dried and gold sputter-coated before observation (Fig. 10.5).
Morphological Characterization of Microparticles
10.3.2 Particle Size Analysis and Distribution
Size analysis of microparticles is performed through the determination of the equivalent diameters. The methods commonly employed to evaluate particle size and size
distribution are electrical stream sensing zone (Coulter counter) and laser light scattering (Staniforth 2002).
Analysis of samples through Counter Coulter requires dispersion of the sample
in an electrolyte to form a highly diluted suspension. Laser light scattering methods
allow determining equivalent diameters such as area diameter, volume diameter,
and volume/area diameter. The equivalent diameter usually calculated in the
dynamic light scattering is the hydrodynamic sphere (Amrite et al. 2006).
Fig. 10.5 Microphotograph of PLGA microspheres unloaded (a) and loaded (b) with vitamin E,
prepared according to the O/W emulsion technique. Vitamin E can be used as additive and as
antioxidant in the PLGA microspheres. Microspheres can contain more than one active substance
(“combo” microparticles)
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Fig. 10.6 Thermograms obtained from PLGA 50:50 (1) and acyclovir (2) as raw materials.
Thermograms from PLGA microspheres loaded with acyclovir before (3) and after (4) sterilization. Adapted from Martínez-Sancho et al. (2004)
10.3.3 Infrared Absorption Spectrophotometry (IR)
IR spectra are recorded on an infrared absorption (IR) spectrophotometer. Scans
of samples are evaluated at a determined resolution over a wave number region.
The absorption bands in a particular region allow characterizing the polymer and
the encapsulated drug.
10.3.4 Differential Scanning Calorimetry (DSC)
DSC is useful to determine physicochemical interactions between the polymer and
the active substance before and after the microencapsulation procedure. Scans are
obtained under established heating conditions and heating rate. Glass transition
temperatures (Tg) and crystalline melting points (Tm) are identified. Crystalline
substances present Tm while amorphous ones display Tg. If an interaction of a
crystalline drug and the polymer exists the Tm decreased with respect to drug raw
material (Fig. 10.6).
10.3.5 X-Ray Diffraction
X-Ray diffraction allows studying the physical structure of the active substance and
polymer separately and in the microspheres (Fig. 10.7).
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Fig. 10.7 RX diffractograms from PLGA 50:50 (a); acyclovir (b) and microspheres loaded with
acyclovir (c). Adapted from Martínez-Sancho et al. (2004)
10.3.6 Gel Permeation Chromatography (GPC)
This technique is used to determine changes in molecular weight of polymer due to
the microencapsulation procedure and in samples of particles at different stages of
the in
vitro release or upon exposure to gamma-irradiation.
Molecular weights are expressed as weight-average molecular weight (Mw) and
number-average molecular weight (Mn) (Martinez-Sancho et
al. 2004).
10.3.7 Determination of Drug Loading Efficiency
Microencapsulation efficiency is calculated as the ratio of the actual active substance content in the microparticles over the theoretical drug loading. Generally, the
amount of encapsulated drug depends on the physicochemical properties of the
polymer and the drug as well as on the technical procedure employed for microencapsulation. The loading of the active substance is usually expressed as amount of
drug (mg) per mg of microspheres.
When microparticles are intended for the administration of a drug in a relatively
isolated area as it is the case of the back of the eye, the amount of injected polymer
must be as low as possible and higher encapsulation efficiencies are preferred.
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10.3.8 “In Vitro” Release Studies
Drug release studies are critical in the development of drug delivery systems and
allow calculating the amount of microparticles to be injected. Release rate assays
are carried out in “sink conditions” to avoid solubility problems that can affect the
drug release rate behavior. Particles can be suspended in the release medium directly
or separated by a cellophane membrane. In the latter case, larger volumes of attack
medium are employed.
Generally, microparticles are suspended in a volume of an aqueous solvent and
placed in a shaker bath with constant agitation and at 37°C. At fixed time intervals,
the supernatant is removed and drug concentration is quantified. The same volume
of fresh medium is replaced to continue the release study (Herrero-Vanrell and
Refojo 2001). The attack medium can contain Tween 80 (0.02%) and/or sodium
azide (0.05%) (Checa-Casalengua et al. 2011).
Release rate of active substances from microparticles involves several mechanisms: diffusion through the polymer matrix and/or through the fluid-filled
pores present in the particles, physical erosion and/or hydrolysis of the polymer,
ion exchange, or several of these mechanisms (Li 1999; Herrero-Vanrell and
Refojo 2001).
The release kinetic of the drug is a function of the polymer structure, molecular
weight and rate of degradation in the case of bioerodible polymers, the physicochemical properties of the drug (mainly solubility and molecular size), drug loading,
size of the particles, and the microencapsulation technique. Usually, the encapsulated drug is released according to a first order kinetic in which the release rate is a
function of the concentration of the substrate. Low molecular weight biodegradable
polymers release the encapsulated drug faster than high molecular weight ones. In
its turn, higher size microparticles, with lower surface area, release the active substance slower than low size particles (Fig. 10.8).
10.3.8.1 Additives in Microspheres
Drug release profile from microparticles can be modulated to some extent by modifying their components. If the solvent evaporation technique is used to prepare
microspheres, additives can be added to the inner or the external phase of the emulsion (Herrero-Vanrell and Refojo 2001; Martinez-Sancho et al. 2003a, b; Herrero-
emulsion it remains in the microparticles. In such cases the additive must be biocompatible and biodegradable (Barcia et
drug encapsulation efficiencies and longer release rate compared with the microspheres without additive (Martinez Sancho et al. 2003a) (Fig. 10.9).
al. 2005). Additives can promote higher
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