Ind. Eng. Chem. Res. 2001, 40, 933-948
933
In Vitro Sustained Release of Human Immunoglobulin G from Biodegradable Microspheres Hsin Min Wong, Jian Jun Wang, and Chi-Hwa Wang* Department of Chemical & Environmental Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576
The in vitro controlled release kinetics of human immunoglobulin G (IgG) of different loadings from microspheres of the biodegradable polymers poly(L-lactide) (PLA) and poly(D,L-lactide-coglycolide) (PLGA) were investigated. The microspheres were prepared by a double-emulsion technique. The release profiles exhibited an initial burst followed by a period of slow release, with PLA microspheres showing a faster rate of release than PLGA microspheres. The release rate increased with an increase in drug loading. Scanning electron microscopy (SEM) observations revealed wide differences in the morphology of microspheres made from different polymers. Drug loading had no significant effect on the morphology of the microspheres. Laser scanning confocal micrographs demonstrated a homogeneous drug distribution within the microspheres. Results from SEM and mass loss studies revealed no significant extent of polymer erosion after 50 days of release. Modeling studies within the first 50 days of incubation suggested that the mechanisms of drug release were mainly diffusion- and dissolution-controlled. 1. Introduction The delivery of therapeutic peptides and proteins has become an important area of research since the feasibility of slow and continuous release of macromolecular bioactive agents was demonstrated.1 Rapid and innovative progress in analytical and biological technology, especially cell and gene technology, has facilitated the successive discovery and mass production of therapeutic peptides and proteins such as hormones, cytokines, regulatory factors, growth factors, and monoclonal antibodies. Currently, large numbers of therapeutic peptides and proteins are used for medical therapy, and drug delivery systems for these drugs using biodegradable polymers are intensively investigated to achieve optimal efficiency and to increase patient compliance. The protein drug investigated in this work is the antibody human immunoglobulin G (IgG). Human immunoglobulin G (IgG) is produced by the body as “on-line defense” against foreign cells such as bacteria and viruses. The molecular weight of this protein is about 150 000. IgG is normally used in replacement therapy for patients suffering from congenital or acquired hypogammaglobulinemia. It is also used in the treatment of secondary IgG deficiencies that are common in patients with cancer, nephropathies, gastrointestinal disturbances, and burns.2 Recently, IgG therapy has been shown to improve relapsing remitting multiple sclerosis3 and to be clinically beneficial in a variety of immune disorders associated with pregnancy.4 Controlled delivery systems are attractive compared to the classical methods of intravenous and intramuscular administrations because of the localized delivery of the drug; the decrease in treatment frequency, which reduces costs; the improvement in patient compliance; and the reduced dose handling, leading to a reduction in side effects. Biodegradable polymer microspheres of polylactide (PLA) and polylactide-co-glycolide (PLGA) have been * Corresponding author. Telephone: 65-8745079. Fax: 65-7791936. E-mail:
[email protected].
investigated extensively for controlled release applications. PLGA and PLA are shown to be biocompatible in physiological environments and to degrade to toxicologically acceptable products that are eventually eliminated from the body.5 They are also among the few synthetic polymers approved for human clinical use,6 and they are commercially available. Microparticles containing therapeutic agents have been configured as microspheres, microcapsules, and liposomes. Microparticles are solid spherical particles ranging from 1 to 1000 µm in size7 and can be administered in a fluidized form with a liquid carrier. This permits their use in intravascular infusion preparations8 and injectable emulsions for both parenteral and enteric administration,9 as well as vaccine dosage forms for use in subcutaneous or intramuscular injection.10 In this work, the sustained release of IgG from microspheres of PLGA and PLA polymers is studied. Drug release profiles can be affected by many factors, including fabrication methods, polymer molecular mass, copolymer ratio, properties of the drug, drug loading, matrix size, and geometry. The main objectives of this study are to design and characterize IgG-loaded PLGA and PLA microspheres fabricated by a W/O/W doubleemulsion technique and investigate the release kinetics of IgG from these microspheres. Specifically, we investigated the effect of the drug content and polymer type and composition on the morphological characteristics of PLGA and PLA microspheres and the release profiles of IgG from these microspheres. The determination of IgG content in the in vitro release study was based on perfusion immunoassay using a high-performance liquid chromatography (HPLC) system. Several techniques were employed for both quantitative and qualitative study of the structure and characteristics of the microspheres. Some of these techniques include the laser diffraction method and the Brunauer-Emmett-Teller (BET) method for quantitative analysis and laser scattering confocal microscopy and scanning electron microscopy for qualitative characterization. Morphological
10.1021/ie0006256 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/12/2001
934
Ind. Eng. Chem. Res., Vol. 40, No. 3, 2001
changes of the microspheres in the course of the release were studied in an attempt to elucidate the release mechanism. The process of drug release often occurs by several, often simultaneous, mechanisms. The main mechanisms that have been assumed for drug release from PLA and PLGA microspheres are diffusion through matrix pores, solid drug dissolution, swelling of the polymer network, and surface or bulk erosion of the polymer matrix.11,12 PLA and PLGA systems undergo a hydration process followed by bulk erosion in an aqueous system.13 Erosion only partly represents a mechanism of drug release. Its other effect is the modification of particle morphology, which subsequently affects diffusional release. The effect of the swelling of the PLA microspheres can be assumed to be negligible, as the water uptake by the polymer was less than 10%.14 Observations from the SEM pictures of IgG-loaded microspheres and data obtained from the mass loss study after 7 weeks of in vitro release verified that the microspheres had not experienced a significant extent of erosion ( 0 C ) C∞ r ) R
(4)
t>0 and
where C∞ is the concentration needed to maintain equilibrium with the surroundings. This value can be zero is some cases. The solution23,25 is given by
936
Ind. Eng. Chem. Res., Vol. 40, No. 3, 2001
Mt MT
)1-
6
∞
∑
1
π2 n)1n2
(
)
n2π2 Dt R2
exp -
where Mt represents the cumulative amount of drug released at time t and MT represents the respective amount after a long time T. The second case considers a finite coefficient of mass transfer on the surface and assumes that the concentration in the surroundings is constant. The boundary conditions are described by eqs 3 and 6
∂C t > 0 -D ) h(Cs - C∞) r ) R ∂r
( )
βn2 6S exp - Dt )12 2 2 MT n)1 β (β + S S) R2 n n ∞
∑
2
(7)
(
)
(14)
(
)
∂2ψ 2 ∂ψ ∂ψ ) + - Diψ ∂τ ∂ξ2 ξ ∂ξ
(17)
∂ψ )0 ξ)0 ∂ξ
(18)
τ>0 ψ)1 ξ)1
(19)
τ>0
and
τ>0
∂ψ ) Sψ ξ ) 1 ∂ξ
(20)
respectively. The solution to case 1, for which release is under perfect sink conditions (infinite mass transfer coefficient on the surface), is given in the form of the cumulative dimensionless quantity of drug release at time t, Ψt.15
(10)
Just as in the first model, two cases in which the mass transfer coefficient on the microsphere surface is either infinite or finite will be considered. The boundary conditions for these two cases are identical to those given for the first model. Equation 10 can be transformed into a dimensionless form by defining the following dimensionless parameters:
(16)
while the initial and boundary conditions in eqs 11, 3, 4, and 6 become
Ψt )
(11)
(15)
This number expresses the relative importance of the dissolution and diffusion terms in relation to the overall release process. With the use of the dimensionless parameters given by eqs 12-15, eq 10 is transformed into
(9)
In this equation, k represents a first-order dissolution constant (in units of time-1), ∈ is the porosity of the microspheres, and Csat denotes the saturated concentration of the drug in the system. Hence, ∈Csat represents the equivalent drug saturation concentration in the pores. The notations for the parameters C, D, t, and r are the same as those given for the first model. The initial condition is given by
t ) 0 C ) ∈Csat 0 < r < R
ψ ) 1 - C/∈Csat
Di ) kR2/D
(8)
Diffusion/Dissolution Model. Drug dissolution cannot be the only mechanism by which a drug is transported out of the microsphere. This transport occurs by subsequent drug diffusion. Hence, a common approach to the characterization of the dissolution effect is to expand on the diffusion equation. The diffusion/dissolution model incorporates a linear first-order dissolution term into the Fickian diffusion equation.15
∂2C 2 ∂C ∂C )D 2 + + k(∈Csat - C) ∂t r ∂r ∂r
(13)
τ)0 ψ)0 0
