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An overview of preparation and evaluation sustained-release injectable

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Article in Journal of Microencapsulation · November 2012

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 Journal of Microencapsulation, 2013; 30(4): 369–382
ß 2013 Informa UK Ltd.
ISSN 0265-2048 print/ISSN 1464-5246 online
DOI: 10.3109/02652048.2012.742158

REVIEW

An overview of preparation and evaluation sustained-release

injectable microspheres
Liandong Hu1,2, Hailei Zhang1,2 and Weihua Song1,2
1
College of Pharmaceutical Sciences, Hebei University, No. 180, Wusi Road, Baoding 071002, P.R. China, and
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13

2
Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Hebei University, Baoding 071002, P.R. China

Abstract
Recently, sustained-release injectable microspheres as a novel parenteral administration system have been
interested on for many years, due to the excellent advantages when compared to traditional dosage forms:
less administration frequency, lower adverse side effects and no need for a surgical procedure. Therefore,
major progresses in the development of another successful marketed sustained-release injectable forma-
tion have been made, but most investigations are merely limited in laboratory levels; in addition, few
reports focus on giving some positive guidance to launch these novel microspheres into market. This
review addressed some commonly used polymers, preparation methods and sterilization processes relating
to biodegradable microspheres. Moreover, the processes for measuring the sustained-release behaviour of
this novel system are summarized in this report, including the methods to determine the in vitro and in vivo
For personal use only.

release behaviours and the strategies to analyse the in vitro and in vivo correlations.
Keywords: sustained-release injectable microspheres, evaluation, preparation, in vitro, in vivo

Introduction degradation of complex gastrointestinal circumvent.

In recent years, there has been a great deal of interest in
developing sustained-release systems, which can prolong
the therapeutic effect, decrease adverse side effects and
reduce administration frequency (Shi and Li, 2005).
Nowadays, the oral sustained-release preparations account
for a larger market share comparing to other sustained-
release forms, mainly because of its noninvasive pain-free
administration. However, the hostile environment of gas-
trointestinal tract and first-pass effect may lead to low bio-
availability of some drugs, such as some proteins, peptides
and hormones (Sinha and Trehan, 2003; Motulsky et al.,
2005). Moreover, some other noninvasive pain-free deliv-
ery approaches, such as transdermal, pulmonary and nasal
drug delivery, are not appropriate for such macromolecular
drugs, mainly because of the low biological membrane
permeability and instability in the delivering environment
(Shi and Li, 2005).
Nowadays, using the parenteral route is the most
common way to avoid the first-pass effect and the
Some formations developed in early years, such as emul-
sions and suspensions are found to have numerous disad-
vantages: nonadjustable release behaviour and instability
on long-term storage. Traditional implants are sometimes
employed as sustained-release preparations due to low sys-
temic levels and toxic effects, but the need for a surgical
procedure makes it more unacceptable for patients.
However, the sustained-release injectable microspheres
possess all over advantages and can overcome the above-
mentioned problems (Sinha and Trehan, 2005). 13
Microspheres are small spherical particles, with diame-
20
ters in the micrometer level. The diameter size to a lesser
degree than 250 mm, ideally 125 mm is suitable for paren-
teral delivery (Jain, 2000). The target activity is the signifi-
cant characteristic differing from traditional dosage forms,
leading to higher bioavailability and lower toxicity. Smaller
microspheres (less than 10 mm) are demonstrated to have
targeted activity via their uptake by macrophages and den-
dritic cells (Singh and O’Hagan, 1999), and their localiza-
tion in lymph nodes (Men et al., 1997; Partidos et al., 1997).

Address for correspondence: Liandong Hu, College of Pharmaceutical Sciences, Hebei University, No. 180, Wusi Road, Baoding 071002, P.R. China.
Tel: þ86 013463689875. Fax: þ86 312 5971107. E-mail: hbupharm@126.com

(Received 2 Jun 2012; accepted 10 Oct 2012)

http://www.informahealthcare.com/mnc
369
 370 L. Hu et al.

Table 1. List of some currently marketed sustained-release injectable microspheres.

Trade name Main drug Company Year of FDA Patent Polymer Production methods
approval number

Lupron Depot Leuprolide acetate Takeda 1985 US5480656 PLGA Emulsion method
Risperdal Consta Risperidone Janssen 1997 US5650173 PLGA Phase separation method
Sandostatin LAR Octreotide acetate Novartis 1998 US5639480 PLGA Emulsion method
Nutropin Depot Growth hormone Genentech 1999 US5213810 PLGA Emulsion method
Trelstar Depot Triptorelin pamoate Watson 2000 US5134122 PLGA Spray drying method
Vivitrol Naltrexone Alkermes 2006 US6306425 PLA Emulsion method
Somatuline Depot Lanreotide acetate Ipsen 2007 US6475507 PLGA Spray drying method
Exenetide LAR Exenetide Amylin/Eli Lilly/Alkermes 2012 US2008146490 PLGA Emulsion method

Table 2. Some detailed comparisons on the marketed sustained-release injectable microspheres.

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Trade name Suitable molecular weights of polymers Suitable L/G Size of particles Weight of active substance (w/w)

Lupron Depot 14 100–18 200 Da 80/10–100/0 1–300 mm 1.5–2.5%

Risperdal Consta About 150 000 Da 50/50–85/15 –* 35–45%
Sandostatin LAR 35 000–60 000 Da 40/60–60/40 1–250 mm 2–8% 1/16
Nutropin Depot 6000–31 000 Da 50/50 1–180–mm 15%
Trelstar Depot –* 45/55–90/10 200 mm 0.1–15%
Vivitrol –* –y 20–100 mm 15–25%
Somatuline Depot –* 75/25 –* 15–25%
Exenetide LAR –* 50/50 –* 0.5–5%

Note: *Information cannot be found and ynot applicable for PLA.

For personal use only.

The first market product (DecapeptylÕ ) of sustained-
release injectable microspheres was developed by Ipsen
in 1986. DecapeptylÕ is a gonadotropin-releasing hormone
agonist, which can decrease pituitary secretion of gonado-
tropins luteinizing hormone and follicle stimulating
hormone. Nowadays, various FDA-approved sustained-
release injectable microspheres are available in the
market (Table 1); in addition, some detailed comparisons
are also introduced in Table 2. Some of the products pos-
sess excellent sustained-release effects, which can main-
tain therapeutic effects for several months in one dose.
Materials
A key factor in designing sustained-release injectable
microspheres is the choice of an appropriate biodegradable
polymer. Generally speaking, the materials employed for
injectable microspheres are divided into two major
groups: natural polymers and synthetic polymers. The
investigations of natural polymers for sustained-release
injectable microspheres have focused on proteins (e.g.
albumin (Porta et al., 2010), gelatin (Habraken et al.,
2009) and collagen (Nagai et al., 2010)) and polysaccha-
rides (e.g. starch (Björses et al., 2010), dextran
(Mohaghegh and Tafaghodi, 2011), cellulose (Shi et al.,
2009) and sodium alginate (Grellier et al., 2009)). The nat-
ural polymers exhibit their advantages in controlling
release behaviours and improving target activities.
However, their applications are still limited by large-scale
commercial manufacturing and difficulties in purification
(Sinha and Trehan, 2003), and the complexity and
variability result in difficulties for defining the standards
of natural polymers.
Some generally used synthetic polymers are listed in
Table 3. Among the above-mentioned synthetic polymers,
polylactic-co-glycolic acid (PLGA) and polylactic acid
(PLA) are used in a host of FDA approved therapeutic
devices and have been widely used in medical and phar-
maceutical fields (Anderson and Shive, 1997; Jain, 2000).
PLGA is composed of one or more of three different
hydroxyl acid monomers (d-lactic, l-lactic and/or glycolic
acids). In general, the polymer can be made to be highly
crystalline or completely amorphous and encapsulate mol-
ecules of virtually any size through changing the monomer
ratio, molecular mass and end-group chemistry (Jiang
et al., 2005). The above-mentioned three parameters can
determine the hydrophobicity and degradation kinetics of
the materials, and thereby, the microencapsulation effi-
ciency, release rate and interactions of the microspheres
with phagocytizing cells are also influenced (Tamber
et al., 2005). For example, increase in polymer molecular
mass or higher lactic acid content can result in longer deg-
radation times and slower release (Jiang et al., 2005).
Molecular mass and monomer ratio can be served as two
major adjustable factors to achieve an expected sustained-
release behaviour (Hutchinson, 1982). The molecular mass
of PLGA often used in microsphere preparation is ranged
from 10 to 100 kDa, and the commonly used ratios of lactic
to glycolic focus on 50:50 up to 100:0.
When used for injectable microspheres, PLGA is
degraded into oligomers and monomers through the
hydrolytic scission of the ester bonds in first stage. In this
period, a large amount of lactic and glycolic acids are pro-
duced resulting in a significant decline of pH values.
 Preparation and evaluation sustained-release injectable microspheres
Table 3. The main types of commonly used synthetic polymers for the sustained-release injectable microspheres.
Class
Polyesters
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Polyorthoesters
Polyanhydrides
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Polyamides
Polyalkylcyanoacrylates
Pseudo-polyamino acids
Polyphosphazenes
Therefore, most drugs used with PLGA in injectable micro-
spheres are relatively stable in acidic media. In the second
stage, the microspheres lose mass and the rate of polymer
chain scission may increase (Fu et al., 2000). Some
researchers summarize the two stages as degradation and
erosion (Tamber et al., 2005). The produced lactic and gly-
colic acids are metabolized in the Krebs cycle to CO2 and
water ultimately (Yoo et al., 2005). Nowadays, PLGA micro-
spheres have a long safety record, and some sustained-
release products are able to control the release of drugs
slowly and continuously from one to four months.
Despite the excellent biocompatibility and widespread
application of PLGA, some mild inflammatory responses
are reported by some researchers (Gupta et al., 1997;
371
General structures
Hermeling et al., 2004). PLA is investigated as a drug deliv-
ery biodegradable material as early as 1971 and applied in
microsphere research works in early years (Brannon-
Peppas, 1995). However, PLA is not able to be appropriate
for encapsulating molecules of virtually any size drugs
through changing self-characters like PLGA, so the PLA
has a limitation in developing modern sustained-release
injectable microspheres.
Due to the need for better delivery formulations, various
types of copolymers have been developed. For example,
PLGA/PEG (poly ethylene glycol) block copolymers have
been processed as diblock (PLGA–PEG) or triblock mole-
cules with PLGA–PEG–PLGA and PEG–PLGA–PEG types
(Jeong et al., 2000; Cheng et al., 2007; Makadia and
 372 L. Hu et al.
Siegel, 2011). Better release kinetics from formulations of
copolymers and the stability of the preparations have been
demonstrated in comparison to PLGA alone, but the addi-
tion of PEG often leads to a reduction of the encapsulation
efficiency. In addition, the triblock copolymers of both
PLGA–PEG–PLGA and PEG–PLGA–PEG types can also act
as thermogels (Makadia and Siegel, 2011).
Recently, inorganic artificial biomaterials such as bio-
ceramics and metallic biomaterials have attracted a lot
attention for medical applications. Some kinds of glass
microspheres and ferrimagnetic ceramic particles have
exhibited great potential in treatment of cancer through
via in situ radiotherapy or hyperthermia of cancer. In
1980s, Day et al. entrapped the Y2O3–Al2O3–SiO2 glass
microspheres into capillary bed of the tumours and the
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90
Y contained in these microspheres could irradiate
tumour tissue locally by emitting -rays with a half-life of
64.1 h (Cianni et al., 2010). In this technique, the invasion
of radiotherapy was significantly decreased. In 1990s,
Kawashita et al. (1999) developed 32P-containing SiO2
glass microspheres for radiotherapy of cancer by implant-
ing Pþ ions into microspheres at high energy. In this
research, colloid particles were replaced by red phospho-
rus colloid particles, and the sustained-release effects were
significantly improved. Moreover, Kawashita et al. (2003)
also prepared YPO4 microspheres composed of crystalline
For personal use only.
YPO4, which exhibit better effects than Y2O3–Al2O3–SiO2
glass microspheres.
Research in the past several years has indicated that the
ferromagnetic microspheres possessed significant superi-
orities for embolic hyperthermia treatment of cancer.
Fe3O4 and -Fe2O3 was frequently used as ferromagnetic
material, such as Fe3O4–SiO2 microspheres (Deng et al.,
2008), Fe3O4–TiO2 microspheres (Li et al., 2008), Fe3O4–
C–Au microspheres (Qi et al., 2010) and the microspheres
composed of small crystals of -Fe2O3 (Kawashita et al.,
2008).
In addition, some investigators also developed some
kinds of microspheres containing different species of mate-
rials. For example, Habraken et al. (2006) developed an
injectable PLGA microsphere/calcium phosphate (CaP)
cement system with sufficient setting/cohesive properties.
Results showed that all physical parameters were well in
range of 10:90–20:80 (PLGA microsphere: CaP cements;
Habraken et al., 2006). Despite many other similar reports
about new type materials, there is a long way to go in devel-
oping another classical material like PLGA and PLA.
Preparation methods
Emulsion methods
The emulsion methods have been widely used because of
the convenience in controlling process parameters, and
there is no need for producing with expensive and complex
instrument. This process is mainly divided into two major
parts: emulsification and removal procedure; in addition, a
brief flow diagram is described in Figure 1. In the first stage,
the emulsion or multiple emulsions are formed in presence
of a suitable emulsifier following the addition of the drug
into polymer solution. The oil-in-water (O/W) method is
the mostly used single emulsion method in preparing
microspheres with water-insoluble drugs (Jalil and Nixon,
1990), and water-in-oil-in-water method is widespread in
multiple emulsions for preparing microsphere with water-
soluble drugs like peptides, proteins and vaccines (Genta
et al., 2001; Rosas et al., 2001). In addition to the two major
methods, some special emulsion methods have also been
employed for microsphere preparation. For example, the
oil-in-oil emulsification method has been developed to
encapsulating water-soluble drugs in the single emulsion
method (Sánchez et al., 2003); solid-in-oil-in-water, solid-
in-oil-in-oil and even solid-in-oil-in-oil-in-water emulsifi-
cation methods have also been developed to avoid dena-
turation in protein drugs (Tobı́o et al., 1999; Wang et al.,
2004; Yuan et al., 2009).
In the second stage, extraction or evaporation is
employed to remove organic solvent and harden the drop-
lets followed by the particles are harvested, washed and
dried or freezing-dried (Freitas et al., 2005). When extrac-
tion is used, the emulsion is transferred to a large quantity
quench medium, and then the solvent associated with the
oil droplets is diffused out. In evaporation process, emul-
sion is exposed to a large amount of water or cosolvent,
often under reduced pressure and increased temperature.
In emulsion methods, the technology can be optimized by
changing a number of factors in process, including: (a) the
organic solvent, emulsifier and polymer used; (b) the drug/
polymer ratio; and (c) the parameters in emulsification
process and extraction or evaporation process. Although
a number of hydrophilic drugs have been successfully
encapsulated by this method, some disadvantages in emul-
sion methods, such as consuming long time, lower loading
capacity, poor encapsulation efficiency and large initial
burst release properties, cannot be ignored still.
Phase separation
The phase separation method is also called coacervation,
and a brief flow diagram is shown in Figure 2. When com-
pared to emulsion methods, the requirement of solvents for
the polymer is less stringent in concentration and this pro-
cess is more suitable for encapsulating water-soluble drugs
(Andrianov et al., 1998; Lim et al., 2000). In this process, the
water-soluble drugs are dissolved in water and then added
into an organic solution with the polymer to form W/O
emulsions, and water-insoluble drugs can be solubilized
or dispersed in the polymer solution. An organic nonsol-
vent is then added into the system to achieve the phase
separation, followed by the polymer solvent is gradually
extracted from the coacervation phase. This organic non-
solvent should be miscible with the polymer solvent and
immiscible with the polymer or the drug, such as vegetable
oils and low molecular weight liquid polybutadiene. As a
result, the soft coacervate droplets are formed to entrap the
drug. The two-phase system is then transferred into a large
 Preparation and evaluation sustained-release injectable microspheres
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For personal use only.
Figure 1. The brief flow diagram of emulsion methods.
volume of an organic hardening agent (different from the
former organic nonsolvent) to harden the droplets and
achieve the final microspheres. The second nonsolvent
should be volatile and easy to wash the first nonsolvent,
such as hexane and heptane.
Several kinetic parameters, such as the phase/organic
phase volume ratio, stirring rate for the drug dispersion in
polymer solution, the addition rate of organic nonsolvent
and the used polymer and nonsolvents, are reported to
have influence on some aspects of the microencapsulation
process. However, due to the absence of emulsion stabi-
lizers in this process, agglomeration is a common problem
in this method (Jain, 2000).
373
Spray drying method
The two methods mentioned above both have fatal weak-
ness in large-scale production. Large quantities of organic
solvent are needed in these methods, and it is difficult to
achieve a favourable encapsulation efficacy. Contrary to
these methods, the spray drying method is relatively
simple and appropriate to large-scale production, involv-
ing mild conditions. Moreover, it has less demand on the
solubility parameter of drug, polymer and solvents
(Giunchedi et al., 2001; Vehring, 2008). In this process,
the polymer is first dissolved in a volatile organic solvent,
and then the drug is dispersed in polymer solution usually
 374 L. Hu et al.
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For personal use only.
Figure 2. The brief flow diagram of the phase separation method.
with high speed homogenization. The dispersion or emul-
sion is atomized in a flow of drying air to vaporize the
organic solvent, followed by the hardened microspheres
that are collected in a deposition chamber. Some types of
PLGA microspheres are successfully prepared through this
method (Mu and Feng, 2001; Gavini et al., 2004).
Some procedure parameters, such as the inlet temper-
ature, outlet temperature, aspirator setting and flow rate of
the pump, can affect the formation of microspheres.
Recently, there have been some improvements in the
spray drying method; for example, a novel double-nozzle
spray-drying technique has been developed to solve the
problem of the adhesion of the microspheres to the
inside wall of the spray-drier apparatus. A major problem
still existing in this method is that the formation of fibres
may lead to difficulties in breaking up the polymer
solution.
Supercritical fluid method
In recent years, the supercritical fluid (SCF) has received
increased attention as an alternative approach to prepare
microspheres. Supercritical carbon dioxide (SCO2) is the
Table 4. Different classifications of the SCF processes when used for
preparing sustained-release injectable microspheres.
Classification Process
Solvent Rapid expansion of supercritical solution
Anti-solvent SCF anti-solvent
Gas anti-solvent
Precipitation with a compressed anti-solvent
Solution enhanced dispersion by SCFs
Solute Particle from gas saturated
solution or suspension
Reaction medium Synthesis of coating material
within supercritical solution
mostly used fluid, mainly due to its mild critical conditions
(TC ¼ 304.1 K, PC ¼ 7.38 MPa; Bahrami and Ranjbarian,
2007). In addition, it is also nontoxic, not flammable, recy-
clable and economical (He et al., 2010; Du et al., 2011).
Previous research works have studied the effect of phase
behaviour on the formation of microspheres in three kinds
of processes, in which SCF acts as solvent, anti-solvent or
solute (Table 4; Bahrami and Ranjbarian, 2007).
The rapid expansion of supercritical solution is the first
SCF method (Tom and Debenedetti, 1991). In this method,
 Preparation and evaluation sustained-release injectable microspheres
the microspheres are formed as a result of a rapid expan-
sion of SCF solvent. However, this technique is mainly
applicable to small molecular weight drugs, because the
drug and the polymer both need sufficiently high solubility
in supercritical CO2.
In supercritical anti-solvent methods, the drug is first
dissolved in an organic solvent and the solution is then
sprayed into SCF. The quick mass transfer between SCFs
and solution reduces the solubility properties, followed by
the drug particles which can be achieved by precipitation
(Reverchon, 1999). In this technique, the microspheres can
be prepared to meet certain size, shape or porosity by vary-
ing necessary process parameters of the system. However,
some microspheres produced by anti-solvent methods are
puzzled by the problem of residual solvent sometimes.
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In addition, sometimes SCO2 can be used as a reaction
medium, in which the coating material synthesizes by a
chemical reaction and coats the surface of particles. This
technique is mainly used to prepare nanospheres.
The particles from gas saturated solutions production
process was a single-step process and has significant
advantages over conventional production methods, as it
requires neither drug particle nor polymer to dissolve in
SCO2, no organic solvents are needed, and the SCO2 is of
low consumption. In this method, SCO2 acts as a molecular
lubricant liquefying the polymer at temperatures signifi-
For personal use only.
cantly below its glass transition temperature. When
exposed to SCO2 in a pressure vessel, the polymers can
liquefy the main drug to be mixed efficiently into the poly-
mer. The mixture is then depressurized through a nozzle
whereby the CO2 evaporates solidifying the polymer
around the drug, resulting in the production of micro-
spheres (Jordan et al., 2010).
Others
With the development of technology, a number of new
methods and equipments are developed to prepare micro-
spheres. Here, the authors give a brief introduction of some
widely recognized novel methods: (a) the ultrasonic atom-
ization method, sometimes called ‘‘one-step’’ operation,
can produce microspheres in different sizes by changing
the nozzle diameter, vibration frequency and the flow rate
of the polymer. Therefore, the drug release behaviours can
also be controlled by these parameters (Fini et al., 2011).
(b) An electrospray system, including a liquid delivery
system (pump), a needle with high electric potential and
a grounded electrode, is employed for preparing micro-
spheres in various sizes by changing the potential differ-
ence of inner and outer needles and the flow rates
(Loscertales et al., 2002).
Sterilization
To enable the application of the preparations into human
or animal settings, the process of sterilization is of signifi-
cant importance. As most of the polymers used in
375
microspheres are heat-sensitive, moist or dry heat sterili-
zation is impossible. In addition, the microspheres are typ-
ically too large to pass through the 0.22 mm sterile filters
(Mohanan et al., 2012). Aseptic technique seems to be
the guarantee for sterility, but it brings significant complex-
ity and costs to large-scale manufacture. In recent years,
sterilization by gamma-irradiation has been widely used in
this field, especially the PLGA microspheres, which can
significantly reduce the cost and complexity in aseptic
technique. However, some reports indicate that the
gamma-irradiation can increase the risk in changing the
structure of both the drug and polymer, therefore,
the release kinetics and even the pharmaceutical effect
may be changed by the influence of gamma-irradiation
(Montanari et al., 1998; Jain et al., 2011). Take PLGA as
an example, the ionization phenomena caused by irradia-
tion can create free radicals and reactive oxygen species
from PLGA (Mohanan et al., 2012).
Several efforts have been made to reduce the probability
of ionizing radiation of microsphere preparations. Some
researchers demonstrated that the use of low temperatures
during the gamma-irradiation sterilization process can
maintain the nature properties of some low molecular
weight drugs in microspheres (Fernandez-Carballido
et al., 2004; Martinez-Sancho et al., 2004). However,
Zbikowska et al. (2006) verified that the low temperatures
were not the guarantee to protect the drug and polymer
from undesirable reactions.
The use of antioxidants has been proved to have effects
to remove the free radical intermediates or inhibit other
oxidation reactions produced by ionizing radiation
(Martinez-Sancho et al., 2004; Mohanan et al., 2012).
Furthermore, Schwach et al. (2003) demonstrated the
inclusion of the active agent in its solid form possessed
more stable properties in the process of gamma-irradiation
sterilization. Some researchers also reported that the use of
the three above-mentioned strategies could achieve a
better protective effect during the gamma-irradiation ster-
ilization process (Checa-Casalengua et al., 2012). However,
there are so many uncertain influences in the process of
gamma-irradiation exposure including the known and
unknown factors. At the present stage, no other strategies
can replace the gamma-irradiation sterilization except for
aseptic technique, which is one of the core problems to
restrict the development of the microsphere technique.
Evaluation of sustained-release behaviour
In vitro release
Besides the polymer characteristics, the in vitro release
behaviour is also dependent on dosage form characteris-
tics, such as particle size, morphology, porosity, residual
solvents and drug loading rate (Anderson and Shive,
1997; Ertl et al., 1999). The in vitro release of sustained-
release injectable microspheres usually exhibits a typical
triphasic profile: (a) an initial burst release; (b) a lag
phase and (c) an apparent zero-order or approximately
 376 L. Hu et al.

Table 5. Advantages and disadvantages of some apparatuses and methods used for sustained-release injectable microspheres.

Apparatus Advantages Disadvantages

Shaker Simple equipment and easy operation (a) Loss in volume by sampling
(b) Deposit of degradation products
(c) Aggregation

Centrifugation Simple equipment, easy operation and a small (a) Loss in volume by sampling
amount of medium (b) Difficulty in maintaining sink conditions
(c) Aggregation

Dialysis Convenience in sampling and media (a) Low in vivo predictability

replacement (b) Loss an accurate analysis of burst effect
(c) The outer media is hard to be agitated
(d) Aperture blocking

Paddle Traditional dissolution apparatus make the (a) Loss in volume by sampling
method more accessible (b) Evaporation of the media in a long assessment
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(c) Difficulty in changing media

Flow-through cell
(a) In situ monitoring at all times (a) Has not been widespread used
(b) Constantly circulating make it more rea- (b) Polymer degradation may lead to a clogging of the filter
sonable to simulate the in vivo environment
(c) A good IVIVC
(d) Minimum evaporation of the media

zero-order release phase (Zolnik and Burgess, 2007; Mitra described in Table 5; in addition, the advantages and dis-
and Wu, 2010; Rawat et al., 2011). The release kinetics from advantages of each method are also described.
biodegradable polymers are controlled via diffusion, poly-
(a) Shaken method: drug-loaded microspheres are sus-
mer erosion or a combination (Mitra and Wu, 2010).
pended into glass vials containing approximate
For personal use only.

The initial burst release of drug from the microsphere

release media followed by the vials shaken on set-
system can be defined as the quantity of drug that escapes
tled rotate speed and temperature. At preselected
from microspheres prior to the onset of polymer erosion-
time, right amounts of samples are withdrawn
mediated drug release. A modest initial burst release can
(Akhtar and Lewis, 1997).
be seen as an inherent property of diffusion-controlled
(b) Centrifugation method: drug-loaded microspheres
drug delivery systems, but an excessive drug release in
are placed in a small amount dissolution medium
the burst phase may be toxic and low robust (Allison,
in screw cap glasses rotated in a metal-block ther-
2008). Up to now, numerous efforts have been made to
mostat with settled rotating speed and tempera-
control burst release and the strategies are mainly divided
ture. At preselected time, right amounts of
into three groups. The first is improving the compatibility
samples are withdrawn (Kissel et al., 1996).
between the drug and polymer, such as changing the salt
(c) Dialysis: drug-loaded microspheres are physically
form of the drug (Zeng et al., 2005), complexation of drug
separated from the media through a dialysis mem-
molecules (Choi and Park, 2000), using cosolvent systems
brane, and the release behaviour is measured by
and using excipients to modify the release behaviour of
tracking the drug-concentration from the outer
drugs (Choi and Park, 2006; Kang et al., 2007). Second,
bulk over time (Kostanski and DeLuca, 2000).
the processing methods and microsphere formulations
(d) Paddle method: a traditional dissolution system
can be altered or optimized to prevent the escape of the
included in USP as an apparatus II (Jameela et al.,
drug (Mao et al., 2007). The third is reducing the drug dif-
1998).
fusion into the solvent extraction liquid (Costantino et al.,
(e) Flow-through cell method: the system consists of a
2004).
reservoir, a pump and flow-through cells. Drug-
Due to the need for a standard in vitro release method,
loaded microspheres are contained in media to
some regulatory authorities and academia introduced
achieve a circulation through a column. The release
some methods to analyse the in vitro release behaviours.
behaviour can be measured by determining the
Some important factors should be considered during
concentration in cells without sampling (Rawat
developing an in vitro release method. These include:
et al., 2011). The authors suggest that the flow-
(a) the dissolution environment should be physiological
through cell method is more suitable to assess the
relevance and in sink conditions; (b) the release method
sustained-release injectable microspheres due to
should be robust enough to be able to assess the in vitro
the attractive advantages, and this method also
release behaviour both in laboratory scale and manufactur-
exhibits huge potentials in establishing quality con-
ing process; (c) the method should be able to predict initial
trol specifications.
burst effect and (d) the measured in vitro release profile
should have a good correlation with in vivo behaviour. Even though an appropriate apparatus has been
Some commonly used apparatuses and methods are selected with a good in vitro and in vivo correlation
 Preparation and evaluation sustained-release injectable microspheres 377

(IVIVC), there is another problem needed to be focused on, increased temperature is based on the stability of the
that is some sustained-release injectable microspheres can drug and the glass transition temperature of the polymer.
provide continuous release for up to several months
in vivo. Therefore, the in vitro release behaviour should
also be tracked as long as several months, in traditional
In vivo release
views and practices, which can bring huge difficulties to
select formulations and establish standards. Recently,
The in vivo assessment consists of not only the release
some researchers have focused on developing accelerated
behaviour, but also some other factors, such as biocompat-
in vitro release test methods aiming to shorten the con-
ibility, undue toxicity and inflammatory response. Three
sumed time and keep a good correlation to normal
stage tissue responses are usually included in vivo:
in vitro or in vivo release behaviours. It is desired that the
(a) local inflammation; (b) thin fibre wall is formed in the
mechanism of drug release should be changed in acceler-
interface between microspheres and organization and (c)
ated release testing so that one-to-one correlations can be
phagocytosis emerges when the microspheres degrade into
established between accelerated and real-time release pro-
certain size (Ali et al., 1994).
files. Therefore, excessively extreme conditions and the
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13

The in vivo releases usually display different behaviours

methods involving the use of organic solvents are not pre-
from in vitro, and the reason is easy to be explained. Some
ferred in these studies.
factors in vivo environment, such as enzymes and lipids,
In general, accelerated in vitro release from PLGA
are nearly impossible to be imitated in vitro assessment.
microspheres can be achieved by increasing PLGA degra-
The absence of the lag phase often emerges in vivo release
dation at elevated temperature or extreme pH conditions
profiles and the in vivo release profiles usually consisted of
(Zolnik et al., 2006; Zolnik and Burgess, 2007). The estab-
two major parts: an initial burst and an apparent zero-
lishment of the accelerated in vitro release method should
order release phase. Some researchers attributed the
be based on the stability of the drug. Rawat et al. (2011)
absence of the lag phase to different erosion behaviours
developed an accelerated in vitro release method for
between in vitro and in vivo release behaviours. The enzy-
RisperdalÕ ConstaÕ using USP apparatus IV. About
matic degradation can lead to an ‘‘outside in’’ erosion dif-
250 mL of 0.05 M phosphate buffer saline (PBS) pH 7.4
ferent from the ‘‘inside out’’ erosion occurring in vitro.
For personal use only.

with 0.1% sodium azide was circulated through the flow-

Moreover, lipids may also lead to an accelerate degrada-
through cells. In this accelerated in vitro release method,
tion. Some researchers divided the factors that affect the
the temperature was increased from 37 C to 45 C and the
in vivo release into two major parts: delivery system inde-
overall duration of the in vitro drug release from the micro-
pendent and delivery system dependent. The former fac-
spheres was decreased from about 40 to 7 days. The corre-
tors include: barriers to drug diffusion, drug partitioning at
lation of real-time and accelerated release profiles was
the site and the fluid volume available at the site; the latter
evaluated by time scaling (scaling factor: 6.5) and linear
factors include: enzymolysis, protein adsorption and
regression. The results showed that the real time can be
phagocytosis (Mitra and Wu, 2010).
overlapped with the accelerated release profile on the same
Numerous animals, such as rats (Woo et al., 2002; Murty
axis, and the equation of linear regression was obtained
et al., 2004; Zolnik and Burgess, 2008; Rhee et al., 2011),
with a correlation coefficient of 0.9929. Zolnik and
rabbits (Jameela et al., 1998; Holger et al., 2011), dogs (Han
Burgess (2007) evaluated the effect of acidic pH on dexa-
et al., 2010), swine (Thompson et al., 1997) and humans
methasone/PLGA microsphere release. For the 25 K formu-
(Gefvert et al., 2005; Vlugt-Wensink et al., 2007), have been
lation, the overall duration of the in vitro drug release was
used reported in literature works for use in such in vivo
decreased from about 30 to 19 days when the pH value
studies; in addition, relating details in recent literature
dropped from 7.4 to 2.4; for the 70 K formulation, the
works are described in Table 6. Here, some special com-
time to reach 80% of drug release was reduced from 84 to
monalities are summarized as follows:
52 days. At pH 2.4, the mechanism of drug release (erosion
controlled) in accelerated release did not change from that (a) The microsphere powders are reconstituted with
under ‘‘real-time’’ conditions, and good linear relation- special sterile media to create a uniform suspension
ships are also established (Zolnik and Burgess, 2007). for subcutaneous or intramuscular injection, in
In recent research works about the investigations of order to prevent any sticking during injection.
accelerated in vitro drug release, the temperature-rising Usually, isoosmotic adjusting agent, anticoagulant
method is mostly used and presents a correlation with or other auxiliary materials are contained in disper-
real-time release profiles. Some researchers reported that sion media. In addition, some intravenous liquid
the morphology of degrading microspheres was signifi- microspheres have also been developed by some
cantly different at extreme pH conditions, compared to researchers to achieve a fast release effect (Gowda
the conditions at pH 7.4 (Zolnik and Burgess, 2007). et al., 2009), so these formulations are not intro-
Moreover, the temperature-rising method has a notable duced in this article.
superiority in shortening the test time. Therefore, the (b) In general, during the first day, there is frequent
authors regard the temperature-rising accelerated in vitro sampling to capture the burst release. Two or
release method as a potential method for quality control three days are often employed as time intervals in
and formulation development. Usually, the setting of the the following half a month, followed by the time
 378 L. Hu et al.
Table 6. Recent reports about operations in vivo release tests for sustained-release injectable microspheres.
Source Animal
Gefvert et al. (2005) Humans
Han et al. (2010) Beagle dogs
Jameela et al. (1998) New Zealand white male
rabbits
Murty et al. (2004) Sprague Dawley rats
Rhee et al. (2011) Rats
Thompson et al. (1997) Swines
Woo et al. (2002) Sprague Dawley rats
Vlugt-Wensink et al. (2007) Humans
Zolnik et al. (2006) Sprague Dawley rats
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13
intervals turn to be larger with the time increased.
Moreover, the sampling time intervals can be over a
month after several months, when the micro-
spheres with an excellent sustained-release effect.
The finish time mainly depends on the detection
limit of main drugs.
(c) Rats are most commonly used due to ease of han-
dling, and fewer resources needed to maintain rats
in a study. However, due to the limitation of the
For personal use only.
physical characteristics, rats are commonly used
to measure in vivo release behaviour in one
month and not appropriate to track a longer release
profile. Beagle dogs and rabbits are more suitable
to serve as model animals to measure in vivo
release profiles of microspheres with excellent sus-
tained-release effects.
(d) One-dose model is usually adopted for this formu-
lation, and the amount is often depended on
animal weight.
However, the target activity of microspheres can lead
to an unbalanced distribution in different organs,
together with the sustained-release effect, which may
result in relative lower plasma concentration in some
positions. This situation may bring difficulties to analyse
the in vivo statistics precisely and build a good connec-
tion to in vitro release results. Zolnik and Burgess (2008)
employed a sacrifice method to measure the in vivo
release behaviours of dexamethasone/PLGA sustained-
release injectable microsphere. In this method, five rats
were sacrificed at each sampling point and the subcuta-
neous tissues containing the microspheres were removed
from the injection site. The amount of released drug was
calculated by the total drug in microspheres minus the
drug content in the degraded microspheres, and the
in vivo curves are closed after plateau stage achieved.
The in vitro and in vivo release behaviours in this
study built a good relationship via linear regression.
This method shows various advantages: there are no lim-
itations on the numbers of sampling points, it makes the
rats possible to measure a longer in vivo behaviour, and
the toxicity can be tracked at the same time. The authors
Sampling Injection Main drug/carrier
Not mentioned Gluteal intramuscular/20G RisperdalÕ ConstaÕ
Forelimb veins/3 mL Gluteal area Pingyangmycin/PLGA
Marginal ear vessels/1 mL Gluteal muscle/23G Progesterone/Chitosan
Tail vein/0.5 mL Subcutaneous injection Octreotide/PLA
Tail-vein nicking method/ Intramuscularly into the rat’s Octreotide/PLGA
0.6–0.8 mL quadriceps
Not mentioned Subcutaneous injection Rismorelin/PLGA
Tail vein Subcutaneously at the back Leuprolide/PLA
of the neck
Not mentioned Subcutaneous injection in Human growth hormone/
the abdomen PLGA
Subcutaneous tissues were Subcutaneous/18G Dexamethasone/PLGA
removed
suggest that this method may achieve good effects in
laboratory conditions, but it is hard to possess signifi-
cance in building standard due to the individual varia-
tions. Blood collection method should still be given the
first consideration to measure the in vivo release behav-
iours of sustained-release injectable microspheres.
Although numerous sites for parental delivery are
adopted in previous studies, currently, the marketed prod-
ucts still rely largely on intramuscular injection and subcu-
taneous injection. The in vivo research works in humans
have also been reported, and the studies are mainly divided
into two parts based on the difference on subjects: patients
with relating disease and healthy volunteers. All the sub-
jects should be passed through pre-study physical exami-
nation and screened for eligibility within several weeks
prior to the institutionalization period. Toxicity tests are
usually conducted before human experiments when
uncommercial microspheres are involved. Here, there are
some differences between the two major subjects. Single
dose is often adopted in healthy volunteers and the
selection principle of sampling times is similar to the
above-mentioned procedure. Other items have no signifi-
cant differences from the traditional formulations (Vlugt-
Wensink et al., 2007). However, multiply dose model is
sometimes used in patients with relating disease and phar-
macodynamics studies are often preceded in the same
period. Concomitant medications that do not interfere
with the metabolism of model drugs are allowed during
the study in case some health problems in a much longer
period than normal tests. Frequent sampling is also
adopted in the first 24 h after each administration, and a
more frequency is carried through after the last injection to
measure the burst effect after continuous administration.
For example, Gefvert et al. (2005) investigated five intra-
muscular injections of Risperdal ConstaTM at the following
time points: first administration (day 1): predose and at 4,
8, 24 and 96 h; second administration (day 15): predose and
at 8 and 24 h; third administration (day 29): predose and at
8 and 24 h; fourth administration (day 43): predose and at 8
and 24 h and fifth administration (day 57): predose and at
4, 8, 24 and 72 h. Blood samples were also taken on days 62,
 Preparation and evaluation sustained-release injectable microspheres
64, 67, 69 and 71 and then weekly to day 113 (Gefvert
et al., 2005).
Here, the authors point out that some difficulties and
problems still exist in the studies of in vivo release behav-
iours of sustained-release injectable microspheres.
(a) Large amount of reports have compared the in vivo
release behaviours among polymers in different
molecular weights or between different doses,
aiming to select a optimize effect with less initial
burst and sustained-release behaviour. However,
some reports merely focus on this point and with
no consideration on effect concentration of main
drugs. It is meaningless to seek an excellent sus-
tained-release behaviour without the effect concen-
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13
tration. Not only the pharmacokinetics, but the
pharmacodynamics research should also be
tracked.
(b) Until now, few researchers have built an appropri-
ate system to evaluate the relation between sus-
tained-release injectable microspheres and
normal injections. Some researchers merely adopt
one-dose model to both two preparations, which is
not accurate to assess their superiority or inferior-
ity. The normal injections should be administrated
in a multiply dose model at least in a period of
sustained-release products, and the total dose
For personal use only.
should be equivalent.
In vitro and in vivo correlation
The main aim of establishing IVIVC is to make the in vitro
drug release methods be used in lieu of in vivo studies for
formulation composition and process optimization. This
section will summarize some IVIVC methods and introduce
some reports of good IVIVC results. For sustained-release
injectable microspheres, it is more meaningful and practi-
cal to develop IVIVC due to its long development cycle.
Traditionally, IVIVC can be classified as follows. Level A is
a point-to-point correlation over the entire release profile;
Level B is the comparison between in vitro dissolution time
and mean residence time or in vivo dissolution time; Level
C is a correlation between a dissolution parameter to a
single-point correlation; multiple Level C involves a corre-
lation at several time points in the release profile and Level
D is a rank–order correlation (Zolnik and Burgess, 2008).
Up to now, three mathematical models have been
widely used and adopted by FDA: deconvolution-based
methods, convolution-based methods and differential
equation-based methods. The deconvolution-based meth-
ods are commonly divided into two major stages: in the first
stage, the absorption part of the in vivo release profiles is
picked out and a curve of fraction absorbed to time is usu-
ally established and in the second stage, the established
curve (fraction absorbed to time) is connected to the
in vitro release profile. The IVIVC can be established in a
point-to-point relationship between the in vivo (fraction
absorbed) and in vitro (fraction dissolved) data at the
379
same time by a simple linear or an exponential function.
The calculation of fraction absorbed relies on compart-
mental models; in addition, the Wagner–Nelson method
and Loo–Riegelman method are commonly used to assay
the fraction absorbed (Wagner and Nelson, 1963; Loo and
Riegelman, 1968). Chu et al. (2006) prepared huperzine A-
loaded PLGA microspheres by an O/W method, and the
pharmacokinetics research of three different formulations
were measured by five beagle dogs. In vitro release studies
were conducted in phosphate buffer (pH 7.4) employing a
shaken method. The Wagner–Nelson procedure and the
Loo–Riegelman method with the linear trapezoidal rule
were used to obtain in vivo cumulative release profiles.
The IVIVC was established through the per cent drug
absorbed (anlysed by Wagner–Nelson procedure and
Loo–Riegelman) versus the amount of drug released
in vitro in a point-to-point model. The results showed a
good linear regression relationship between the per cent
in vitro dissolution in PBS at 37 C and the per cent absorp-
tion: R2 ¼ 0.974–0.990 for the Wagner–Nelson method and
R2 ¼ 0.959–0.993 for the Loo–Riegelman method.
Different from deconvolution-based methods, convolu-
tion-based methods can directly relate the in vivo release to
the in vitro release in a one-stage model. The rationale and
equations for these methods have been described in detail
in some publications (O’Hara et al., 2001). Some other
functional dependencies may be adopted due to the pres-
ence of time delay for the in vivo release. Time scaling is
also frequently used due to the differences in total times
between in vivo release and in vitro release. Zolnik and
Burgess (2008) prepared two PLGA microsphere formula-
tions with different polymer molecular weights (13 and
28 K). A USP apparatus IV was used for in vitro testing
and Sprague Dawley rats were employed for pharmacoki-
netics studies (Zolnik and Burgess, 2008). The secondary
zero-order phase of both formulations was selected out to
establish a relationship between the in vitro and in vivo
release data. Time scaling was used in this study because
the time to reach the plateau was different between the
in vitro and in vivo profiles. The scaling standard was
achieved by plotting in vitro time on the x-axis and
in vivo time on the y-axis and a linear relationship was
assessed. The slope was served as time scaling parameters
(0.4984 and 0.5008 for 13 and 28 K) and intercept was
served as time shifting (1.4276 and 3.8255 for 13 and
28 K). The in vitro and in vivo relationship established for
the 28 K formulation was utilized to predict the in vivo
release of dexamethasone from the 13 K formulation. The
IVIVC of the 28 K formulation was achieved by a linear
regression: percentage of cumulative in vivo release ¼
1.6381 (percentage of cumulative in vitro release)24.457
(R2 ¼ 0.9862). A difference factor (f1) and a similarity factor
(f2) were utilized to compare in vivo data between pre-
dicted curves and experimental curves. The difference
factor (f1) and the similarity factor (f2) were calculated as
16 and 58, suggesting equivalence of the two profiles.
One or two compartment pharmacokinetic models are
frequently used in differential equation based methods.
The plasma concentrations are directly related to in vitro
 380 L. Hu et al.
fraction-dissolved data through clear models and corre-
sponding assumptions of distribution and elimination.
Time scaling and time shifting are also allowed in this
method (Buchwald, 2003). However, due to the complexity
of the in vivo release behaviours of sustained-release
injectable microspheres, it is hard to analyse pharmacoki-
netic statistics in simple compartmental models and
directly relate the plasma concentration to fraction-dis-
solved data.
Conclusion
This review focuses on preparing sustained-release micro-
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13
spheres and evaluating their release behaviours. Some
commonly used polymers, preparation processes and eval-
uation methods are described within this article; in addi-
tion, the advantages and disadvantages for each method
are compared. These discussions provide some guiding
principles on how to develop such a parenteral drug deliv-
ery system and select appropriate methods to measure the
release behaviours. Moreover, some details, such as the
accelerated in vitro test methods, the principle on selecting
sampling points and the procedures on establishing IVIVC,
are also introduced, aiming to shorten development cycles
and measure more accurate release profiles.
For personal use only.
Acknowledgements
This work was supported by the Talent Introduction
Program of Hebei University (no. y2005064) and by a
grant of the Medical and Engineering Science Research
Center of Hebei University (no. BM201109).
Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of this
article.
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