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An overview of preparation and evaluation sustained-release injectable

microspheres

Article in Journal of Microencapsulation · November 2012

DOI: 10.3109/02652048.2012.742158 · Source: PubMed

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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
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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.

Some formations developed in early years, such as emul-
In recent years, there has been a great deal of interest in sions and suspensions are found to have numerous disad-
developing sustained-release systems, which can prolong vantages: nonadjustable release behaviour and instability
the therapeutic effect, decrease adverse side effects and on long-term storage. Traditional implants are sometimes
reduce administration frequency (Shi and Li, 2005). employed as sustained-release preparations due to low sys-
Nowadays, the oral sustained-release preparations account temic levels and toxic effects, but the need for a surgical
for a larger market share comparing to other sustained- procedure makes it more unacceptable for patients.
release forms, mainly because of its noninvasive pain-free However, the sustained-release injectable microspheres
administration. However, the hostile environment of gas- possess all over advantages and can overcome the above-
trointestinal tract and first-pass effect may lead to low bio- mentioned problems (Sinha and Trehan, 2005). 13
availability of some drugs, such as some proteins, peptides Microspheres are small spherical particles, with diame-
20
and hormones (Sinha and Trehan, 2003; Motulsky et al., ters in the micrometer level. The diameter size to a lesser
2005). Moreover, some other noninvasive pain-free deliv- degree than 250 mm, ideally 125 mm is suitable for paren-
ery approaches, such as transdermal, pulmonary and nasal teral delivery (Jain, 2000). The target activity is the signifi-
drug delivery, are not appropriate for such macromolecular cant characteristic differing from traditional dosage forms,
drugs, mainly because of the low biological membrane leading to higher bioavailability and lower toxicity. Smaller
permeability and instability in the delivering environment microspheres (less than 10 mm) are demonstrated to have
(Shi and Li, 2005). targeted activity via their uptake by macrophages and den-
Nowadays, using the parenteral route is the most dritic cells (Singh and O’Hagan, 1999), and their localiza-
common way to avoid the first-pass effect and the 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- variability result in difficulties for defining the standards
release injectable microspheres was developed by Ipsen of natural polymers.
in 1986. DecapeptylÕ is a gonadotropin-releasing hormone Some generally used synthetic polymers are listed in
agonist, which can decrease pituitary secretion of gonado- Table 3. Among the above-mentioned synthetic polymers,
tropins luteinizing hormone and follicle stimulating polylactic-co-glycolic acid (PLGA) and polylactic acid
hormone. Nowadays, various FDA-approved sustained- (PLA) are used in a host of FDA approved therapeutic
release injectable microspheres are available in the devices and have been widely used in medical and phar-
market (Table 1); in addition, some detailed comparisons maceutical fields (Anderson and Shive, 1997; Jain, 2000).
are also introduced in Table 2. Some of the products pos- PLGA is composed of one or more of three different
sess excellent sustained-release effects, which can main- hydroxyl acid monomers (d-lactic, l-lactic and/or glycolic
tain therapeutic effects for several months in one dose. 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
Materials et al., 2005). The above-mentioned three parameters can
determine the hydrophobicity and degradation kinetics of
A key factor in designing sustained-release injectable the materials, and thereby, the microencapsulation effi-
microspheres is the choice of an appropriate biodegradable ciency, release rate and interactions of the microspheres
polymer. Generally speaking, the materials employed for with phagocytizing cells are also influenced (Tamber
injectable microspheres are divided into two major et al., 2005). For example, increase in polymer molecular
groups: natural polymers and synthetic polymers. The mass or higher lactic acid content can result in longer deg-
investigations of natural polymers for sustained-release radation times and slower release (Jiang et al., 2005).
injectable microspheres have focused on proteins (e.g. Molecular mass and monomer ratio can be served as two
albumin (Porta et al., 2010), gelatin (Habraken et al., major adjustable factors to achieve an expected sustained-
2009) and collagen (Nagai et al., 2010)) and polysaccha- release behaviour (Hutchinson, 1982). The molecular mass
rides (e.g. starch (Björses et al., 2010), dextran of PLGA often used in microsphere preparation is ranged
(Mohaghegh and Tafaghodi, 2011), cellulose (Shi et al., from 10 to 100 kDa, and the commonly used ratios of lactic
2009) and sodium alginate (Grellier et al., 2009)). The nat- to glycolic focus on 50:50 up to 100:0.
ural polymers exhibit their advantages in controlling When used for injectable microspheres, PLGA is
release behaviours and improving target activities. degraded into oligomers and monomers through the
However, their applications are still limited by large-scale hydrolytic scission of the ester bonds in first stage. In this
commercial manufacturing and difficulties in purification period, a large amount of lactic and glycolic acids are pro-
(Sinha and Trehan, 2003), and the complexity and duced resulting in a significant decline of pH values.
 Preparation and evaluation sustained-release injectable microspheres 371

Table 3. The main types of commonly used synthetic polymers for the sustained-release injectable microspheres.

Class General structures

Polyesters
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Polyorthoesters

Polyanhydrides
For personal use only.

Polyamides

Polyalkylcyanoacrylates

Pseudo-polyamino acids

Polyphosphazenes

Therefore, most drugs used with PLGA in injectable micro- Hermeling et al., 2004). PLA is investigated as a drug deliv-
spheres are relatively stable in acidic media. In the second ery biodegradable material as early as 1971 and applied in
stage, the microspheres lose mass and the rate of polymer microsphere research works in early years (Brannon-
chain scission may increase (Fu et al., 2000). Some Peppas, 1995). However, PLA is not able to be appropriate
researchers summarize the two stages as degradation and for encapsulating molecules of virtually any size drugs
erosion (Tamber et al., 2005). The produced lactic and gly- through changing self-characters like PLGA, so the PLA
colic acids are metabolized in the Krebs cycle to CO2 and has a limitation in developing modern sustained-release
water ultimately (Yoo et al., 2005). Nowadays, PLGA micro- injectable microspheres.
spheres have a long safety record, and some sustained- Due to the need for better delivery formulations, various
release products are able to control the release of drugs types of copolymers have been developed. For example,
slowly and continuously from one to four months. PLGA/PEG (poly ethylene glycol) block copolymers have
Despite the excellent biocompatibility and widespread been processed as diblock (PLGA–PEG) or triblock mole-
application of PLGA, some mild inflammatory responses cules with PLGA–PEG–PLGA and PEG–PLGA–PEG types
are reported by some researchers (Gupta et al., 1997; (Jeong et al., 2000; Cheng et al., 2007; Makadia and
 372 L. Hu et al.

Siegel, 2011). Better release kinetics from formulations of the emulsion or multiple emulsions are formed in presence
copolymers and the stability of the preparations have been of a suitable emulsifier following the addition of the drug
demonstrated in comparison to PLGA alone, but the addi- into polymer solution. The oil-in-water (O/W) method is
tion of PEG often leads to a reduction of the encapsulation the mostly used single emulsion method in preparing
efficiency. In addition, the triblock copolymers of both microspheres with water-insoluble drugs (Jalil and Nixon,
PLGA–PEG–PLGA and PEG–PLGA–PEG types can also act 1990), and water-in-oil-in-water method is widespread in
as thermogels (Makadia and Siegel, 2011). multiple emulsions for preparing microsphere with water-
Recently, inorganic artificial biomaterials such as bio- soluble drugs like peptides, proteins and vaccines (Genta
ceramics and metallic biomaterials have attracted a lot et al., 2001; Rosas et al., 2001). In addition to the two major
attention for medical applications. Some kinds of glass methods, some special emulsion methods have also been
microspheres and ferrimagnetic ceramic particles have employed for microsphere preparation. For example, the
exhibited great potential in treatment of cancer through oil-in-oil emulsification method has been developed to
via in situ radiotherapy or hyperthermia of cancer. In encapsulating water-soluble drugs in the single emulsion
1980s, Day et al. entrapped the Y2O3–Al2O3–SiO2 glass method (Sánchez et al., 2003); solid-in-oil-in-water, solid-
microspheres into capillary bed of the tumours and the in-oil-in-oil and even solid-in-oil-in-oil-in-water emulsifi-
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90
Y contained in these microspheres could irradiate cation methods have also been developed to avoid dena-
tumour tissue locally by emitting -rays with a half-life of turation in protein drugs (Tobı́o et al., 1999; Wang et al.,
64.1 h (Cianni et al., 2010). In this technique, the invasion 2004; Yuan et al., 2009).
of radiotherapy was significantly decreased. In 1990s, In the second stage, extraction or evaporation is
Kawashita et al. (1999) developed 32P-containing SiO2 employed to remove organic solvent and harden the drop-
glass microspheres for radiotherapy of cancer by implant- lets followed by the particles are harvested, washed and
ing Pþ ions into microspheres at high energy. In this dried or freezing-dried (Freitas et al., 2005). When extrac-
research, colloid particles were replaced by red phospho- tion is used, the emulsion is transferred to a large quantity
rus colloid particles, and the sustained-release effects were quench medium, and then the solvent associated with the
significantly improved. Moreover, Kawashita et al. (2003) oil droplets is diffused out. In evaporation process, emul-
also prepared YPO4 microspheres composed of crystalline sion is exposed to a large amount of water or cosolvent,
For personal use only.

YPO4, which exhibit better effects than Y2O3–Al2O3–SiO2 often under reduced pressure and increased temperature.
glass microspheres. In emulsion methods, the technology can be optimized by
Research in the past several years has indicated that the changing a number of factors in process, including: (a) the
ferromagnetic microspheres possessed significant superi- organic solvent, emulsifier and polymer used; (b) the drug/
orities for embolic hyperthermia treatment of cancer. polymer ratio; and (c) the parameters in emulsification
Fe3O4 and -Fe2O3 was frequently used as ferromagnetic process and extraction or evaporation process. Although
material, such as Fe3O4–SiO2 microspheres (Deng et al., a number of hydrophilic drugs have been successfully
2008), Fe3O4–TiO2 microspheres (Li et al., 2008), Fe3O4– encapsulated by this method, some disadvantages in emul-
C–Au microspheres (Qi et al., 2010) and the microspheres sion methods, such as consuming long time, lower loading
composed of small crystals of -Fe2O3 (Kawashita et al., capacity, poor encapsulation efficiency and large initial
2008). burst release properties, cannot be ignored still.
In addition, some investigators also developed some
kinds of microspheres containing different species of mate-
rials. For example, Habraken et al. (2006) developed an Phase separation
injectable PLGA microsphere/calcium phosphate (CaP)
cement system with sufficient setting/cohesive properties. The phase separation method is also called coacervation,
Results showed that all physical parameters were well in and a brief flow diagram is shown in Figure 2. When com-
range of 10:90–20:80 (PLGA microsphere: CaP cements; pared to emulsion methods, the requirement of solvents for
Habraken et al., 2006). Despite many other similar reports the polymer is less stringent in concentration and this pro-
about new type materials, there is a long way to go in devel- cess is more suitable for encapsulating water-soluble drugs
oping another classical material like PLGA and PLA. (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
Preparation methods emulsions, and water-insoluble drugs can be solubilized
or dispersed in the polymer solution. An organic nonsol-
Emulsion methods vent is then added into the system to achieve the phase
separation, followed by the polymer solvent is gradually
The emulsion methods have been widely used because of extracted from the coacervation phase. This organic non-
the convenience in controlling process parameters, and solvent should be miscible with the polymer solvent and
there is no need for producing with expensive and complex immiscible with the polymer or the drug, such as vegetable
instrument. This process is mainly divided into two major oils and low molecular weight liquid polybutadiene. As a
parts: emulsification and removal procedure; in addition, a result, the soft coacervate droplets are formed to entrap the
brief flow diagram is described in Figure 1. In the first stage, drug. The two-phase system is then transferred into a large
 Preparation and evaluation sustained-release injectable microspheres 373
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Figure 1. The brief flow diagram of emulsion methods.

volume of an organic hardening agent (different from the Spray drying method
former organic nonsolvent) to harden the droplets and
achieve the final microspheres. The second nonsolvent The two methods mentioned above both have fatal weak-
should be volatile and easy to wash the first nonsolvent, ness in large-scale production. Large quantities of organic
such as hexane and heptane. solvent are needed in these methods, and it is difficult to
Several kinetic parameters, such as the phase/organic achieve a favourable encapsulation efficacy. Contrary to
phase volume ratio, stirring rate for the drug dispersion in these methods, the spray drying method is relatively
polymer solution, the addition rate of organic nonsolvent simple and appropriate to large-scale production, involv-
and the used polymer and nonsolvents, are reported to ing mild conditions. Moreover, it has less demand on the
have influence on some aspects of the microencapsulation solubility parameter of drug, polymer and solvents
process. However, due to the absence of emulsion stabi- (Giunchedi et al., 2001; Vehring, 2008). In this process,
lizers in this process, agglomeration is a common problem the polymer is first dissolved in a volatile organic solvent,
in this method (Jain, 2000). and then the drug is dispersed in polymer solution usually
 374 L. Hu et al.
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Figure 2. The brief flow diagram of the phase separation method.

with high speed homogenization. The dispersion or emul-

Table 4. Different classifications of the SCF processes when used for
sion is atomized in a flow of drying air to vaporize the preparing sustained-release injectable microspheres.
organic solvent, followed by the hardened microspheres
that are collected in a deposition chamber. Some types of Classification Process
PLGA microspheres are successfully prepared through this Solvent Rapid expansion of supercritical solution
method (Mu and Feng, 2001; Gavini et al., 2004). Anti-solvent SCF anti-solvent
Some procedure parameters, such as the inlet temper- Gas anti-solvent
ature, outlet temperature, aspirator setting and flow rate of Precipitation with a compressed anti-solvent
Solution enhanced dispersion by SCFs
the pump, can affect the formation of microspheres.
Solute Particle from gas saturated
Recently, there have been some improvements in the solution or suspension
spray drying method; for example, a novel double-nozzle Reaction medium Synthesis of coating material
spray-drying technique has been developed to solve the within supercritical solution
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 mostly used fluid, mainly due to its mild critical conditions
may lead to difficulties in breaking up the polymer (TC ¼ 304.1 K, PC ¼ 7.38 MPa; Bahrami and Ranjbarian,
solution. 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
Supercritical fluid method behaviour on the formation of microspheres in three kinds
of processes, in which SCF acts as solvent, anti-solvent or
In recent years, the supercritical fluid (SCF) has received solute (Table 4; Bahrami and Ranjbarian, 2007).
increased attention as an alternative approach to prepare The rapid expansion of supercritical solution is the first
microspheres. Supercritical carbon dioxide (SCO2) is the SCF method (Tom and Debenedetti, 1991). In this method,
 Preparation and evaluation sustained-release injectable microspheres 375

the microspheres are formed as a result of a rapid expan- microspheres are heat-sensitive, moist or dry heat sterili-
sion of SCF solvent. However, this technique is mainly zation is impossible. In addition, the microspheres are typ-
applicable to small molecular weight drugs, because the ically too large to pass through the 0.22 mm sterile filters
drug and the polymer both need sufficiently high solubility (Mohanan et al., 2012). Aseptic technique seems to be
in supercritical CO2. the guarantee for sterility, but it brings significant complex-
In supercritical anti-solvent methods, the drug is first ity and costs to large-scale manufacture. In recent years,
dissolved in an organic solvent and the solution is then sterilization by gamma-irradiation has been widely used in
sprayed into SCF. The quick mass transfer between SCFs this field, especially the PLGA microspheres, which can
and solution reduces the solubility properties, followed by significantly reduce the cost and complexity in aseptic
the drug particles which can be achieved by precipitation technique. However, some reports indicate that the
(Reverchon, 1999). In this technique, the microspheres can gamma-irradiation can increase the risk in changing the
be prepared to meet certain size, shape or porosity by vary- structure of both the drug and polymer, therefore,
ing necessary process parameters of the system. However, the release kinetics and even the pharmaceutical effect
some microspheres produced by anti-solvent methods are may be changed by the influence of gamma-irradiation
puzzled by the problem of residual solvent sometimes. (Montanari et al., 1998; Jain et al., 2011). Take PLGA as
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In addition, sometimes SCO2 can be used as a reaction an example, the ionization phenomena caused by irradia-
medium, in which the coating material synthesizes by a tion can create free radicals and reactive oxygen species
chemical reaction and coats the surface of particles. This from PLGA (Mohanan et al., 2012).
technique is mainly used to prepare nanospheres. Several efforts have been made to reduce the probability
The particles from gas saturated solutions production of ionizing radiation of microsphere preparations. Some
process was a single-step process and has significant researchers demonstrated that the use of low temperatures
advantages over conventional production methods, as it during the gamma-irradiation sterilization process can
requires neither drug particle nor polymer to dissolve in maintain the nature properties of some low molecular
SCO2, no organic solvents are needed, and the SCO2 is of weight drugs in microspheres (Fernandez-Carballido
low consumption. In this method, SCO2 acts as a molecular et al., 2004; Martinez-Sancho et al., 2004). However,
lubricant liquefying the polymer at temperatures signifi- Zbikowska et al. (2006) verified that the low temperatures
For personal use only.

cantly below its glass transition temperature. When were not the guarantee to protect the drug and polymer
exposed to SCO2 in a pressure vessel, the polymers can from undesirable reactions.
liquefy the main drug to be mixed efficiently into the poly- The use of antioxidants has been proved to have effects
mer. The mixture is then depressurized through a nozzle to remove the free radical intermediates or inhibit other
whereby the CO2 evaporates solidifying the polymer oxidation reactions produced by ionizing radiation
around the drug, resulting in the production of micro- (Martinez-Sancho et al., 2004; Mohanan et al., 2012).
spheres (Jordan et al., 2010). 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
Others sterilization. Some researchers also reported that the use of
the three above-mentioned strategies could achieve a
With the development of technology, a number of new better protective effect during the gamma-irradiation ster-
methods and equipments are developed to prepare micro- ilization process (Checa-Casalengua et al., 2012). However,
spheres. Here, the authors give a brief introduction of some there are so many uncertain influences in the process of
widely recognized novel methods: (a) the ultrasonic atom- gamma-irradiation exposure including the known and
ization method, sometimes called ‘‘one-step’’ operation, unknown factors. At the present stage, no other strategies
can produce microspheres in different sizes by changing can replace the gamma-irradiation sterilization except for
the nozzle diameter, vibration frequency and the flow rate aseptic technique, which is one of the core problems to
of the polymer. Therefore, the drug release behaviours can restrict the development of the microsphere technique.
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 Evaluation of sustained-release behaviour
a grounded electrode, is employed for preparing micro-
spheres in various sizes by changing the potential differ- In vitro release
ence of inner and outer needles and the flow rates
(Loscertales et al., 2002). Besides the polymer characteristics, the in vitro release
behaviour is also dependent on dosage form characteris-
tics, such as particle size, morphology, porosity, residual
Sterilization solvents and drug loading rate (Anderson and Shive,
1997; Ertl et al., 1999). The in vitro release of sustained-
To enable the application of the preparations into human release injectable microspheres usually exhibits a typical
or animal settings, the process of sterilization is of signifi- triphasic profile: (a) an initial burst release; (b) a lag
cant importance. As most of the polymers used in 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 Sampling Injection Main drug/carrier

Gefvert et al. (2005) Humans Not mentioned Gluteal intramuscular/20G RisperdalÕ ConstaÕ
Han et al. (2010) Beagle dogs Forelimb veins/3 mL Gluteal area Pingyangmycin/PLGA
Jameela et al. (1998) New Zealand white male Marginal ear vessels/1 mL Gluteal muscle/23G Progesterone/Chitosan
rabbits
Murty et al. (2004) Sprague Dawley rats Tail vein/0.5 mL Subcutaneous injection Octreotide/PLA
Rhee et al. (2011) Rats Tail-vein nicking method/ Intramuscularly into the rat’s Octreotide/PLGA
0.6–0.8 mL quadriceps
Thompson et al. (1997) Swines Not mentioned Subcutaneous injection Rismorelin/PLGA
Woo et al. (2002) Sprague Dawley rats Tail vein Subcutaneously at the back Leuprolide/PLA
of the neck
Vlugt-Wensink et al. (2007) Humans Not mentioned Subcutaneous injection in Human growth hormone/
the abdomen PLGA
Zolnik et al. (2006) Sprague Dawley rats Subcutaneous tissues were Subcutaneous/18G Dexamethasone/PLGA
removed
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13

intervals turn to be larger with the time increased. suggest that this method may achieve good effects in
Moreover, the sampling time intervals can be over a laboratory conditions, but it is hard to possess signifi-
month after several months, when the micro- cance in building standard due to the individual varia-
spheres with an excellent sustained-release effect. tions. Blood collection method should still be given the
The finish time mainly depends on the detection first consideration to measure the in vivo release behav-
limit of main drugs. iours of sustained-release injectable microspheres.
(c) Rats are most commonly used due to ease of han- Although numerous sites for parental delivery are
dling, and fewer resources needed to maintain rats adopted in previous studies, currently, the marketed prod-
in a study. However, due to the limitation of the
For personal use only.

ucts still rely largely on intramuscular injection and subcu-

physical characteristics, rats are commonly used taneous injection. The in vivo research works in humans
to measure in vivo release behaviour in one have also been reported, and the studies are mainly divided
month and not appropriate to track a longer release into two parts based on the difference on subjects: patients
profile. Beagle dogs and rabbits are more suitable with relating disease and healthy volunteers. All the sub-
to serve as model animals to measure in vivo jects should be passed through pre-study physical exami-
release profiles of microspheres with excellent sus- nation and screened for eligibility within several weeks
tained-release effects. prior to the institutionalization period. Toxicity tests are
(d) One-dose model is usually adopted for this formu- usually conducted before human experiments when
lation, and the amount is often depended on uncommercial microspheres are involved. Here, there are
animal weight. some differences between the two major subjects. Single
However, the target activity of microspheres can lead dose is often adopted in healthy volunteers and the
to an unbalanced distribution in different organs, selection principle of sampling times is similar to the
together with the sustained-release effect, which may above-mentioned procedure. Other items have no signifi-
result in relative lower plasma concentration in some cant differences from the traditional formulations (Vlugt-
positions. This situation may bring difficulties to analyse Wensink et al., 2007). However, multiply dose model is
the in vivo statistics precisely and build a good connec- sometimes used in patients with relating disease and phar-
tion to in vitro release results. Zolnik and Burgess (2008) macodynamics studies are often preceded in the same
employed a sacrifice method to measure the in vivo period. Concomitant medications that do not interfere
release behaviours of dexamethasone/PLGA sustained- with the metabolism of model drugs are allowed during
release injectable microsphere. In this method, five rats the study in case some health problems in a much longer
were sacrificed at each sampling point and the subcuta- period than normal tests. Frequent sampling is also
neous tissues containing the microspheres were removed adopted in the first 24 h after each administration, and a
from the injection site. The amount of released drug was more frequency is carried through after the last injection to
calculated by the total drug in microspheres minus the measure the burst effect after continuous administration.
drug content in the degraded microspheres, and the For example, Gefvert et al. (2005) investigated five intra-
in vivo curves are closed after plateau stage achieved. muscular injections of Risperdal ConstaTM at the following
The in vitro and in vivo release behaviours in this time points: first administration (day 1): predose and at 4,
study built a good relationship via linear regression. 8, 24 and 96 h; second administration (day 15): predose and
This method shows various advantages: there are no lim- at 8 and 24 h; third administration (day 29): predose and at
itations on the numbers of sampling points, it makes the 8 and 24 h; fourth administration (day 43): predose and at 8
rats possible to measure a longer in vivo behaviour, and and 24 h and fifth administration (day 57): predose and at
the toxicity can be tracked at the same time. The authors 4, 8, 24 and 72 h. Blood samples were also taken on days 62,
 Preparation and evaluation sustained-release injectable microspheres 379

64, 67, 69 and 71 and then weekly to day 113 (Gefvert same time by a simple linear or an exponential function.
et al., 2005). The calculation of fraction absorbed relies on compart-
Here, the authors point out that some difficulties and mental models; in addition, the Wagner–Nelson method
problems still exist in the studies of in vivo release behav- and Loo–Riegelman method are commonly used to assay
iours of sustained-release injectable microspheres. the fraction absorbed (Wagner and Nelson, 1963; Loo and
Riegelman, 1968). Chu et al. (2006) prepared huperzine A-
(a) Large amount of reports have compared the in vivo
loaded PLGA microspheres by an O/W method, and the
release behaviours among polymers in different
pharmacokinetics research of three different formulations
molecular weights or between different doses,
were measured by five beagle dogs. In vitro release studies
aiming to select a optimize effect with less initial
were conducted in phosphate buffer (pH 7.4) employing a
burst and sustained-release behaviour. However,
shaken method. The Wagner–Nelson procedure and the
some reports merely focus on this point and with
Loo–Riegelman method with the linear trapezoidal rule
no consideration on effect concentration of main
were used to obtain in vivo cumulative release profiles.
drugs. It is meaningless to seek an excellent sus-
The IVIVC was established through the per cent drug
tained-release behaviour without the effect concen-
absorbed (anlysed by Wagner–Nelson procedure and
Journal of Microencapsulation Downloaded from informahealthcare.com by Monash University on 05/02/13

tration. Not only the pharmacokinetics, but the

Loo–Riegelman) versus the amount of drug released
pharmacodynamics research should also be
in vitro in a point-to-point model. The results showed a
tracked.
good linear regression relationship between the per cent
(b) Until now, few researchers have built an appropri-
in vitro dissolution in PBS at 37 C and the per cent absorp-
ate system to evaluate the relation between sus-
tion: R2 ¼ 0.974–0.990 for the Wagner–Nelson method and
tained-release injectable microspheres and
R2 ¼ 0.959–0.993 for the Loo–Riegelman method.
normal injections. Some researchers merely adopt
Different from deconvolution-based methods, convolu-
one-dose model to both two preparations, which is
tion-based methods can directly relate the in vivo release to
not accurate to assess their superiority or inferior-
the in vitro release in a one-stage model. The rationale and
ity. The normal injections should be administrated
equations for these methods have been described in detail
in a multiply dose model at least in a period of
in some publications (O’Hara et al., 2001). Some other
sustained-release products, and the total dose
For personal use only.

functional dependencies may be adopted due to the pres-

should be equivalent.
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
In vitro and in vivo correlation Burgess (2008) prepared two PLGA microsphere formula-
tions with different polymer molecular weights (13 and
The main aim of establishing IVIVC is to make the in vitro 28 K). A USP apparatus IV was used for in vitro testing
drug release methods be used in lieu of in vivo studies for and Sprague Dawley rats were employed for pharmacoki-
formulation composition and process optimization. This netics studies (Zolnik and Burgess, 2008). The secondary
section will summarize some IVIVC methods and introduce zero-order phase of both formulations was selected out to
some reports of good IVIVC results. For sustained-release establish a relationship between the in vitro and in vivo
injectable microspheres, it is more meaningful and practi- release data. Time scaling was used in this study because
cal to develop IVIVC due to its long development cycle. the time to reach the plateau was different between the
Traditionally, IVIVC can be classified as follows. Level A is in vitro and in vivo profiles. The scaling standard was
a point-to-point correlation over the entire release profile; achieved by plotting in vitro time on the x-axis and
Level B is the comparison between in vitro dissolution time in vivo time on the y-axis and a linear relationship was
and mean residence time or in vivo dissolution time; Level assessed. The slope was served as time scaling parameters
C is a correlation between a dissolution parameter to a (0.4984 and 0.5008 for 13 and 28 K) and intercept was
single-point correlation; multiple Level C involves a corre- served as time shifting (1.4276 and 3.8255 for 13 and
lation at several time points in the release profile and Level 28 K). The in vitro and in vivo relationship established for
D is a rank–order correlation (Zolnik and Burgess, 2008). the 28 K formulation was utilized to predict the in vivo
Up to now, three mathematical models have been release of dexamethasone from the 13 K formulation. The
widely used and adopted by FDA: deconvolution-based IVIVC of the 28 K formulation was achieved by a linear
methods, convolution-based methods and differential regression: percentage of cumulative in vivo release ¼
equation-based methods. The deconvolution-based meth- 1.6381 (percentage of cumulative in vitro release)24.457
ods are commonly divided into two major stages: in the first (R2 ¼ 0.9862). A difference factor (f1) and a similarity factor
stage, the absorption part of the in vivo release profiles is (f2) were utilized to compare in vivo data between pre-
picked out and a curve of fraction absorbed to time is usu- dicted curves and experimental curves. The difference
ally established and in the second stage, the established factor (f1) and the similarity factor (f2) were calculated as
curve (fraction absorbed to time) is connected to the 16 and 58, suggesting equivalence of the two profiles.
in vitro release profile. The IVIVC can be established in a One or two compartment pharmacokinetic models are
point-to-point relationship between the in vivo (fraction frequently used in differential equation based methods.
absorbed) and in vitro (fraction dissolved) data at the The plasma concentrations are directly related to in vitro
 380 L. Hu et al.

fraction-dissolved data through clear models and corre- Brannon-Peppas L. Recent advances on the use of biodegradable micro-
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Checa-Casalengua P, Jiang C, Bravo-Osuna I, Tucker BA, Molina-
of the in vivo release behaviours of sustained-release Martinez IT, Young MJ, Herrero-Vanrell R. Preservation of biological
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Conclusion sodium oleate for sustained release from biodegradable polymeric
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223–8.
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spheres and evaluating their release behaviours. Some

Chu DF, Fu XQ, Liu WH, Liu K, Li YX. Pharmacokinetics and in vitro and
commonly used polymers, preparation processes and eval- in vivo correlation of huperzine A loaded poly(lactic-co-glycolic acid)
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tion, the advantages and disadvantages for each method Cianni R, Urigo C, Notarianni E, Saltarelli A, D’Agostini A, Iozzino M,
Dornbusch T, Cortesi E. Radioembolisation using yttrium 90 (Y-90) in
are compared. These discussions provide some guiding patients affected by unresectable hepatic metastases. Radiol Medica,
principles on how to develop such a parenteral drug deliv- 2010;115:619–33.
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drug particle size and initial release of recombinant human growth hor-
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accelerated in vitro test methods, the principle on selecting Deng Y, Qi D, Deng C, Zhang X, Zhao D. Superparamagnetic high-magne-
sampling points and the procedures on establishing IVIVC, tization microspheres with an Fe3O4@ SiO2 core and perpendicularly
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For personal use only.

introduced by an enhanced mixer for micronization of lysozyme: Particle

morphology, size and protein stability. Int J Pharm, 2011;421:258–68.
Ertl B, Platzer P, Wirth M, Gabor F. Poly(D, L-lactic-co-glycolic acid) micro-
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Fernandez-Carballido A, Herrero-Vanrell R, Molina-Martinez IT,
This work was supported by the Talent Introduction Pastoriza P. Sterilized ibuprofen-loaded poly(D, L-lactide-co-glycolide)
Program of Hebei University (no. y2005064) and by a microspheres for intra-articular administration: Effect of gamma-irradia-
tion and storage. J Microencapsul, 2004;21:653–65.
grant of the Medical and Engineering Science Research Fini A, Cavallari C, Ospitali F, Gonzalez-Rodriguez ML. Theophylline-
Center of Hebei University (no. BM201109). loaded compritol microspheres prepared by ultrasound-assisted atomi-
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Freitas S, Merkle HP, Gander B. Microencapsulation by solvent extraction/
evaporation: Reviewing the state of the art of microsphere preparation
Declaration of interest process technology. J Control Release, 2005;102:313–32.
Fu K, Pack DW, Klibanov AM, Langer R. Visual evidence of acidic environ-
ment within degrading poly(lactic-co-glycolic acid)(PLGA) micro-
The authors report no conflicts of interest. The authors spheres. Pharm Res, 2000;17:100–6.
alone are responsible for the content and writing of this Gavini E, Chetoni P, Cossu M, Alvarez MG, Saettone MF, Giunchedi P.
PLGA microspheres for the ocular delivery of a peptide drug, vancomy-
article.
cin using emulsification/spray-drying as the preparation method:
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