Vaishya 2014

Review
Long-term delivery of protein

therapeutics
Ravi Vaishya, Varun Khurana, Sulabh Patel & Ashim K Mitra

1. Introduction University of Missouri-Kansas City, Pharmaceutical Sciences, Kansas City, MO, USA
2. In situ injectable gel/implants

Introduction: Proteins are effective biotherapeutics with applications in
3. Microparticles diverse ailments. Despite being specific and potent, their full clinical potential
4. Nanoparticles/nanospheres has not yet been realized. This can be attributed to short half-lives, complex
5. Miscellaneous modes of structures, poor in vivo stability, low permeability, frequent parenteral admin-
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 02/04/15
protein delivery istrations and poor adherence to treatment in chronic diseases. A sustained
release system, providing controlled release of proteins, may overcome
6. Expert opinion
many of these limitations.
Areas covered: This review focuses on recent development in approaches,
especially polymer-based formulations, which can provide therapeutic levels
of proteins over extended periods. Advances in particulate, gel-based formu-
lations and novel approaches for extended protein delivery are discussed.
Emphasis is placed on dosage form, method of preparation, mechanism of
release and stability of biotherapeutics.
Expert opinion: Substantial advancements have been made in the field of
extended protein delivery via various polymer-based formulations over last
decade despite the unique delivery-related challenges posed by protein bio-
For personal use only.
logics. A number of injectable sustained-release formulations have reached

market. However, therapeutic application of proteins is still hampered by
delivery-related issues. A large number of protein molecules are under clinical
trials, and hence, there is an urgent need to develop new methods to deliver
these highly potent biologics.
Keywords: formulation, hydrogel, implant, microparticle, nanoparticle, protein delivery, release

mechanism, stability, sustained release, thermosensitive gel
Expert Opin. Drug Deliv. [Early Online]
1. Introduction
Proteins are large biomolecules with complex tertiary (30) and quaternary (40) struc-
tures. These macromolecules participate in a number of biological pathways and
have diverse functionalities such as enzymes, hormones, IFNs and antibodies. The
complex 30 and 40 structures impart important properties including selectivity,
specificity and high potency. Abundance in various biological functions,
specificity and potency are most important parameters. Newer targets are being
discovered at a rapid rate due to advanced understanding of pathology such as can-
cer and autoimmune diseases. Precise number of proteins expressed in the human
body is not known yet. But various reports suggest that there are at least
84,000 -- 200,000 different proteins in the human body [1,2]. Protein--protein inter-
action plays a key role in most biological pathways. It may be targeted by designing
agonist or antagonist peptides and proteins [3,4]. Advancement in high-throughput
screening (HTS) technology such as hot-spot determination has also catalyzed
development of novel biotherapeutics [3,5]. Therapeutic protein development and
production have reached advanced stages owing to innovations in manufacturing
technology [6], process control [7] and protein characterization [8,9]. Additionally,
protein biotherapeutics have shown to be very efficacious and specific, such as anti-
bodies. However, we are yet to take full advantage of these potent biotherapeutics
10.1517/17425247.2015.961420 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 1

All rights reserved: reproduction in whole or in part not permitted
R. Vaishya et al.
degradation, complex structure and immunogenicity [12].

Article highlights. Large molecular size and hydrophilicity hinder permeation
. Proteins are important biotherapeutics owing to of proteins across biological barriers such as GI mucosa lead-
advantages such as specificity, selectivity and potency. ing to poor absorption following oral administration. Extreme
. Numerous protein therapeutics including antibodies, gastric pH and digestive enzymes degrade proteins prior to
IFNs, enzymes and vaccines are in clinical trials for the
oral absorption. Following GI absorption, the first-pass
treatment of various diseases.
. Most formulation strategies to achieve long-term metabolism eliminates a significant fraction of absorbed
delivery of peptides cannot be applied directly for biomolecules. Hence, protein delivery via most favored --
proteins biologics. This is due to unique challenges oral route -- is highly challenging, and a large portion of
posed by proteins such as stability, immunogenicity, approved and investigational protein molecules is adminis-
complex structure (30 & 40), high molecular weight,
tered via parenteral routes (intravenous [i.v.], intramuscular
poor permeability across biological membrane and poor
[i.m.] or subcutaneous [s.c.]). Proteins also suffer from a
half-life.
. Protein instability may arise at many stages including number of physical, chemical and biological instability due
formulation development and during release. to their complex secondary, tertiary and quaternary structures.
. Proteins are susceptible to aggregation and denaturation Any alteration in active confirmation may lead to loss of
via exposure to organic solvents, oil--water interface and
activity and irreversible aggregation of proteins. Vulnerability
at high concentration in aqueous solution. Solvent-free
techniques may be useful in overcoming the towards enzymatic degradation under in vivo condition results
above-mentioned issues. into short half-lives even with parenteral administration.
. Protein may denature during release leading to Many protein therapeutics are intended for chronic ailments
incomplete release, hence influence of excipients and such as bevacizumab for age-related macular degeneration
polymers on protein stability should be explored before
(AMD). Short half-lives of proteins require frequent paren-
the formulation development.
. Release mechanism of protein from matrix type teral administrations to maintain therapeutic levels. However,
biodegradable formulations is generally combination of frequent parenteral administrations are not patient compliant.
erosion and diffusion. In addition, frequent administrations may not be well toler-
. Dissolution-controlled release mechanism is not a ated. For example, frequent intravitreal injections have shown
favorable strategy in formulation development in order
to be associated with many complications including cataract,
to achieve long-term drug release.
. Burst release, release rate and duration can be retinal hemorrhage and detachment [13].
controlled by polymer molecular weight, polymer Invasive and noninvasive routes have been investigated with
composition, porosity and additives such as salts and various formulations to deliver proteins. As mentioned earlier,
sugar. most frequently used invasive routes are i.v., i.m. and s.c.
. Several injectable sustained release protein formulations
Noninvasive routes include oral, rectal, transdermal and inha-
are in development, which may be available as
marketed product in coming years. lation. Of these, the most preferred noninvasive route is per-
oral administration, which is also the Holy Grail of protein
This box summarizes key points contained in the article. delivery. It is highly challenging among the noninvasive routes
due to the limitations discussed earlier. Major approaches
used for oral protein delivery include use of absorption
despite the fast-paced advancements in protein therapeutics enhancers such as surfactants, enzyme inhibitors, polymer-
R&D and production. US biopharmaceutical industry is --inhibitor conjugates, mucoadhesive particles, thiolated poly-
one of most innovative and research-intensive enterprise as mers, nanoparticles, microparticles and emulsion-based
noted by Congressional Budget Office [10]. As per recent mar- formulations [14-17]. Nevertheless, all these approaches suffer
ket survey, pharmaceutical/biotech industry invested nearly a from certain drawbacks and side effects. For example, cost
$50 billion every year from 2011 to 2013 in R&D of new of effective enzyme inhibitors, adverse effects due to frequent
medicines and most of it was spent for biopharmaceutical oral administrations in chronic diseases, poor stability of
R&D [11]. As a result, there are > 907 biotherapeutics in var- formulations such as liposomes in GI track, poor loading effi-
ious phases of clinical trials. Of these biotherapeutics, major- ciency of protein in particles owing to hydrophilicity, polydis-
ity are protein biotherapeutics including 338 monoclonal persed size distribution and particle aggregation [17]. Similar
antibodies, 93 recombinant proteins, 20 IFNs and 250 vac- limitations are also associated with noninvasive routes of
cines for various conditions such as cancer, autoimmune dis- delivery [18,19]. Moreover, it is challenging to achieve con-
eases and infectious diseases [1]. A detailed representation of trolled protein delivery for extended duration via patient
the protein biotherapeutics currently in clinical trial is compliant noninvasive routes. A potential reason for the fail-
depicted in [1]. ure of treatment strategy in chronic diseases may be lack of
Proteins pose a number of delivery-related challenges due adherence to a given treatment regimen by patient. Therefore,
to a myriad of factors. Some of them are large size, hydrophi- frequent administration is not a recommended strategy for
licity, poor permeability across biological membranes (such as chronic ailments. As per an estimate by WHO, only 50% of
gastrointestinal [GI] tract), susceptibility to enzymatic patients suffering from chronic diseases stick to the treatment
2 Expert Opin. Drug Deliv. (2014) 12(3)

Long-term delivery of protein therapeutics
regime in developed countries [20]. In developing countries, extended duration [23,24]. One of the reasons for wide spread
adherence to prolonged therapy is even lower [20]. Therefore, use of these systems is the simplicity. The system is liquid
proteins are suitable candidate for sustained release formula- or injectable semisolids, which turn to gel/implant upon
tions. Advantages of sustained protein delivery formulation injection. A number of mechanisms govern the process of
include better adherence to chronic therapy, in vivo stability, implant/gel formation. Broadly, these formulations can be
local delivery, fewer side effects, reduction in dose and dosing classified as in situ phase inversion and crosslinking systems
frequency and improved patient compliance. Furthermore, based on mechanism of implant/gel formation. Classification
parenteral routes, although not very patient compliant, may of in situ forming implant/gel systems is represented in
be a simpler answer to limitations and drawbacks associated Figure 1. In situ implant formation via solvent extraction has
with noninvasive administrations. Extended duration of pro- been thoroughly investigated and is successful with two mar-
tein delivery with lower frequency of administration may keted formulations. Temperature-dependent sol-to-gel sys-
improve patient compliance significantly. tems have a number of advantages over the previously
Ideal protein delivery formulation should be able to pro- developed systems. Several pH- and temperature-dependent
vide controlled release to maintain therapeutic levels for an hybrid polymers have also been developed to provide better
extended duration and ensure stability of encapsulated pro- control over the process of implant formation. Temperature,
tein. Components of delivery system must be biodegradable phase inversion and pH change are most investigated triggers
and/or biocompatible, nonimmunogenic, nontoxic and pref- to form in situ gel and are discussed.
erably FDA approved for human use. If the formulation is
intended via parenteral route, it should be possible to inject 2.1 Solvent extraction/phase inversion systems
it through a narrow gauge needle to minimize pain. In addi- Injectable implant consists of polymer solution in suitable
tion, in a few cases, it is impractical to use higher gauge nee- water-miscible organic solvent, where in protein is suspended
dles. For example, in case of intravitreal injections, a 27G to in organic solvent. Drug-loaded implant is formed upon
30G needle is recommended to avoid damage to intraocular in vivo injection. Following administration, water-miscible
tissues. Volume of injection should also be considered care- organic solvent diffuses out in the aqueous environment of
fully depending on the route of administration. Protein struc- surrounding tissue, resulting in precipitation of water-
ture and activity should not be altered during the preparation insoluble polymer. During precipitation, protein is entrapped
of delivery system, and protein must be in active conforma- inside the polymer matrix. Typical polymers used for these
tion following in vivo release. In particulate- and hydrogel- systems include polyesters such as poly(lactic-co-glycolic
based systems, initial burst release is a serious concern. In acid) (PLGA), poly(lactic acid) (PLA), Poly(glycolic acid)
the case of particle-based systems, burst release is due to (PGA) and polycaprolactone (PCL). A number of solvents
high surface adsorption of protein. Higher initial burst release with varying polarity have been examined including N-meth-
of proteins may shorten total duration of release and result in ylpyrrolidone (NMP), DMSO, ethanol, ethyl benzoate, tria-
dose-dependent adverse effects. Therefore, the formulation cetin, ethyl acetate, benzyl benzoate, PEG500-dimethylether
must be optimized to achieve minimal burst release. Above and glycofurol for dissolving polymers. NMP and DMSO
all these factors, method of formulation preparation should are widely employed despite the associated toxicities.
be scalable, robust and reproducible. Statistical methods Recently, PEG-alkyl ether and glycofurol have been investi-
such as quality-by-design, design of experiments [21] and in- gated for PLGA implants. Both solvents have been indicated
process quality control methods such as six-sigma may be to be well tolerated and biocompatible in vivo [25-27]. Glyco-
able to aid development of high-quality product with robust furol has been shown to be compatible with PLGA polymer
and reproducible method of manufacturing [7,22]. causing minimal polymer degradation. The objective of the
Despite all these challenges, substantial advancements have system is to deliver proteins over a long period to lower dosing
been made in the field of extended protein delivery via various frequency and provide stability to protein. The rate and dura-
polymer-based formulations over last decade. A number of tion of release for phase inversion systems/implants are influ-
injectable sustained-release formulations have reached market. enced by variables such as gelling rate, porosity of implant and
Nonetheless, there is room for further development. In the burst release. These factors are again dependent on the
present review, recent advancements in particulate- and gel- polymer type, concentration and molecular weight, solvent
based formulations and other novel approaches for controlled system, polymer crystallinity and additives [28-32].
extended protein delivery are discussed. Emphasis is placed on One of the widely used solvents for injectable implants is
dosage form, method of preparation, mechanism of release NMP. The rate of phase inversion leading to implant forma-
and stability of biotherapeutics. tion is very rapid due to its relatively high polarity compared
to solvents such as ethyl benzoate and benzyl benzoate. Short-
2. In situ injectable gel/implants coming of rapid phase inversion is formation of porous
implants with highly evolved network of interconnected pores.
Injectable implants have been designed to deliver a variety of Porous implants result in a significant burst release of peptides
therapeutics, including small molecules and proteins for and proteins. For example, 40% of lysozyme was released from
Expert Opin. Drug Deliv. (2014) 12(3) 3

R. Vaishya et al.
Trigger
Type
Solvent
extraction
In situ
pH change
solidification
Temperature
In situ forming
implant/gel
Photo-induced
In situ cross-
Chemical
linking
Physical
Figure 1. Classification of in situ forming implant/gel systems based of mechanism of formation.
80
(< 5 -- 10% of initial dose) (Figure 2) [29]. Depending on the sol-
70
ubility of a protein or peptide, it may be dispersed or solubi-
Percent lysozyme released
60 lized in the polymer solution during preparation. It has been

shown that when proteins are in dispersed state, the release
50
rate is higher [33]. When dispersed protein is released from the
40 implant, the porosity of the implant is increased tremendously
resulting in higher release rate. Similarly, type of additive also
30
influences the release rate and burst release. Addition of a
20 water-soluble additive results in high release rate, even from
an implant formed using triacetin (Figure 3A) [33]. Release rate
10 was found to be directly proportional to concentration of man-
0 nitol. The effect was very prominent during late phase. Again,
0 5 10 15 20 it was due to formation of porous network upon dissolution
Time (days) of mannitol during release, similar to a system where protein
is dispersed in implant. In contrast, addition of hydrophobic
additive such as linoleic triglyceride (Miglyol 818) resulted
Figure 2. Lysozyme release rate from 50 wt% PLGA/solvent in a much slower release rates (Figure 3B) [33]. Similar results
solutions; NMP (); triacetin (&); ethyl benzoate (~) are reported in an in vivo study, where PLGA implants contain-
quenched into a PBS solution. ing human growth hormone (hGH) were prepared using
Reproduced with permission from [29].
PLGA: Poly(lactic-co-glycolic acid).
NMP, triacetin, ethyl benzoate and benzyl benzoate [34]. Depot
formed by benzyl benzoate resulted in sustained levels of hGH
a PLGA implant prepared in NMP, followed by very slow levels in the plasma within a therapeutic window [34]. In order
release of remaining dose [29]. Conversely, less polar solvents to further lower the burst effect and control overall release pro-
such as triacetin and ethyl benzoate produce implants with file, physical properties of the hGH were also manipulated.
less porosity resulting in significantly reduced burst effects hGH was modified by preparing complex with Zn and

A. burst effect from PLGA implants, surfactants (triton X and

10 PVA) have also been incorporated in implants [36]. Addition
myoglobin (0.45%) of surfactants influenced mechanical stability of PLGA, which
myoglobin (0.45%) + mannitol (0.40%) in turn reduced extent of burst release.
8 myoglobin (0.45%) + mannitol (1.25%)
myoglobin (0.45%) + mannitol (2.50%) In order to further control the release profile and improve
Cum. mg released
peptide loading in implants, a few modified techniques have

6 been investigated. One such alternative injectable implant
system is referred to as SABER developed by Durect
Corp. Instead of polyesters such as PLGA or PLA, SABER
4
employs Sucrose Acetate Isobutyrate (SAIB) to form matrix.
SAIB is dissolved in organic solvent, such as ethanol or benzyl
2 benzoate, and injected to form a depot with slower phase
inversion. Okumu et al. utilized the SABER system to delivery

0
hGH with a few modification [37]. PLA (1.0 & 10% w/w) was
0 3 6 9 12 15 added along with SAIB in ethanol or benzyl benzoate and
Time (days) lyophilized hGH was suspended in organic solvent. Higher
drug loading (10%) along with injectability through a narrow
B.
gauge needle (25G) were advantages of the system. During
100 in vitro release from SABER system without PLA, a huge
10% gel with HSA, phenol, NaCI
5% gel
burst release (at 24 h) of 78% of total dose was observed.
80 10% gel Interestingly, when SABER system was modified by adding
Cum. percent released
10% gel with miglyol B12 1 and 10% of PLA, burst release of only 2 and 0.2% hGH
was released, respectively. However, a significant amount of
60 protein was degraded during release due to the presence of
organic solvent exposing proteins to organic-aqueous inter-

40 face. In order to minimize protein denaturation in SABER
system, Pechenov et al. investigated encapsulation of proteins
in crystal form [38]. a-Amylase was used as a model protein
20
and suspended in SAIB with ethanol (SAIB/ethanol) or
PLGA solution in acetonitrile (PLGA/acetonitrile) system,
0 in crystal or amorphous states. A very high protein loading
0 2 4 6 8 10 12 14
(> 30%) was obtained when protein was used in crystal state
Time (days)
in both systems. Long-term storage stability at 4 C suggested
that protein in crystal form was more stable in SAIB/ethanol
Figure 3. A. Effect of mannitol on release of myoglobin from and PLGA/acetonitrile systems with nearly 100% activity of
PLGA formulations. B. Effect of polymer concentration and protein. A negligible amount of initial burst was observed
additives on myoglobin release from PLGA formulations. during in vitro release from PLGA/acetonitrile system having
Reproduced with permission from [33]. crystalline amylase. Crystal shape also influenced the release
profile. Rod-shaped crystals had a significant high burst com-
pared to absence of burst effect with grain-shaped crystals.
densification of protein. A substantial control over release pro- As a variation to injectable forming implant system, an
file and negligible burst effect was observed with Zn-complexed injectable in situ forming microparticle system has been
and densified hGH compared to lyophilized and densified developed [39-41]. In situ microparticle (ISM) forming system
hGH [34]. A reduction in the initial burst was attributed to consists of an o/w or o/o emulsion prepared by dissolving
reduced aqueous solubility following complex formation and polymer, typically PLGA, in organic solvent such as NMP
densification of hGH protein. The influence of polymer molec- or triacetin. Following injection, microparticles are formed
ular weight, drug loading and polymer concentration on effi- in situ via solvent extraction. The emulsion system typically
cacy of leuprolide from PLGA implants prepared in has less viscosity compared to plain oil-based system, hence
NMP [35]. Neither drug loading (3 -- 6%) nor polymer concen- can be injected through a narrow gauge needle. Influence of
tration (40 -- 50%) had any influence on efficacy of leuprolide various process parameters on performance of injectable
implant in rat model. However, as expected, low--molecular- ISM (o/o PLGA emulsion system) has been investigated using
weight PLGA had shorter duration of action compared to cytochrome c (Mw: 12,327 Da) and myoglobin (Mw:
high-molecular-weight polymer. In canine model, one of the 16,950) [41]. PLGA (RESOMER grade RG 502 H) was dis-
formulation resulted in suppression of testosterone levels over solved in triacetin along with drug in PEG 400, where migloyl
3 months following single injection [35]. In an attempt to lower 812 was continuous phase. The dispersion was stabilized by

R. Vaishya et al.
addition of Tween 80 and Span 80. Upon injection, disper- aqueous solution along with polymer, which is encapsulated
sion forms solid matrix-type microparticles entrapping upon formation of gel. The characteristics of gel depend on
the drug (in situ formed microspheres) [42]. Similar to PLGA the concentration of polymer, molecular weight and type of
injectable implants, release rate was dependent on porosity polymer forming hydrophobic block. An advantage with ther-
of microspheres, molecular weight of PLGA and encapsula- mosensitive gel system over injectable implant is absence of
tion efficiency. A burst release of 30 -- 40% was observed for organic solvent, which is responsible for protein degradation
cytochrome c with ISMs. An increase in burst release was and in vivo toxicity. Hence, thermosensitive gel system is
observed for myoglobin release upon addition of mannitol. more compatible with tissue and therapeutic protein. In addi-
Presence of mannitol (5.4%) in ISMs significantly enhanced tion, aqueous solution-based formulation can be easily filter
burst release from 30 to 70%. The encapsulated myoglobin sterilized and injected easily via narrow gauge needles.
was extracted from ISM. The structure of extracted myoglo- Thermosensitive gel system composed of PLGA-PEG-
bin was found to be intact. However, with all modifications PLGA polymer is one of the widely investigated system and
in ISMs system, the maximum length of release was only commercially available under ReGel. ReGel forms clear
2 weeks. Influence formulation parameters such as molecular aqueous solution at room temperature at ~ 23% w/w that
weight of PLGA and PLA, polymer blending and polymer turns to gel at body temperature following injection. Sol-to-
concentration on in situ microsphere formation and leupro- gel transition in ReGel is reversible; hence, the gel turns
lide release rate and duration has also been investigated [39]. back to liquid upon decrease in temperature. ReGel has
Low-molecular-weight PLGA resulted in lower initial burst been investigated extensively to deliver peptide and proteins
compared to high-molecular-weight PLGA. It was attributed including lysozyme, porcine growth hormone (pGH), granu-
to slower rate of solvent extraction (NMP) in external aqueous locyte colony-stimulating factor, insulin and recombinant
media, which resulted in less porous dense microspheres. Fur- hepatitis B surface antigen [44-47].
thermore, PLGA with free acid end groups resulted in slower Influence of molecular weight of PLGA and PEG segments
rate of release compared to end-esterified PLGA, presumably on sol-to-gel transition and release characteristics of proteins is
due to electrostatic interactions between polymer and leupro- well documented [47]. Two ReGel polymers with PEG Mw
lide. High leuprolide loading of 15% was obtained in an ISM 1000 Da [ReGel-1] and 1450 Da [ReGel-2] were synthesized
system containing a polymer blend (PLA R 203H and R with a total molecular weight of 4200. It was shown that the
202H at ratio of 1:1, polymer concentration 30%). It released ReGel with PEG 1000 Da had lower critical gelling tempera-
leuprolide over 6 months (in vitro) with minimal burst ture compared to PEG 1450 Da at all concentrations. Sol-to-
release. gel transition was also reversible. In vitro release studies were
Despite the advancements in achieving extended release performed with Zn-insulin, granulocyte colony-stimulating
and near success in a few cases, application of injectable factor and pGH from ReGel 1 and 2 at 23% w/w. In all the
implants is mostly limited to the peptides. It is presumably cases, total release duration was 2 -- 3 weeks with a burst release
due to fragile 30 and 40 structures of proteins that are suscep- of 25 -- 40%, except for Zn-insulin from ReGel-2. A drastically
tible to degradation following exposure to organic solvents slow release rate with negligible burst of < 10% was observed
and oil-aqueous interface. Widely explored solvent NMP for Zn-insulin from ReGel-2 compared to ReGel-1. No expla-
has been shown to cause significant aggregation of protein nation was provided for this phenomenon. It was also shown
as discussed earlier. Although injectable PLGA depot system that complexation of insulin and pGH with Zn resulted in
(Eligard Injection Kit containing leuprorelin acetate in low initial burst. In an in vivo hypophysectomized rat model,
NMP) has reached market, cases of systemic allergic dermati- single injections of ReGel-1/pGH or ReGel-1/Zn-pGH
tis due to NMP have been reported [43]. To achieve extended (dose: 1 ml of solution containing 70 mg/ml pGH) was nearly
duration of release, a formulation must have higher drug load- as efficacious as daily injections of 5 mg of pGH for 14 days.
ing. With most of the systems investigated so far, drug loading Influence of polymer compositions and concentrations on
ranged from ~ 3 to 12%. Nevertheless, use of polymeric blend release characteristics has also been investigated using a model
systems (ISMs) and crystalline protein may provide better protein lysozyme [44]. Thermosensitive triblock copolymers
drug loading. (PLGA-PEG-PLGA) with different block lengths of PLGA
(PEG Mw 1000 Da) were synthesized. It was observed that
2.2 Thermosensitive gels the sol-to-gel and gel-to-sol transitions were dependent on
Thermosensitive systems are composed of aqueous polymer the molecular weight of each block and concentration. Lower
solution at room temperature. The solution undergoes sol- critical gelling temperature rises and upper critical gelling
to-gel transition to form a solid gel structure when injected temperature was falls with increase in molecular weight
due to change in temperature. Typical thermosensitive poly- (Figure 4) [44]. Moreover, the process of sol-to-gel transition
mers are amphiphilic block copolymers, where the hydropho- is not reversible for higher molecular weight polymers. In vitro
bic segment consists of PLGA, PLA, PCL or PPO. PEG is release studies were performed using lysozyme as a model pro-
widely explored as hydrophilic block due to its excellent water tein at 30% w/v polymer concentration. With increase in
solubility and biocompatibility. Protein can be dissolved in polymer molecular weight, the initial burst effect was also

60
Precipitate
50
40
Temp. (C)
30 Gel
20
10
Sol
0
0 5 10 15 20 25 30 35
Polymer (wt%)
Figure 4. The phase diagram of PLGA--PEG--PLGA triblock copolymer. Key: ( ) PLGA 995 --PEG 1000 --PLGA 995 ,
(----) PLGA1125--PEG1000--PLGA1125, (&) PLGA1350--PEG1000--PLGA1350, and (~) PLGA1400--PEG1000--PLGA1400.
PEG: Poly(ethylene glycol); PLGA: Poly(lactic-co-glycolic acid).
reduced from 41.2 5.4% (Mw 1602 Da) to 16.1 3.9% to-gel transition. These hydrogels were capable of sustaining
(Mw 7859 Da). Following initial burst, all the block copoly- the release of model proteins, BSA and horseradish peroxi-
mers exhibited slow release of lysozyme over a period of dase, for more than a month under in vitro conditions. Tak-
4 weeks. Similarly, initial burst effect was also shown to be ing the advantage of aqueous solution-based system, it was
inversely proportional to the polymer concentration. Triblock possible to prepare formulation with high protein loading
copolymer PLGA-PEG-PLGA (MW 1400-1000-1400) was (10% w/w). The rate and total duration of release were depen-
also utilized to deliver pGH [48]. In vitro release at 0.12 and dent on the polymer molecular weight and concentration.
0.42% w/v pGH exhibited nearly zero-ordered release at The release rate was slower for hydrogels with high molecular
30% w/v polymer concentration suggesting that diffusion weight with < 20% of total burst release. Similarly, release rate
was major mechanism of release along with some influence was lowered considerably upon increasing the polymer con-
of polymer degradation. At higher pGH concentration, the centration from 15 to 25 wt%. Duration of release was
release reached plateau phase after 4-week suggesting incom- more than a month with high-molecular-weight polymer
plete release. However, nearly 100% was released from gel (Figure 5) [57]. However, it is also worth noting that, in all sys-
containing 0.12% w/v pGH. Similar results were observed tems with high-molecular-weight gels, 40 -- 60% of BSA was
in rabbit model following s.c. injections at 0.12 and 0.42% released within first 15 days followed by an incomplete release
w/v doses. Bioavailability at low-dose formulation was 86%, in vitro. Only hydrogels from low--molecular-weight polymer
in contrast to 38% at higher dose. Poor bioavailability at (Mw 2600 Da) exhibited complete release within 20 days.
0.42% could be due to incomplete release of pGH from ther- In vivo gel formation and degradation of PCL-PEG-PCL
mosensitive gel. Incomplete release was attributed to protein copolymers indicated that the hydrogel lasted for > 45 days
aggregation, which occurs at higher proteins concentration in mice following s.c. injection. Gong et al. synthesized a
in aqueous solution [49-51]. Recently, it has been shown that series of triblock copolymers with different block arrangement
protein aggregation can be minimized in PLGA implants (PEG-PCL-PEG) having molecular weights 2992 -- 5046
using sugars such trehalose and histidine-HCl buffer using Da [58] and performed in vitro release with BSA as a model
bovine serum albumin (BSA) and model protein [52]. protein. The authors investigated influence of total protein
Another class of thermosensitive triblock polymers is based loading on release. Hydrogel with higher protein loading
on PCL rather than lactide or glycolide-based PLGA, PLA or resulted in lower initial burst and slower release rate. Total
PGA. One advantage in utilizing caprolactone-based polymer duration of release was 2 weeks. However, no formulation
is absence of acidic byproducts such as lactic acid and glycolic exhibited complete release of BSA. Formulation with less
acid, which are formed following degradation of PLGA. loading showed only 65% of release. Stability of released
These byproducts turn mirco-environment acidic, which BSA was examined by SDS--PAGE analysis. However, no
may be deleterious to proteins resulting in loss of activ- data on stability of BSA in the formulation after 2 weeks of
ity [53,54]. Hence, proteins released from PCL-based implants release was shown. PEG-PCL-PEG-thermosensitive polymer
have shown better stability comparatively [55,56]. Ma et al. syn- was utilized to deliver basic fibroblastic growth factor
thesized a series of PCL-PEG-PCL gelling polymers with (bFGF). bFGF-loaded hydrogel maintained strong humoral
average molecular weights ranging from 2600 to 4100 Da immune response for > 12 weeks in BALB/c mice. Release
[57]. All the polymers exhibited temperature-dependent sol- rate and initial burst were influenced by initial protein loading

R. Vaishya et al.
A. GL)1880-PEG1540-P(CL-GL)1880] triblock copolymer was

120 studied. In an in vitro study, polymer degradation occurred
at glycolide residues and the pH of medium remained near
Cumulative release (%)
100 neutral for initial 8 weeks [59]. Polymer can be expected to

P1
80 P2 have faster degradation compared to in vitro due to presence
of enzymes; nonetheless, it was not studied. BSA was used as
P3
60 model protein to study release pattern at low drug loading
P4
of 0.1% w/v in 25 wt% gel. A total of 80% protein release
40
was obtained over a period of a month via diffusion-
20 controlled release mechanism.
Thermosensitive hydrogel is simple, elegant and biocompat-
0
ible aqueous systems, which has shown a few promising results
0 5 10 15 20 25 30 35
as a standalone system to deliver protein over a period of
Time (day)
B.
2 -- 4 weeks. There are few important aspects, which should
90
be considered during preclinical development. One of the
important aspect of protein delivery is to gain high drug load-
80
Cumulative release (%)
ing in formulation. High drug loading can ensure therapeutic

70
concentration over a longer duration. Thermosensitive hydro-
60
gel can accommodate proteins in solution form at higher
50
concentration because it is aqueous solution-based system.
40 The system is also devoid of oil--water interface, which have
15 wt% 10% w/w
30
20 wt% 10% w/w been shown to be detrimental to protein structural integrity.
20 Nonetheless, numerous studies have reported incomplete pro-
25 wt% 10% w/w
10 tein release at higher drug loading. Irreversible protein aggrega-
0 tion (formation of trimers and tetramers) and degradation may

0 5 10 15 20 25 30 35
be possible explanation of incomplete in vitro release and
Time (day)
< 100% bioavailability in vivo. Such results are observed with
a variety of proteins at higher concentration. Hence, it is advis-
Figure 5. In vitro release of BSA from PCL-PEG-PCL hydrogels able to have lower drug loading in aqueous solution-based
in PBS at 37 C with marked copolymer and drug loadings. A. hydrogels. Another key aspect that may limit the clinical success
Effect of different compositions on the BSA release. The of thermosensitive gels is handling of gel during injection,
copolymer concentration was 20 wt%, and the drug loading which is complicated. The polymer is in solution phase at
was 10.0% (w/w). B. Effect of varying polymer concentra- 4 C with good injectability and low viscosity. The solution
tions on the BSA release from copolymer P2 formulations. forms gel at body temperature. However, viscosity of the solu-
Each point represents the mean s.d., n = 3. Key: P1 PCL1100- tion increases as it approaches the room temperature making it
PEG1500-PCL1100, P2 PCL1250-PEG1500-PCL1250, P3 PCL1500-
difficult to inject [58]. Sol-to-gel transition is a rapid process.
PEG1500-PCL1500, P4 PCL750-PEG1000-PCL750.
Hence, holding the vial or syringe containing thermosensitive
BSA: Bovine serum albumin. solution may result in accidental formation of gel, which can-
not be injected. Regel hydrogels exhibit reversible sol-to-gel
transition, but it is an impractical approach in clinical setting.
as shown earlier for BSA-loaded hydrogels. PCL-based ther- Besides, there are numerous reports available indicating incom-
mosensitive polymers provide advantage over lactide- and patibility of PLGA with proteins. A sophisticated device that
glycolide-based polymers because it degrades slowly due to maintains temperature of polymer solution should be devel-
its hydrophobic and semicrystalline nature resulting in gener- oped for practical clinical application.
ation of less acidic by products. The gel could last more than a
month in vivo. However, with most PCL-based systems total
duration of release is not > 3 weeks. Slower degradation may 2.3 pH-dependent in situ gels
lead to accumulation of empty delivery vehicle at the sight As the name suggest, implant formation and protein release
of injection. Several investigators have attempted to lower from these implants triggered by change in pH. The systems
crystallinity of PCL segment to achieve faster degradation is composed of polyionic macromolecules (polycation or
in vivo. In one such approach, triblock polymer with random polyanion) having pH-dependent aqueous solubility. Polymer
copolymer of glycolide and caprolactone as hydrophobic seg- is soluble in water due to ionization of functional groups.
ment were synthesized [59]. Degradation and drug release pat- Following administration, the pH change leads to precipita-
tern of hydrogel of poly(e-caprolactone-co-glycolide)-poly tion of polymer leading to gel formation. Such polymers
(ethylene glycol)-poly(e-caprolactone-co-glycolide) [P(CL- include alginates and chitosan (CS). Poly(methacrylic acid)

(+)
1 2 (-)
(+)
(-)
(+)
Scaffold
4 3
Figure 6. Schematic of LbL architecture shows that a 3DP scaffold (or glass surface) is repeatedly dipped with tetralayer units
consisting of (1) poly 2 (positively charged), (2) chondroitin sulphate (negatively charged), (3) BMP-2 (positively charged) and
(4) chondroitin sulfate. This tetralayer structure was repeated 100 times for all LbL films.
3DP: 3-dimensionally printed; BMP: Bone morphogenetic protein; LbL: Layer-by-layer.
is a polyanionic polymer, which forms hydrogel at acidic pH A number of pH-sensitive polymers have been developed
(pH < 5.8) [60]. and investigated for protein delivery via oral route [68,69]. How-
Layer-by-Layer (LbL) coating approach has been utilized to ever, chemical cross-linking may lead to toxic side effects due
develop protein-eluting implants [61,62]. Bone morphogenetic to cross-linking agents or unwanted reactions with proteins
protein 2 (BMP-2) was loaded on implants using polyelectro- drugs [70]. Dergunov et al. utilized g-irradiation to form
lyte coating with poly(b-aminoester)/poly2 (polycation) and cross-linked hydrogels consisting of CS and PVP for pH-
chondroitin (polyanion) (Figure 6) [61]. Implant released sensitive oral delivery of proteins [71]. The cross-linked hydro-
BMP-2 over a period of 2 weeks at physiological pH and gels showed pH-dependent swelling with marked swelling
showed promising results in vivo. In a similar study, poly under acidic pH. BSA exhibited highest binding with CS at
(L-lysine)/hyaluronic acid-coated granules were utilized to pH 5. Absorption of BSA in gel was also directly proportional
deliver BMP-2 [63]. Using similar technique, surface of anod- to porosity of gel, which in turn is dependent on the extent of
ized titanium implant was modified to achieve pH-dependent cross-linking and PVP content. BSA loading in cross-linked
protein release [64]. Implant surface was modified by polyelec- gel was driven by charge--charge interactions between CS and
trolyte coating of poly-L-histidine (polycation) and poly(meth- BSA molecules. Hence, negligible amount of BSA was released
acrylic acid) (polyanion). Fluorescently labeled poly-L-lysine (< 10%) from CSPVP hydrogels at acidic pH (pH 1.5), com-
(15 - 30 kDa) was utilized as model protein to study release pared to 40 -- 90% released at pH 7.4 within 24 h (Figure 7) [71].
from coated implant. LbL-coated implant exhibited pH- Similarly, in order to facilitate oral protein delivery and pre-
dependent release, with maximum release at pH 5 - 6. It also vent protein degradation at acidic gastric pH, Zhang et al.
demonstrated low levels of sustained release at physiological developed pH-responsive gels using p(PEGMA-g-MAA)
pH (pH 7 - 8). Length of release and burst release were found with triethylene glycol dimethacrylate as cross-linking agent
to be dependent on molecular weight of poly(methacrylic acid) [72]. Hydrogels also exhibited negligible release of model
and method of coating. Later, using the same technology, it protein BSA at acidic pH and nearly complete release within
was shown that LbL-coated implant could sustain the release 12 h, at pH 7.4, suggesting possible application of pH-
of BMP-2 and basic fibroblast growth factor over 25 days responsive hydrogels in oral protein delivery. No data on pro-
with nearly identical release profile for both proteins [65]. tein stability following encapsulation or release was presented.
CS is a biocompatible and biodegradable polyamine poly- As discussed, pH-controlled implants have not been able
saccharide [66]. It is soluble at acidic pH owing to ionization to provide extended duration of protein release. Hence,
of amine groups. However, at physiological pH, ionized hydrophobic polymer was incorporated along with pH
amines undergo deprotonation thereby precipitating the poly- sensitive segments to develop pH- and temperature-sensitive
mer to form gel. The gel structure formed by CS is porous due polymers [73-79]. A pH- and temperature-sensitive polymer
to crystallinity of polymer that degrades rapidly under in vivo was developed by grafting pH-sensitive sulfamethazine
conditions. Hence, it has limited success in achieving sustained oligomers (SMOs) to thermosensitive poly(e-caprolactone-
release of proteins. To overcome this limitation, CS was cross- co-lactide)--poly(ethylene glycol)--poly(e-caprolactone-co-
linked with polyvinylpyrrolidone (PVP) using glutaraldehyde lactide) (PCLA--PEG--PCLA) polymer [73]. The resulting
to lower crystallinity of CS and form gel at physiological polymer SMO--PCLA--PEG--PCLA--SMO remained as solu-
pH [67]. Hydrophobic modification with PVP also provided tion at high pH (pH 8) or at temperatures (70 C). Thus, it
mechanical strength to the gel structure. was possible to inject the solution at pH 8 with low chance

R. Vaishya et al.
100 degradation products, and neither should it alter any pharma-

cological properties of the active ingredient. It should also be
80 nontoxic, nonirritant and biocompatible if not biodegradable
[81]. Table 1 summarizes cases with various polymers selected
Release %
60 to prepare microspheres for controlled delivery of peptide

and proteins including degradation mechanisms.
40 Formulation of microspheres from biodegradable polymer
is initiated by selecting an appropriate encapsulation method
20 in order to achieve desired/ideal controlled release. Particle
size and polydispersity is another important aspect as it
0 directly influences syringeability, which is one of the impor-
0 4 8 12 16 20 24 tant requirement for an ideal microsphere system. During
t, h microsphere formulation process, there should be no alter-

ation in biological activity of encapsulated protein. Methods
in which exposure of protein/peptide in strong denaturing
Figure 7. BSA release profiles from CSPVP hydrogels at 0.1 M
solvent are not employed [82].
ionic strength. pH 7.4 at 37 C, CSPVP1 (&),CSPVP2 (D),
Spray drying method has also been employed in formulat-
CSPVP3 (.),CSPVP4 (&), release of BSA from hydrogels at
pH 1.5 (*), at pH 9 (~) and in water (x). Key: CSPVP1: Cs:PVP ing microspheres. Volatile organic solvents such as dichloro-
1:1 w/w crosslinked at 3.2 kGy, CSPVP2: Cs:PVP 1:1 w/w methane or acetone are used to dissolve biodegradable
crosslinked at 5 kGy, CSPVP3: Cs:PVP 1:2 w/w crosslinked at polymer. Then, the drug is dispersed into the polymer solu-
3.2 kGy and CSPVP4: Cs:PVP 1:2 w/w cross-linked at 5 kGy. tion in solid form by high-speed homogenization. Volatile
Reproduced with permission from [71]. organic solvent is then evaporated yielding microspheres of
1 -- 100 mm size range. Proteins such as recombinant human
of accidental gel formation during handling. The solution rap- erythropoietin were encapsulated in microsphere using spray
idly formed gel upon s.c. injection under physiological condi- drying method in nitrogen atmosphere [83]. Release of active
tions (pH 7.4 and 37 C) in rats. Injected gel was ingredient from microspheres occurs via different mechanisms
biocompatible and degraded in 6 weeks with no sign of inflam- such as diffusion, polymer degradation or combination of
mation. The SMO--PCLA--PEG--PCLA--SMO polymer was them [84]. Graphical representation of different mechanism
studied as a potential injectable scaffold and protein delivery responsible for drug release through microspheres is shown
system [74]. The polymeric hydrogel was capable of encapsulat- in (Figure 8).
ing human mesenchymal stem cells and recombinant human Chronic ulcerations of the skin have been reported due to
BMP-2 with high encapsulation efficiency. The formulation the deficiency of prolidase enzyme. This enzyme is involved
was examined in vivo and was shown to be effective over in the later stages of protein catabolism. To overcome this
7 weeks following injection. problem, PLGA microspheres encapsulating prolidase were
formulated by w/o/w multiple emulsion technique. Results
obtained from ex vivo studies demonstrated that microencap-
3. Microparticles sulation imparted stability to protein inside the polymer
matrix leading to release of active enzyme from the formula-
Controlled release of protein from parenteral formulation can tion. This technique can be employed for enzyme replace-
be achieved by a matrix-type delivery system. Microsphere ment therapy [85].
formulation holds distinct advantages and has gained popu- Similarly, another formulation of PLGA microspheres has
larity in recent years [80]. More than 200 studies have been been prepared with b-lacto globulin (BLG). Newborns are
published per year pertaining to biodegradable microsphere prone to allergies related to milk proteins, which can be pro-
formulations for peptide and protein delivery. Advantage of hibited by prompting oral tolerance to these proteins. PLGA
employing microspheres formulation for delivery of proteins microspheres encapsulating a major allergenic protein, BLG,
is that it offers stability for molecules, which are rapidly were formulated by w/o/w double emulsion technique. Con-
degraded or eliminated in vivo. Encapsulation in microsphere trolled release of proteins and higher encapsulation efficiency
prevents contact of protein from cells or enzymes such as pro- was reported after introduction of Tween 20 in the formula-
teases/esterases in surrounding tissues, until it has been tion. Improved encapsulation efficiency and controlled release
released from microsphere. Microsphere formulation can be resulted in the reduction of dose required for specific anti-
easily delivered to the target sites via i.v., i.m. or oral route. BLG IgE response following oral administration [86].
Owing to the recent advancements in polymer science and A microsphere formulation encapsulated with IFN-a and
synthesis technology, development of controlled release comprising of calcium alginate core surrounded by PELA
microsphere formulations has gained immense popularity. (poly D, L-lactide-poly ethylene glycol) was prepared by
The polymers should not produce harmful side effects or toxic w/o/w multiple emulsion technique. Coated microspheres

Table 1. Various polymers used to prepare microspheres for controlled delivery of peptide and proteins.
Polymer Material Degradation Biodegradation Active pharmaceutical Ref.

nature mechanism ingredient
Natural Starch Amylase Biodegradable Insulin [130]

Alginate pH, enzymes Biodegradable Protein [131]
Chitin pH, enzymes Biodegradable BSA [132]
Chitosan pH, enzymes Biodegradable Antigens, BSA, salmon [133]
calcitonin
Collagen/gelatin Collegenase Biodegradable Hydroxyapatite [134]
Corn protein (zein) Enzymes Biodegradable Ivermectin [135]
Cross-linked albumin Enzymes Biodegradable Virus antigen [136]
Hydroxyapatite Dissolves by the time Biodegradable Bone morphogenic [137,138]
protein, recombinant
human
glucocerebrosidase
Hyaluronic acid Hyaluronidase Biodegradable BSA [139]
Synthetic Azo-cross-linked copoly- Reduction of azo bonds Partially degradable Insulin and vasopressin [140]
mer of by
styrene and HEMA-coated microflora in large
particles intestine
Maleic anhydride/poly Enzymes Partially degradable Dextran [141]
(N-isopropylacrylamide)
hybrid hydrogels
Poly sebacic anhydrides Hydrolysis Biodegradable Rhodamin B [142]
Polyesters/poly lactides Ester hydrolysis by Biodegradable Somatostatin analogues [143]
esterases
Polyorthoesters Hydrolysis Biodegradable BSA
[144]
PLGA Hydrolysis Biodegradable Leuprolide acetate, [89,145]
goserelin acetate,
triptorelin, integrilin,
insulin
Polycaprolactones Hydrolysis Biodegradable BSA, insulin, nerve [146]
growth factor
Poly etilen oksit/amino Enzymes Biodegradable Poly(L-aspartic acid), [147]
acids plasmid, DNA,
cyclophosphamide
Polyphosphazenes Hydrolysis, dissolution Biodegradable Naproxen, BSA [148,149]
BSA: Bovine serum albumin; PLGA: Poly(lactic-co-glycolic acid).
impart stability to IFN-a in the PELA matrix. These micro- 4. Nanoparticles/nanospheres

spheres also demonstrated enhanced encapsulation efficiency
and retention of biological activity as compared to micro- Diameter of smallest capillaries in human body is around
spheres produced by conventional method [81]. In another 5 -- 6 mm. Polymeric nanoparticles have size of < 1 m in
study, PLGA microspheres encapsulated with salmon calci- diameter. These are prepared from natural or synthetic poly-
tonin exhibited 5 -- 9 days release following s.c. injection mers. In order to avoid deleterious effects (such as embolism)
in rats [87]. Blends of PEG with PLA homopolymer and due to particle aggregation, the size of particles being admin-
PLGA copolymer were utilized to prepare insulin-loaded istered in bloodstream should be < 5 m [90]. Ability to deliver
microspheres by w/o/w multiple emulsion technique. The comprehensive range of drugs to different target sites over
resulting microspheres exhibited high entrapment efficiency prolonged period made nanoparticles as a carrier of choice.
and provided controlled release of insulin for 28 days [88]. Different routes can be selected depending on the target site
In another study, ZnO-PLGA microspheres containing in order to deliver a wide variety of therapeutics via
insulin demonstrated rapid and long-lasting suppression of nanoparticles.
glucose levels for 9 days following s.c. administration in Insulin-containing poly (ethyl cyanoacrylate) nanoparticles
rats [89]. have been investigated to study the effect of several formula-
Characteristics of various protein-encapsulated micro- tion variables. The results obtained demonstrate high yield
spheres formulated with various different methods are shown (90%) with low polydispersity index for nanoparticles. Size
in Table 2. Table 3 summarizes the clinically approved of nanoparticles ranged between 130 and 180 nm, which
sustained-release microparticle formulations. was influenced by the mass of monomer used in nanoparticle

R. Vaishya et al.
Diffusion short duration (6 h), and released insulin was biologically

Polymer degradation
active [98].
Drug release Combination of diffusion and erosion PLGA is one of the most widely used biocompatible and
biodegradable polymer to prepare nanoparticles. It is also
available with varying lactide and glycolide content. However,
Enzymatic it causes instability of the encapsulated protein/peptide mole-
Hydrolysis cule. For example, release of BSA from PLGA implants
Polymer prepared by hot-melt extrusion was incomplete. Incomplete
Combination of enzymatic and hydrolysis
degradation
release was because of acylation of BSA [99]. Gaudana et al.
successfully formulated and characterized hydrophobic ion
pairing (HIP) complex of lysozyme (15 kDa), by employing
Surface erosion
dextran sulfate, a sulfated polysaccharide complexing agent
Bulk degradation
Hydrolysis [100]. Spontaneous emulsion solvent diffusion method was uti-
lized to prepare nanoparticles. Sustained release of lysozyme
up to 30 days without significant burst release was observed
from the prepared nanoparticles. Released lysozyme as well
as lysozyme-dextran sulfate complex showed no change in
Figure 8. Drug release mechanism from microspheres. their enzymatic activity. Sustained release of larger protein
molecules such as antibodies can be achieved by HIP com-
formulation. Similarly, the release rate was also influenced by plexation method [100]. In another study, Gaudana et al. suc-
mass of monomer used in nanoparticle formulation, showing cessfully formulated and characterized HIP complex of BSA
zero-ordered release over 10 days [91]. (66 kDa), by employing dextran sulfate. Solid-in-oil-in-water
Proteins and peptides exhibit instability in PLGA due (S/O/W) emulsion method was used to prepare nanoparticles.
to hydrophobic nature of PLGA and acidity of PLGA degra- No change in the secondary and tertiary structures of BSA was
dation products [92]. In addition, burst release of proteins/ reported due to HIP complexation as well as nanoparticle
peptides from PLGA matrices is considered as a major draw- preparation. These results were confirmed by circular dichro-
back. These concerns have been addressed by a number of ism and intrinsic fluorescence analysis [101,102].
investigators by modifying the properties of PLGA matri-
ces [93,94]. In a study, BSA was encapsulated in PLGA-PVA 5. Miscellaneous modes of protein delivery
composite nanoparticles [91]. The nanoparticles were having
nonporous surface and exhibited extended release of BSA 5.1 Nanoparticles-in-gel composite systems
over 2 months. The presence of terminal carboxyl group in A considerable attention has been paid to sustained delivery of
PLGA leads to enhanced protein loading and continuous protein therapeutics via encapsulating in nano/micro par-
release of protein over 20 days from PLGA nanoparticles [95]. ticles. Nano/micro particles provide a stable environment for
In contrast, presence of esterified carboxyl end groups in peptide/protein against catalytic enzymes, allowing improved
PLGA leads to diminished protein loading and release over biological half-lives. However, protein-loaded particulate
14 days from nanoparticles [95]. drug delivery systems exhibited major disadvantage of burst
Fishbein et al. reported release kinetics of nanoparticles effect (dose dumping), which may result in severe dose-related
encapsulated with PDGF-receptor b (PDGFR-b) tyrphostin toxicity. Nanoparticle-in-gel composite systems have been
inhibitor, AG-1295. Spontaneous emulsification/solvent investigated to minimize the burst release of proteins and
displacement technique was employed to formulate AG-1295- provide localized delivery [103,104].
loaded poly(DL-lactide) (PLA) nanoparticles. Nanoparticle of Posterior segment ocular diseases such as wet-AMD,
120 nm size showed 25% of burst release followed by gradual diabetic retinopathy, and diabetic macular edema are sight-
release of AG-1295 (50%) over a period of 30 days [96]. threatening disorders mainly observed in elderly patients.
Kawashima et al. reported a novel delivery system for a physio- Many protein therapeutics such as bevacizumab, ranibizumab
logically active peptide encapsulated in PLGA nanoparticles and aflibercept are repeatedly injected (intravitreally) for the
(diameter 400 nm). These nanoparticles were formulated by a treatment of above-mentioned ocular diseases. Repeated
modified emulsion solvent diffusion method. Glucose level in intravitreal injections lead to many complications such as
guinea pigs reduced significantly over 48 h after pulmonary endophthalmitis, retinal detachment and retinal hemorrhage,
administration of aqueous suspension of PLGA nanoparticles. and more importantly patient noncompliance. Recently,
Formulation produced initial burst release of 85% followed Mitra and Mishra have described protein encapsulation in
by a sustained release of the remaining drug for a few hours [97]. pentablock (PB) polymer nanoparticles dispersed in PB-
With the phase-inversion nanoencapsulation technique, zinc- thermosensitive gel for the sustained delivery of proteins
insulin was encapsulated using various polyester and polyanhy- following intravitreal delivery [105]. PB copolymers exhibited
dride nanoparticle formulations. Insulin release lasted for a excellent biocompatibility with negligible toxicity. A

Table 2. Examples of protein-encapsulated microspheres formulated using different methods.
Release profile and Burst Particle size Polymer Protein encapsulated Activity of released Ref.
characteristics release (%) (mm) protein
W/O/W method
Slow release to 55% in 15 days 38 75 PLGA Staphylokinase variant Retain ~ 85% activity on [150]
K35R (DGR) day 15
Slow release to 65 -- 70% in 20-55 0.2-167 PLGA BSA, VEGF - [151,152]
14 days
Extremely slow release (linearly) 3 46 PLGA BSA - [153]
to 12% in 30 days
Extremely slow release (linearly) 9 19 PLGA Lysozyme - [153]
to 16% in 30 days
Extremely slow release to 7.5% 5 15 PLA Ovalbumin - [154]
in 25 days
Slow release to 29% in 10 2-8 PLA Tetanus toxoid (TT) In vivo serum highest [155]
120 days anti-TT antibody tires
164 g/ml
Slow release to 35% in 40 days 7 - PLGA/PLA Insulin - [156]
Gradual release to ~ 55% in 20 2 PLGA Human chorionic Serum IgG antibody [157]
60 days gonadotropin (hCG) responses up to 12 weeks
Gradual release to ~ 85% in 33 28 PLGA BSA - [158]
35 days
Gradual release to 90 -- 100% 25-55 17-20 PLGA BSA Dematan sulfate reduced [159]
in 24 days insoluble BSA formation
Gradual release to 45 -- 70% in 20-40 17-24 PLGA Insulin Chondroitin sulfate A [160]
34 days preserved secondary
structure of insulin up to
Expert Opin. Drug Deliv. (2014) 12(3)

20 days
Gradual release to 60% in 22 75 PLA Recombinant human Gastric ulcer was cured [161]
11 days epidermal growth factor 82% at day 11 after
administration with dose
of 220 g/kg
Gradual release to 63 -- 84% in 25-43 46-110 PLGA IFN-a2b - [162]
30 days
Gradual release to 80% in 41 1.6-2 PLGA SPf66 malarial antigen Retain in vivo activity up [163]
190 days to week 27
Continuous release <1 78 PLGA and chitosan BSA - [164]
to 45% in 45 days
Continuous release to 87 -- 95% 4-26 35-105 Acetylated pullulan Exenatide No peptide degradation [165]
in 20 days up to day 16
Nearly zero-order release to <5 24 PLGA Lysozyme Retain 90% of bioactivity [166]
95% in 65 days on day 60
A slow and sustained release of - 0.4-1.6 PLGA Matrix metalloproteinase- Sustained degradation of [167]
the protein over 20 days 3 (MMP-3) fibronectin up to 10 days
BSA: Bovine serum albumin; PLA: Poly(lactic acid); PLGA: Poly(lactic-co-glycolic acid).
13
14
Table 2. Examples of protein-encapsulated microspheres formulated using different methods (continued).
without a significant initial burst

R. Vaishya et al.
release
S/O/W method
Initial burst release of ~ 20%, 20 40-100 PLGA/PLA Erythropoietin (EPO)- [168]
followed by sustained release till loaded dextran
60 days
Gradual release to 50% in 26 89 PLGA Insulin In vivo glucose [169]
30 days concentration was below
20 mmol/l for 48 h
Gradual release to 90% in 38 5 PLGA a-Chymotrypsin - [170]
35 days, no further release in
next 25 days
Gradual release to ~ 95% in 35 31 PLGA Recombinant human - [171]
16 days growth hormone
Gradual release to 100% in 30-35 40-100 PLGA BSA, horse myoglobin - [172]
62 days
Gradual release to 82% in 22 - PLGA g-Chymotrypsin Retain 40% activity on [173]
30 days, slow release to 100% day 7
in next 50 days
Gradual release to 60% in 10 - PLGA Insulin In vivo blood glucose level [174]
15 days drop to normal between
days 8 and 10
Gradual release to 97% in 7 11 PLGA Ornitide acetate - [175]
57 days
Nearly zero-order release 1 < 10 PLGA, PEG and PLA Bovine superoxide Retain 100% activity in [176]

to ~ 75% in 28 days dismutase (bSOD) dried microspheres
Nearly zero-order release 10 52 PLA Leuprolide In vivo testosterone levels [177]
to ~ 90% in 150 days were suppressed to
0.5 ng/ml from day 4 to
day 50
Gradual release to 100% in 20 21-24 PLGA Bovine insulin - [178]
130 days
W/O/O (Coacervation) and S/O/O Method
Very slow release to 17% in 12 44 PLGA Tenanus toxoid In vivo tetanus toxin [179]
50 days IgG antibody remained
~ 2 AU/ml for 4 weeks
Fast release to 75% for 25 days 25 2 PLGA Insulin Secondary structure was [180]
followed by slow release to maintained after
90% for next 50 days encapsulation
Gradual release to ~ 75% in 20 84 PLGA Cytochrome c - [181]
15 days
6-11 123 PLGA BSA - [182]
Table 2. Examples of protein-encapsulated microspheres formulated using different methods (continued).
Slow release to 2 -- 14% in

18 days and gradual release to
63% for next 55 days
Gradual release to 19 -- 41% in 4-13 18-49 PLGA Endostatin - [183,184]
28 days
Gradual release to ~ 35% in 5 52 PLGA BSA - [185]
20 days
Spray drying method
Fast release to ~ 98% in 8 days 30 - PLGA BSA - [186]
Gradual release to 80% in 31 - PLGA Vapreotide acetate - [143]
28 days
Gradual release to 94% in 8 10 PLGA Insulin - [187]
35 days
Gradual release to ~ 100% in 26 - PLGA Recombinant human - [188]
30 days insulin-like growth
factor-1 (rhIGF-1)
Gradual release to > 90% in < 10 - PLGA Recombinant human In vivo generate a [189]
35 days vascular endothelial significant angiogenic
growth factor (rhVEGF) response
Gradual release to ~ 100 in 1 - PLGA Recombinant human - [190]
25 days nerve growth factor
(rhNGF)
Nearly zero-order release < 10 12 PLGA Insulin - [191]
to ~ 80% in 45 days

Ultrasonic atomization method
Slow release to 20% in 24 days 5 85 PLGA Lysozyme - [192]
Gradual release to ~ 75% in 28 - PLGA Vapreotide pamoate - [193]
28 days, no further release in
following days
Gradual release to ~ 95% in 33 - PLA BSA - [193]
98 days
Gradual release to ~ 95% in - 36 PLGA BSA - [194]
98 days
Other methods
Fast release to 42 -- 75% in 38-53 26-128 PLGA/PLA Recombinant human Retain 100% activity after [195]
4 days growth hormone (rhGH) encapsulation
Slow release to ~ 28% in 3 days <5 40 PLA BSA - [196]
Nearly zero-order release <1 20 PLGA BSA - [197]
to ~ 70% in 38 days
Nearly zero-order release 6.2 - PLGA/Pluronic F127 rhGH - [198]
to ~ 75% in 38 days
Gradual release to ~ 100% in 22 20 PLA BSA - [199]
72 days
15
R. Vaishya et al.
Table 3. Clinically approved sustained-release microparticle formulations.
Trade name Drug Indications Delivery system Length of release Approval

year
Lupron Depot (TAP) Leuprolide Prostate cancer, Injectable PLGA 1-, 3- and 4-month 1989
endometriosis microparticles formulations
(intramuscular injection)
Sandostatin LAR Octreotide Acromegaly Injectable PLGA 1-month 1998
(Novartis) microparticles
Somatuline LA (Ipsen) Lantreotide Acromegaly Injectable PLGA 10 -- 14 days 1998
microparticles
Nutropin Depot Human growth Growth hormone Injectable PLGA 1 month 1999
(Alkermes/Genentech) hormone (hGH) deficiency microparticles
(subcutaneous injection)
Trelstar Depot Triptorelin Prostate cancer Injectable PLGA 1- and 3-month 2000
(Debiopharm) microparticles formulations
Risperdal Consta Risperidone Schizophrenia Injectable PLGA 2 weeks 2003
(Alkermes/Janssen) microparticles
composite formulation (protein-encapsulated PB NPs dis- eliminate the requirement of repeated dosing and possible
persed in PB-thermosensitive gel) exhibited nearly zero-order dose-related toxicity. Promising preliminary results observed
release with no burst effect. Recently, we have discussed the with the composite delivery system in the treatment of
applicability of a PB polymeric nanoparticles and nanopar- chronic diseases may prove to be a cost-effective, highly
ticles-in-gel composite formulation for the sustained delivery patient-compliant therapy.
of proteins in the treatment of posterior segment ocular dis-
eases [104,106]. Results demonstrated that model proteins 5.2 Non-biodegradable implants
(BSA, IgG and bevacizumab) -loaded PB composite formula- Application of non-biodegradable implant for protein and
tions exhibited nearly zero-order release up to 45 -- 60 days. peptide delivery is not prevalent relative to other modes of
Burst effect in nanoparticles is observed due to immediate delivery. Very few systems have been developed so far, despite
release of surface adsorbed proteins. However, when the the systems such as silicone elastomer (non-biodegradable
NPs are dispersed in thermosensitive gel matrix, an additional hydrophobic polymer) and osmotic implants are known for
diffusion layer provided by gelling polymer hinders the release a long time. One limitation with these systems is that it
of surface adsorbed drug resulting in elimination of burst requires surgery to administer the implant, which may be
effect. In addition, biological activity of the protein molecules complicated or simple procedure depending on the type of
was confirmed by in vitro experiments. disease. Surgical removal of non-biodegradable implant is
Administration of VEGF in appropriate dose may prove as necessary once the release is complete or in case of any unfa-
a promising treatment for the deficient bladder reconstruction vorable/complicated conditions such as infection.
therapy. However, short half-lives and high instability due to Polysiloxanes, silicones or siloxanes are class of organo-
deamidation, diketopiperazine formation and oxidation of silicon synthetic materials. They have been useful in a wide
proteins (VEGF) resulted in disappointing clinical trials. range of biomedical applications due to their biocompatible
Due to high instability, large dose of protein is required, nature, low toxicity and inertness. Polymer can be synthesized
which often attributed to sever side effects such as progression to impart wide range of properties, ranging from water-like
of malignant vascular tumors [107]. In order to achieve liquid, heavy oil-like fluids, greases, rubbers or solid resins
sustained release with minimum burst release of VEGF, depending on the molecular weight of polymer and organic
Geng et al. prepared VEGF-encapsulated PLGA nanoparticles groups attached to silicon atoms. Due to ease of fabrication,
and dispersed them in pluronic-F127-thermosensitive poly- a number of delivery systems including matrix, reservoir and
mer solutions [108]. VEGF-NPs exhibited up to ~ 40% of drug eluting stents system have been developed to provide
burst release within the first 2 days, which is significantly controlled release of various therapeutic agents. Hsieh et al.
reduced to ~ 15% with the formulation of VEGF-NPs first demonstrated applicability of silicon-based polymer
dispersed in thermosensitive gel (Figure 9) [108]. Controlled implants in protein delivery with model protein BSA [109].
delivery of VEGF from a biocompatible delivery system may Kajihara et al. have suggested applicability of silicon-based

100
90
80
VEGF cumulative released (%)

70
60
50
Released from F127 gel
40
Released from NPs

30
Released from NPs-F127
20 gel embedded in matrix
10
0
0 10 20 30 40 50 60
Time (days)
Figure 9. In vitro release of VEGF from pluronic-F127 gel, PLGA-NPs and PLGA-NPs dispersed in pluronic-F127 gel.
implant systems for highly potent protein, which would following a s.c. administration. Tumor suppression was
require low dose using IFN as an example [110]. IFN was observed for > 100 days [110]. Maeda et al. also developed cov-
encapsulated in the silicon formulation in solid particle ered-rod-type formulation with silicone polymer to investi-
form. Interestingly, release rate was found to be directly pro- gate controlled release of small and protein (BSA)
portional to the particle size of encapsulated protein. Release molecules [112]. Sucrose was used as additive and encapsulated
rate was also dependent on protein loading. Release profile along with BSA in the implants. Again, the release was very
was continuous zero-ordered over a month with a total 40% tunable in these systems [112]. In another study, matrix type
protein release. Higher release rate at large protein particle and covered-rod-type injectable implants were compared to
size and loading was attributed to formation of a large number deliver protein using either the model antigen avidin or
of interconnected channels upon protein release. In addition, Clostridium tetani and Clostridium novyi toxoids in sheep [113].
it was shown that the release rate could be further accelerated The matrix type implants delivered antigen in vitro in a first-
by simple addition of glycine to the IFN particles. Presence of ordered manner over a period of a month. In contrast,
glycine, as low as 2%, drastically enhanced release rates. Drug covered-rod-type implant released low amounts of antigen at
release mechanism was attributed to the formation of inter- zero-order for longer duration. Covered-rod-type implants
connected channels in implant upon initial protein release. were shown to produce better immune response from antigen
These channels allowed dissolution of protein inside implant exposure for longer duration and favored production of
causing a rise in osmotic pressure leading to formation of both IgG1 and IgG2 isotypes. Thus, silicon-based implants
cracks, which further facilitated release. To further control have shown promising results in controlling protein release
the release of IFN, covered-rod-type implants were devel- over a period of months, both in vitro and in vivo. Most of
oped [111]. IFN release from these implants was highly tunable these systems also exhibited nearly complete release of
with addition of presence of various additives, protein content proteins, which could be a significant advantage in addition
(% loading) and surface area of implant. Covered-rod-type to biocompatible and nontoxic nature of these systems. Fur-
formulation released IFN at a constant rate over 30 -- thermore, implants can be prepared under mild conditions
100 days in vitro without significant initial burst. The release without heat and organic solvents, which are known to
rate was correlated to the osmotic pressure, that is, higher the degrade proteins [111].
osmotic pressure, higher the release rate. Pharmacokinetic Zero-ordered delivery is the most desired property of an
studies in mice showed detectable levels of IFN after extended-release formulation. Zero-ordered release ensures
60 days [110]. IFN released from this formulation exhibited that drug concentration will remain within the therapeutic win-
very high antitumor activity in nude mice tumor model dow, without reaching toxic or subtherapeutic levels.

R. Vaishya et al.
A. 1 year at constant rate at 37 C in unstirred PBS [116]. Similar

in vitro release profiles have been observed with sufentanil
Membrane Reservoir from the DUROS chronogesic system [120] and IFN from the
Piston Orifice OMEGA DUROS system [114]. Nevertheless, most impressive
character of these implants is the IVIVC of release data, which
simplifies the process of implant development. Figure 10B illus-
trates in vivo/in vitro performance comparison for cumulative
drug delivery in rats [116]. The results showed a good agreement
Osmotic engine Drug
Diffusion between cumulative delivery in vivo and in vitro over a period of
formulation
moderator 12 months. Moreover, the release rates were also very compara-
B.
ble between in vitro and in vivo release from different batches.
Similar studies in canines also showed similar trends. Good
Cumulative leuprolide delivered
120%
lot 1 agreement in IVIVC may be attributed to the stability of pep-

100% lot 2
tide throughout the release period. Stability of protein/peptide
lot 3
80% for such extended duration is very critical for the success of
such implant systems.
60%
40% 5.3 Photoactivated depots

A novel delivery system to control delivery of insulin external
20% stimulus has been developed [121]. Daily multiple administra-
tion of insulin is a primary treatment strategy for type 1
0% In vivo In vitro In vivo In vitro In vivo In vitro In vivo In vitro diabetes. It is possible to self-administer insulin; nonetheless,
3 6 9 12
multiple injections every day is a significant lifetime burden
Time (months)
on patients. In order to minimize number of administrations
and control plasma insulin levels, photoactivated depot

Figure 10. A. Cross-sectional diagram of the DUROS (PAD) was developed. PAD consists of insulin chemically
implant. B. In vivo/in vitro performance comparison for conjugated to an insoluble, biodegradable resin via photo-
cumulative drug delivery in rats. labile group (Figure 11A) [121]. PAD can be injected s.c., and
Reproduced with permission from [116]. insulin release can be controlled externally using irradiation.
In an in vitro release study, investigators showed that insulin
Osmotically controlled, mechanical devices, release was completely dependent on irradiation (light-
nonbiodegradable implants have been designed for extended emitting diode at 365 nm) (Figure 11B) [121]. In absence of
delivery at a constant rate. These systems have been shown to irradiation, no insulin release was observed. Insulin is suitable
be biocompatible and effective to provide constant therapeutic to this approach, since only a small amount is required daily.
concentrations in a number of animal and human trials [114-119]. It may be difficult to utilize such technique for larger proteins.
Some of the osmotically driven implants include Duros Drug loading may be an issue for peptides, which require
(DURECT), ALZET (veterinary medicine, research applica- higher plasma concentrations. In this case, the size of the
tion) and Viadur (leuprolide acetate, marketed formula- depot may be larger and hence irradiation at longer wave-
tion) [119]. Precise control over drug release for extended length may be required to cause deep tissue penetration.
duration (months to year) is achieved by osmotic pressure.
For example, Duros implant is a cylindrical (4 mm in OD 6. Expert opinion
and 45 mm in length), developed for human use and can be
implanted s.c. [116]. As illustrated in Figure 10A, the implant Proteins are promising bio-therapeutics. A number of protein
consists of an osmotic engine (chamber containing sodium therapeutics is currently marketed, and it is projected to
chloride, osmotic agent), a piston and a reservoir chamber for acquire a major share of pharmaceutical market in the future.
drug [116]. A semipermeable rate-controlling membrane covers As discussed earlier, proteins suffer from a number of
the side of cylinder housing osmotic engine. Presence of delivery-related issues resulting in poor in vivo half-life lead-
osmotic agent causes water to permeate through the semiperme- ing to multiple parenteral administrations. Sustained and
able membrane. The osmotic pressure then pushes the piston controlled delivery of proteins can address some of these
towards drug chamber that in turn allow drug release through issues. A number of novel approaches have been investigated
a small opening on the reservoir side of implant. The volume to deliver proteins at sustained rate in controlled manner.
of drug reservoirs is ~ 150 l, and thus implant is best suited In situ gel, microparticles and osmotically driven implants
for potent therapeutics. Viadur is an adaptation of the DUROS have been successful and hence, clinically available for various
implant technology to deliver leuprolide acetate for the treat- ailments. Nevertheless, it has been acknowledged by a number
ment of prostate cancer. Leuprolide release was observed for of investigators that development of practical sustained

A. lactide- and glycolide-based copolymers are widely used poly-

hv mers for sustained protein delivery. However, degradation
products of PLGA lead to acidic microenvironment, which
is detrimental to the stability of peptide and protein. Protein
release from formulations is either dissolution, diffusion or
Shallow injection of insulin
linked to insoluble matrix degradation controlled. Dissolution-controlled release mecha-
hv
nism cannot sustain protein release for longer duration
(> 2 weeks). On the other hand, encapsulation of protein in
polymer matrix (injectable implants and particles) may pro-
Insoluble matrix + vide long-term delivery of proteins. Protein instability is one
of the major obstacles in the clinical development of protein
Photocleavable linker drug delivery systems [12]. There are many stages of process
Immediate diffusion development, such as encapsulation and release, where protein

Insulin Slow biodegradation
of matrix of insulin into body instability may arise. Use of organic solvent during the prep-
B. aration of these matrix formulations has been shown to dete-
riorate protein structure. Protein degradation is often due to
20 exposure to organic solvent or aqueous/organic interface, as
noted by numerous studies [122,123]. In an ideal delivery sys-
15 tem, protein structural integrity should be maintained
throughout the release period. Aqueous solution-based tech-
nmoles released
niques have been examined to avoid use of organic solvents

10
but there is limited success. It has been well documented
that the proteins at high concentration in aqueous solution
5 undergoes irreversible aggregation (formation of trimers and
tetramers) resulting in degradation. Hence, many of the

0 in situ gel systems with high loading have reported incomplete
0 8 16 24 32 40 48 56 64 release of protein in vitro as well as in vivo. Various solvent-
Total time/min free techniques such as use of supercritical CO2 [124,125] are
under development and may provide answer to this problem.
Hydrolysis of proteins at the acidic pH within microenviron-
Figure 11. A. Overall approach to the photoactivated depot ment of polymer matrix, in case of PLGA polymers, has also
(PAD). A drug, insulin in this case, is linked to an insoluble been indicated for protein degradation. Moreover, numerous
but biodegradable resin, through a photocleavable linker.
reports are available indicating covalent modification of
The conjugate is injected in a shallow depot cutaneously or
protein with polymer degradation products within acidic
subcutaneously. Irradiation breaks the link of insulin from
the resin, thereby allowing it to diffuse away from the resin
microclimate. Burst release is another negative feature of the
and be absorbed by the systemic circulation. Ultimately, the polymeric delivery systems such as in situ implants, micro-
resin is biodegraded. B. Stepwise photolysis of the photo- and nanoparticles. Burst release is due to surface adsorption
activated insulin depot. Cumulative moles of insulin released of protein in case of particles. For injectable implants, it is
from the modified resin when using an LED point source dependent on several factors such as solvent polarity and
that was turned on and off repeatedly. Light and dark bars rate of solvent diffusion. Burst release results in loss of signif-
indicate periods of irradiation and darkness. icant amount of protein, which may lead to dose-dependent
Reproduced with permission from [121]. toxicity and lower duration of release. A number of strategies
have been examined such as use of less polar organic solvent in
controlled delivery formulation is challenging. Important var- case of injectable implants and nanoparticle-in-gel approach
iables affecting development of product delivering proteins to minimize the burst effect. In case of matrix type polymeric
for extended duration include method of encapsulation (use delivery systems, drug release is controlled by diffusion and
of organic solvents, water--organic interface and scale up), erosion of polymer matrix. Polymer degradation can be con-
protein loading, stability (during encapsulation, within for- trolled by manipulating composition, molecular weight and
mulation and after release), burst release, release pattern, pro- hydrophobicity. However, it is challenging to achieve com-
tein--polymer and protein--excipient interaction/compatibility plete control over the release pattern from these systems.
and IVIVC for release profiles. Furthermore, proteins impose A few approaches such as nanoparticle-in-gel have shown
unique challenges due to their complex structure, large molec- promising results. An ideal sustained release formulation
ular weight and volume leading to slow diffusion. Hence, one should provide extended duration of release at a constant
should be wary that approaches applicable to peptide and rate that is, zero-ordered release. Non-biodegradable silicon-
small molecules might not work for proteins. For example, based polymer implants and osmotically driven implants

R. Vaishya et al.
have shown substantial promise in achieving sustained release solution of proteins is used, the solution has to be concen-
at constant zero-order for several proteins and peptides. trated to accommodate large dose in limited space of drug
Despite being biocompatible, limitation with these systems chamber (volume ~ 150 l). Tendency of proteins to form
is that they require surgery to insert and remove the implants. irreversible aggregates at higher concentration may limit
Nevertheless, these are the most successful systems until now applicability of such approach. Hence, success of osmotically
to deliver proteins for extended duration (months). In case driven implants in delivering proteins may be dependent on
of biodegradable polymer-based systems, one major issue is inherent stability of proteins against stresses such as organic
poor IVIVC for release studies, which may substantially solvent, concentrated aqueous solution and aqueous/organic
slow down the progress of formulation development, as interface. A few smart techniques to control the release, such
in vitro release rate does not correlate well with in vivo data. as pH-/temperature-sensitive formulations [126], microfabrica-
It can be attributed to variable degradation rates of polymer tion technique [127,128] and PAD [129] have been developed.
under in vitro and in vivo condition. Faster degradation Despite limitations associated with every approach, delivery
in vivo may lead to rapid breakdown of polymer matrix and of proteins for extended duration in controlled manner is
generation of more degradation byproducts that may achievable. Significant advancements have been made in last
adversely affect protein stability. IVIVC for release profile of decade. Several, formulations based on biodegradable poly-
leuprolide acetate peptide from osmotic implants Viadur is mers (injectable implants and microparticles) are in clinical
excellent. It may be due to excellent stability of peptide in use as a result. However, therapeutic application of proteins
the implant system (72 mg leuprolide acetate in 104 mg of is still hampered by delivery-related issues. A large number
DMSO). In addition, the driving force for drug release is of protein molecules are under clinical trials, and hence, there
osmotic pressure, which can be systemically optimized to is an urgent need to develop new methods to deliver these
achieve desired rate and may not alter under in vivo condition. highly potent biologics.
These implants employ organic and aqueous solvent to hold
the therapeutic agent in the drug chamber. Despite the success Declaration of interest
in delivering peptide for extended durations, these systems
may have limited application in case of large proteins since All the authors were supported by NIH Grant
these biomolecules are known to have poor stability in organic R01EY010659-14. The authors have no other relevant affili-
solvents. In addition, following the release of protein in ations or financial involvement with any organization or
organic solvent, an aqueous/organic interface may form entity with a financial interest in or financial conflict with
depending on the miscibility of solvent with water. Such the subject matter or materials discussed in the manuscript
interface is also responsible for degrading proteins. If aqueous apart from those disclosed.
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Review

Long-term delivery of protein

2. In situ injectable gel/implants

logics. A number of injectable sustained-release formulations have reached

Keywords: formulation, hydrogel, implant, microparticle, nanoparticle, protein delivery, release

Expert Opin. Drug Deliv. [Early Online]

10.1517/17425247.2015.961420 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 1

degradation, complex structure and immunogenicity [12].

2 Expert Opin. Drug Deliv. (2014) 12(3)

Expert Opin. Drug Deliv. (2014) 12(3) 3

Figure 1. Classification of in situ forming implant/gel systems based of mechanism of formation.

60 lized in the polymer solution during preparation. It has been

4 Expert Opin. Drug Deliv. (2014) 12(3)

A. burst effect from PLGA implants, surfactants (triton X and

peptide loading in implants, a few modified techniques have

inversion. Okumu et al. utilized the SABER system to delivery

organic solvent exposing proteins to organic-aqueous inter-

Expert Opin. Drug Deliv. (2014) 12(3) 5

6 Expert Opin. Drug Deliv. (2014) 12(3)

Expert Opin. Drug Deliv. (2014) 12(3) 7

A. GL)1880-PEG1540-P(CL-GL)1880] triblock copolymer was

100 neutral for initial 8 weeks [59]. Polymer can be expected to

ing in formulation. High drug loading can ensure therapeutic

0 tion (formation of trimers and tetramers) and degradation may

8 Expert Opin. Drug Deliv. (2014) 12(3)

Expert Opin. Drug Deliv. (2014) 12(3) 9

100 degradation products, and neither should it alter any pharma-

60 to prepare microspheres for controlled delivery of peptide

t, h microsphere formulation process, there should be no alter-

10 Expert Opin. Drug Deliv. (2014) 12(3)

Polymer Material Degradation Biodegradation Active pharmaceutical Ref.

Natural Starch Amylase Biodegradable Insulin [130]

BSA: Bovine serum albumin; PLGA: Poly(lactic-co-glycolic acid).

impart stability to IFN-a in the PELA matrix. These micro- 4. Nanoparticles/nanospheres

Expert Opin. Drug Deliv. (2014) 12(3) 11

Diffusion short duration (6 h), and released insulin was biologically

12 Expert Opin. Drug Deliv. (2014) 12(3)

Table 2. Examples of protein-encapsulated microspheres formulated using different methods.

Expert Opin. Drug Deliv. (2014) 12(3)

without a significant initial burst

Expert Opin. Drug Deliv. (2014) 12(3)

Table 2. Examples of protein-encapsulated microspheres formulated using different methods (continued).

Slow release to 2 -- 14% in

Expert Opin. Drug Deliv. (2014) 12(3)

Table 3. Clinically approved sustained-release microparticle formulations.

Trade name Drug Indications Delivery system Length of release Approval

PLGA: Poly(lactic-co-glycolic acid).

16 Expert Opin. Drug Deliv. (2014) 12(3)

VEGF cumulative released (%)

Released from NPs

PLGA: Poly(lactic-co-glycolic acid).

Expert Opin. Drug Deliv. (2014) 12(3) 17

A. 1 year at constant rate at 37 C in unstirred PBS [116]. Similar

lot 1 agreement in IVIVC may be attributed to the stability of pep-

40% 5.3 Photoactivated depots

and control plasma insulin levels, photoactivated depot

18 Expert Opin. Drug Deliv. (2014) 12(3)

A. lactide- and glycolide-based copolymers are widely used poly-

Immediate diffusion development, such as encapsulation and release, where protein

niques have been examined to avoid use of organic solvents

tetramers) resulting in degradation. Hence, many of the

Expert Opin. Drug Deliv. (2014) 12(3) 19

20 Expert Opin. Drug Deliv. (2014) 12(3)

A. 1 year at constant rate at 37 C in unstirred PBS [116]. Similar