International Journal of Pharmaceutics 503 (2016) 163–173

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

Annealing as a tool for the optimization of lyophilization and ensuring

of the stability of protein-loaded PLGA nanoparticles
Pedro Fontea,b,**, Paulo Roque Linoc , Vítor Seabrab , António J. Almeidac, Salette Reisa ,
Bruno Sarmentob,d,e,*
a
UCIBIO, REQUIMTE, Department of Chemical Sciences, Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228,
4050-113 Porto, Portugal
b
CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde and Instituto Universitário de Ciências da Saúde, Rua Central de
Gandra 1317, 4585-116 Gandra-Prd, Portugal
c
iMed.ULisboa, Faculty of Pharmacy, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisboa, Portugal
d
i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal
e
INEB—Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Article history: The aim of this work was to use annealing as a tool to optimize the lyophilization cycle by decreasing its
Received 4 February 2016 duration time, and simultaneously preserve the stability of poly(lactic-co-glycolic acid) nanoparticles
Received in revised form 7 March 2016 and with upmost importance, maintain the structural stability of loaded insulin, used as model
Accepted 9 March 2016
therapeutic protein. The contribution of a cryoprotectant in this preservation process was also evaluated.
Available online 10 March 2016
Insulin-loaded nanoparticles co-encapsulated with and without trehalose as cryoprotectant were
produced, resulting in a particle size of about 250–300 nm, a PdI around 0.25 and a zeta potential in the
Chemical compounds studied in this article:
range of
20 to
24 mV. The insulin association efficiency was higher than 90%, and the loading capacity
Acetonitrile (PubChem CID: 6342)
Chloroform (PubChem CID: 6212)
was of 11–12%. The use of annealing allowed the decrease of duration time of primary drying in about
Dichloromethane (PubChem CID: 6344) 38%, representing a global decrease of lyophilization time of around 26%. The residual moisture content of
Insulin (PubChem CID: 70678557) all lyophilizates was around 1%, and the reconstitution of lyophilizates obtained using annealing was
Poly(DL-lactic-co-glycolic acid) (PubChem even faster than those without annealing. The co-encapsulated trehalose better preserved the
CID: 23111554) nanoparticle size throughout the lyophilization process using annealing, compared to formulation
Polyvinyl alcohol (PubChem CID: 11199) containing no cryoprotectant. Fourier-transform infrared spectroscopy showed that the trehalose-
Trehalose (PubChem CID: 7427) containing nanoparticles presented higher insulin structural maintenance, compared to nanoparticles
Trifluoroacetic acid (PubChem CID: 6422)
without cryoprotectant, presenting an insulin structural maintenance of 85.3  0.7% and 86.0  1.0% for
annealing and no annealing, respectively. This formulation also presented the closest structural similarity
Keywords:
Annealing with native insulin. Interestingly, the structural features of insulin loaded into nanoparticles upon
FTIR lyophilization with and without annealing were practically identical, showing that annealing had no
Insulin detrimental effect in insulin structure. Circular dichroism and fluorescence spectroscopy confirmed these
Lyophilization results. Overall, this work gave rise to the importance of annealing in decreasing the duration time of
Nanoparticle lyophilization of protein-loaded poly(lactic-co-glycolic acid) nanoparticles, and simultaneously ensuring
Protein the stability of the carrier and loaded protein.
PLGA ã 2016 Elsevier B.V. All rights reserved.

1. Introduction

The freezing step is considered the most important one in

lyophilization, since it influences the size of ice crystals and
consequently the duration of the drying steps (Patapoff and
* Corresponding author at: i3S, Instituto de Investigação e Inovação em Saúde, Overcashier, 2002). Generally, the optimization of lyophilization
Universidade do Porto, Portugal. cycle is mainly focused in decreasing the time needed for primary
** Corresponding author at: UCIBIO, REQUIMTE, Department of Chemical drying, which is the longest step in lyophilization. The shelf
Sciences, Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Rua
temperature and chamber pressure are the main lyophilization
de Jorge Viterbo Ferreira 228, 4050-113 Porto, Portugal.
E-mail addresses: [email protected] (P. Fonte), parameters that may improve the sublimation rate. However, if
[email protected], [email protected] (B. Sarmento).

http://dx.doi.org/10.1016/j.ijpharm.2016.03.011
0378-5173/ ã 2016 Elsevier B.V. All rights reserved.
164 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173

they are not properly adjusted it may lead to undesired product 2.2. Preparation of PLGA nanoparticles
overheating and collapse.
Annealing is a processing step in lyophilization in which Nanoparticles were produced following a previously developed
samples are kept at a determined subfreezing temperature above and optimized protocol to co-encapsulate insulin and trehalose
the Tg’, during a period of time (Searles et al., 2001a). This process into PLGA nanoparticles (Fonte et al., 2015). Briefly, 200 mg of PLGA
influences the size distribution of ice crystals, leading to their 50:50 were dissolved in 2 mL of dichloromethane, and then added
growth. Their size distribution during annealing is ruled by with 0.2 mL of a 150 mg/mL insulin solution in HCl 0.1 M containing
Ostwald ripening, which states that crystals smaller than a critical trehalose at a concentration of 10% (w/v). The primary emulsion
size decrease, whereas the larger ones grow (Searles et al., 2001a). was obtained by sonication at 70% of amplitude for 30 s, using a
Therefore, the increase of size of ice crystals during annealing Bioblock vibracell 75186 sonicator from Fischer Bioblock Scientific
may accelerate the sublimation rate by increasing the diameter of (Rungis Complexe, France). The emulsion was then poured into
pores, in spaces that were previously occupied by ice crystals, and 25 mL of PVA 2% (w/v) at pH 7.4, and sonicated again using the
by a decrease of the mass transfer resistance by the dried layer same previous conditions. The organic solvent was removed by
(Abdelwahed et al., 2006a). The annealing may also decrease the evaporation for 3 h under magnetic stirring. The production
heterogeneity of the drying rate between samples, in order to method was also used to formulate nanoparticles without co-
obtain a more inter-vial homogeneous cake structure (Nail et al., encapsulated cryoprotectant and unloaded nanoparticles. After
2002; Tang and Pikal, 2004). Overall, annealing may be a useful production, nanoparticles were washed three times with milli-Q
tool to obtain a dried cake with good quality in a shorter period of water, and collected after each washing step by centrifugation for
time. 30 min at 23,000g using a Heraeus Megafuge 1.0 R centrifuge
The annealing step has been applied in the lyophilization of (Thermo Scientific). Finally, nanoparticles were redispersed in
protein formulations. In a rhIFN-g formulation, the application of water.
an annealing step avoided the cracking of the cakes, originated
more native-like secondary structure of protein in the solid 2.3. Insulin association efficiency, loading capacity and retention
product and prevented protein aggregation upon reconstitution efficiency
(Webb et al., 2003). Regarding nanoparticle formulations, just a
few works have been focusing on the influence of annealing on The association efficiency (AE) and loading capacity (LC) was
the stability of nanoparticles (Abdelwahed et al., 2006a,c; Shi calculated using the following formulas:
et al., 2012). It was demonstrated that annealing accelerated the
Total amount of insulin
Free insulin in supernatant
sublimation, with simultaneous size maintenance of polymeric AE ¼
Total amount of insulin
nanoparticles (Abdelwahed et al., 2006c). However, the influence  100 ð1Þ
of annealing in the structure and stability of loaded proteins has
not been studied so far. The possible modification of the structure
of loaded proteins during annealing needs to be properly
Total amount of insulin
Free insulin in supernatant
regarded, since it has been demonstrated that the freezing step LC ¼
Total dry weight of nanoparticles
in lyophilization influences the structure of proteins loaded into  100 ð2Þ
nanoparticles (Fonte et al., 2016).
Using insulin and poly(lactic-co-glycolic acid) (PLGA) nano- The nanoparticles suspension was centrifuged at 40,000g at 4  C
particles, as models of therapeutic protein and nanocarriers, for 30 min in a Beckman Optima TL ultracentrifuge from Beckman
respectively, the main aim of this study was to develop an Coulter (Brea, CA, USA.), and the supernatant containing free
optimized lyophilization cycle using annealing, in order to insulin was collected. Centrifuged nanoparticles were lyophilized
decrease the time needed to lyophilize protein-loaded polymeric to obtain the dry mass of nanoparticles. Upon reconstitution of
nanoparticles, assuring simultaneously the stability of nano- lyophilized nanoparticles, the suspension was centrifuged and the
particles and the structural maintenance of the loaded protein. supernatant was collected for insulin quantification and determi-
Additionally, it was assessed the influence of the cryoprotectant nation of insulin retention efficiency. The insulin free in superna-
effect during annealing, in the stability of nanoparticles and with tant was quantified using a HPLC-UV method previously validated
upmost importance, in the stability of the encapsulated protein (Sarmento et al., 2006), in a Merck-Hitachi LaChrom HPLC
upon lyophilization. instrument equipped with a LiChrospher 100 RP-18 guard column,
5 mm particle size from Merck (Whitehouse Station, NJ, U.S.A.) and
2. Materials and methods also a XTerra RP 18 column, 5 mm particle size, 4.6 mm internal
diameter x 250 mm length from Waters (Milford, MA, U.S.A.). All
2.1. Materials samples were run in triplicate.

PLGA 50:50 Resomer1 RG 503 H (Mw 24,000–38,000; Tg 44– 2.4. Particle size and zeta potential analysis

48 C) from Evonik Industries AG (Essen, Germany) was used for
nanoparticles production. Polyvinyl alcohol (PVA), dichlorome- To perform the analysis of mean particle size, polydispersity
thane, recombinant human insulin and trehalose were from index (PdI) and zeta potential, nanoparticles in suspension and
Sigma-Aldrich (Steinheim, Germany). For the high performance reconstituted after lyophilization were diluted with milli-Q water
liquid chromatography HPLC analysis, it was used acetonitrile to achieve a suitable concentration. These analyses were
HPLC Gradient Grade from Fischer Scientific (Loughborough, UK) performed by dynamic light scattering using a 90Plus Particle
and trifluoroacetic acid from Acros Organics (Morris Plains, NJ, Size Analyser, and by phase analysis light scattering using a
USA). Chloroform from Sigma-Aldrich (Steinheim, Germany) was ZetaPALS Zeta Potential Analyser, from Brookhaven Instruments
used for protein extraction from nanoparticles. The milli-Q water Corporation (Holstville, NY, USA). The lyophilization ratio was
was produced in-house and other reagents were of analytical calculated as the ratio between the mean particle size after
grade. lyophilization, and that before lyophilization.
 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173 165

2.5. Lyophilization of nanoparticles 2.9. Circular dichroism analysis

The insulin-loaded PLGA nanoparticles suspensions were Insulin was extracted from PLGA nanoparticles using chloro-
poured into semi-stoppered glass vials at a maximal height of form and HCl 0.01 M to dissolve insulin, prior to CD analysis. All
10 nm, and lyophilized in a VirTis Advantage Plus Benchtop samples were analyzed at 25  C with the lamp housing continu-
lyophilizer from SP Scientific (Warminster, PA, USA). Thus, samples ously purged with nitrogen, in a Jasco J-815CD Spectrometer from
were frozen by ramped cooling at
40  C during 4 h, and primary Jasco Inc. (Easton, MD, USA). The CD spectra were obtained using a
drying occurred at
32  C and 150 mtorr for 24 h, followed by a 0.1 cm cell in the range of 190–250 nm with an average of 5 scans,
secondary drying at 20  C and 50 mtorr for 6 h, at a condenser with a step size of 0.5 nm, a bandwidth of 1.5 nm, and an averaging
surface temperature of
60  C (n = 3). When the thermal treatment time of 5 s. The spectrum of reference sample was subtracted from
of annealing was included in the lyophilization cycle, the the test sample spectra. The molar ellipticity of insulin was
nanoparticle formulations were frozen by ramped cooling at calculated as CD signal  MRW (mean residual weight of each

40  C and the temperature was raised to the annealing insulin residue, 116 Da) [insulin concentration (mg/mL)cell path
temperature of
10  C and held for 2 h. Then, samples were length (0.1 cm)]. The insulin concentration was determined by UV
cooled to
40  C and held for another 2 h. The primary drying absorption at 280 nm in a NanoDrop 2000c UV–vis Spectropho-
occurred at
32  C and 150 mtorr for 15 h, followed by a secondary tometer (Thermo Scientific, Wilmington, DE, USA), using a molar
drying at 20  C and 50 mtorr for 6 h, at a condenser surface extinction coefficient of 6200 M
1 cm
1 for 1.0 mg/mL. As control
temperature of
60  C (n = 3). sample of native insulin, it was used a 0.2 mg/mL insulin solution in
HCl 0.01 M.
2.6. Macroscopic evaluation, residual moisture content and
reconstitution of lyophilizates 2.10. Fluorescence spectroscopy analysis

The lyophilizates were visually inspected to assess the aspect of Insulin was extracted from PLGA nanoparticles using chloro-
the cake, and the residual moisture content was evaluated using an form and HCl 0.01 M to dissolve insulin, prior to fluorescence
A&D MX-50 moisture analyzer from A&D Company Ltd. (Tokyo, spectroscopy analysis. The emission spectra were obtained at 25  C
Japan). Thus, an equal amount of lyophilizate of each formulation in the range of 290–450 nm with 1 nm step in a Jasco FP-
was analyzed, and the residual moisture content was quantified as 6500 Spectrofluorometer from Jasco Inc. (Easton, MD, USA). The
percentage of containing moisture. The lyophilizates were excitation occurred at 280 nm with both emission and excitation
reconstituted by adding milli-Q water on the inside wall of the slits set to 3 nm, with an integration time per data point of 0.1 s and
vials to guarantee the cake wetting. Finally, formulations were an average of 5 scans. The spectrum of reference sample was
swirled until complete homogenization. subtracted from the sample spectra, and normalized based on
insulin concentration. As control sample of native insulin, it was
2.7. Scanning electron microscopy analysis used a 0.2 mg/mL insulin solution in HCl 0.01 M.

The morphology of insulin-loaded PLGA nanoparticles was 2.11. Thioflavin T assay

evaluated using scanning electron microscopy (SEM), in a FEI Quanta
400 FEG scanning electron microscope from FEI (Hillsboro, Oregon, Samples of extracted insulin were incubated with thioflavin T
USA). Both nanoparticle suspensions and lyophilizates were and analyzed in a Jasco FP-6500 Spectrofluorometer from Jasco Inc.
mounted on metal stubs, and coated under vacuum with a layer (Easton, MD, USA) at 25  C, and the final concentration of insulin
of gold/palladium with a 15 mA current for 60 s prior to observation. and thioflavin T were 11 mM and 25 mM, respectively. They were
excited at 450 nm, and the fluorescence intensity was determined
2.8. Attenuated total reflectance-Fourier transform infrared at 485 nm with slit widths of 5 nm. The reference sample spectrum
spectroscopy analysis was subtracted from the test sample spectra. As a positive control it
was used insulin fibrillated by incubation at 60  C during 12 h.
The secondary structure of insulin loaded into PLGA nano-
particles was assessed by attenuated total reflectance-Fourier 2.12. Statistical analysis
transform infrared spectroscopy (ATR-FTIR). Samples were ana-
lyzed in an ABB MB3000 FTIR spectrometer from ABB (Zurich, The statistical analyses were performed in the OriginPro
Switzerland) equipped with a MIRacle triple reflection ATR 8 software from OriginLab Corporation (Northampton, MA,
accessory from PIKE Technologies (Madison, WI, USA). The FTIR USA), using a one-way ANOVA Tukey post hoc test, with a
spectra were collected in the 4000–600 cm
1 region after 256 scans significance level of p < 0.05.
at 4 cm
1 resolution. The insulin spectra were obtained by a double
subtraction method (Dong et al., 1990), followed by a Savitzky- 3. Results and discussion
Golay second derivative of 15 points, and a 3–4 point baseline
correction in the amide I region (1710–1590 cm
1). This spectral It is generally accepted that the annealing process influences
treatment was performed using the Horizon MB FTIR software the sublimation rate and the quality of the lyophilized protein drug
from ABB, and all the spectra were area-normalized for further product. However, its impact in the stability of the protein loaded
comparison. The area of overlap (AO) algorithm was used to assess into nanoparticles was not investigated so far. In addition, the
the quantitative similarity of the area normalized second- influence of the cryoprotectant effect in the stabilization of the
derivative amide I spectra, between native insulin and insulin loaded protein upon lyophilization using annealing needs to be
loaded into PLGA nanoparticles (Kendrick et al., 1996). A 30 mg/ml scrutinized.
insulin solution in HCl 0.1 M was analyzed and used as the native Insulin was used as a model therapeutic protein, and was
insulin spectrum to determine the percentage of spectral co-encapsulated into PLGA nanoparticles together with a cryopro-
similarity, since it is considered that the stability of a solid protein tectant, since this approach demonstrated to preserve better the
formulation increases with the increase of similarity to its spectra protein structure upon lyophilization, compared to the conven-
in solution (Maltesen et al., 2009). tional cryoprotectant addition method (Fonte et al., 2015).
166 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173

Trehalose was used because it demonstrated to be a versatile increased when the annealing temperature was well above the Tg’
cryoprotectant to stabilize protein-loaded nanoparticles upon of the formulation (Abdelwahed et al., 2006a). It has been also
lyophilization using different freezing methods (Fonte et al., 2016). reported when annealing occurred below the Tg’, the sublimation
Trehalose was used at a concentration of 10% (w/v) to obtain the rate was not increased (Searles et al., 2001a). Therefore, a
highest acceptable cryoprotectant effect, since the level of temperature of annealing of
10  C was selected for being well
stabilization provided is concentration dependent (Abdelwahed above the Tg’ of formulations. When this occurs the ice melts, and
et al., 2006d). To characterize the influence of annealing in the the smaller ice crystals melt faster than the larger ones. The size
lyophilized formulations, the properties of nanoparticles and distribution of ice crystals during annealing is ruled by the Ostwald
lyophilizates were assessed, as well as the structural maintenance ripening phenomenon, in which the crystals smaller than a critical
of the loaded protein. size decrease, whereas those larger than that critical size increase
in size (Searles et al., 2001b)
3.1. Particle size, zeta potential, association efficiency and loading Overall, it is recognized that the morphology of ice crystals is
capacity analyses directly related to the sublimation rate (Searles et al., 2001b).
Since annealing promotes the growth of ice crystals and
Insulin-loaded nanoparticles co-encapsulated with trehalose increases the sublimation rate due to the increase of pore size
(insulin-trehalose 10%-PLGA nanoparticles) and without cryopro- of the product, this approach was used to decrease the duration
tectant encapsulated (insulin-PLGA nanoparticles) were produced of primary drying, which is the longest step in lyophilization.
allowing the comparison and assessment of the stabilization effect This possibility is only feasible, if both the nanoparticle features
provided by the cryoprotectant. and the stability of loaded protein are preserved upon
The mean particle size, PdI and zeta potential characterization lyophilization.
of insulin-loaded PLGA nanoparticles is shown in Table 1. Both
formulations presented a close particle size in the range of about 3.3. Visual inspection, reconstitution and residual moisture content of
250–300 nm and a low PdI, demonstrating the robustness of the lyophilizates
encapsulation protocol (Fonte et al., 2016). Considering the zeta
potential analysis, it was observed that nanoparticles had a The lyophilizates obtained with and without annealing were
negative surface charge, characteristic of the acidic PLGA polymer inspected, presenting a cotton-like texture and white color. The
(Danhier et al., 2012). lyophilizates occupied the same volume of the original frozen mass
The insulin AE and LC of formulations are also depicted in and no shrinkage or cake collapse was observed, demonstrating
Table 1. Both formulations presented an AE higher than 90%, which that particularly lyophilization with an annealing step yielded
was a good achievement considering the hydrophilic nature of good lyophilizates. The cake collapse is a problem to avoid, because
insulin and the hydrophobic nature of the polymer. The AE and LC it originates prolonged reconstitution times and high residual
results were similar to those obtained in previous works (Fonte moisture content (Bozdag et al., 2005; Sameti et al., 2003). All
et al., 2016, 2015), further supporting the robustness of the lyophilizates were easily reconstituted obtaining suspensions with
encapsulation protocol, and its ability to co-encapsulate cryopro- no visual aggregates. The complete reconstitution of formulations
tectants together with the protein. lyophilized with annealing was in average about 10 s faster, than
those lyophilized with no annealing. This property may be
3.2. Design of the lyophilization cycle explained by the larger pores of the lyophilizates, that were
previously occupied by larger ice crystals formed during annealing
The lyophilization cycle needs to be designed taking into (Abdelwahed et al., 2006a). Such pores allowed a faster
consideration the engineering principles of the process and the reconstitution of lyophilizates obtained by lyophilization with
physicochemical properties of nanoparticles, to obtain a lyophi- annealing.
lizate with high quality and to properly preserve the nanoparticles The residual moisture content of lyophilizates is depicted in
features (Abdelwahed et al., 2006b,c,d). The knowledge of Table 2. The nanoparticles lyophilized using annealing showed a
physicochemical properties of formulations, such as the Tg’ and similar residual moisture content of those lyophilized without
Tc, is necessary to design an optimal lyophilization cycle (Pikal annealing, ca.1%, demonstrating that annealing allowed the
et al., 1990; Tang and Pikal, 2004). In a previous work, these decrease of the primary drying time in about 38%, without
formulation parameters were characterized to design an optimal increasing the residual moisture content. These results showed the
lyophilization cycle that was also used in the present work (Fonte lyophilization process was effective on removing water from
et al., 2016), but including an annealing step. formulations, since they need to have very low residual moisture
To assess the effect of annealing in the formulations and content, which for pharmaceutical products should be around 1%
simultaneously decrease the time of primary drying, an annealing (Williams and Polli, 1984). This result is also explained by the faster
step of 2 h at
10  C was included in the freezing stage that kept its sublimation rate promoted by the porous structure of annealed
duration time of 4 h, and the duration of primary drying was formulations. Another possible explanation for a faster sublima-
shorten from 24 h to 15 h. This modification represented a decrease tion rate that allowed a decrease in the duration time of primary
of primary drying duration of about 38%, and the reduction of drying, without compromising the low residual moisture content
duration of the lyophilization cycle of about 26%. Previously, of lyophilizates, was the ability of annealing to eliminate the thin
annealing was applied in the lyophilization of polymeric nano- skin layer on the top surface of the cake (Nemati et al., 1992). Such
particles, and it was observed that the sublimation rate was thin layer occurs due to migration of nanoparticles and

Table 1
Mean particle size, polydispersity index, zeta potential, and insulin AE and LC characterization of insulin-loaded PLGA nanoparticles (n = 3, mean  SD).

Formulation Mean particle size (nm) PdI Zeta potential (mV) AE (%) LC (%)

Insulin-PLGA nanoparticles 294  15 0.26  0.02
20.4  2.5 92.6  0.7 12.1  0.6
Insulin-trehalose 10%-PLGA nanoparticles 249  14 0.23  0.08
23.8  2.5 90.5  0.5 11.2  0.4
 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173 167

Table 2
Mean particle size, PdI, zeta potential, lyophilization ratio, insulin retention efficiency and residual moisture content characterization of insulin-loaded PLGA nanoparticles
after lyophilization with and without annealing (n = 3, mean  SD). Results are significantly different (p < 0.05) from the respective formulation prior lyophilization, when
marked with *.

Formulation Mean particle size PdI Zeta potential Lyophilization Insulin retention efficiency Residual moisture content
(nm) (mV) ratio (%) (%)
No annealing
Insulin-PLGA nanoparticles 280  10 0.26  0.01
29.2  3.5* 0.95  0.07 99.1  0.4 1.33  0.41
Insulin-trehalose 10%-PLGA 245  18 0.25  0.02
31.6  2.5* 0.98  0.08 99.0  0.5 1.45  0.35
nanoparticles

Annealing
Insulin-PLGA nanoparticles 270  4* 0.21  0.01
33.1  1.8* 0.91  0.04 98.5  0.9 1.55  0.27
Insulin-trehalose 10%-PLGA 250  23 0.26  0.07
30.8  1.5* 1.00  0.09 98.7  0.4 1.48  0.37
nanoparticles

cryoprotectant during ice crystallization, hampering the water particle size and lyophilization ratio results clearly showed the
removal from the product. nanoparticle stability was maintained upon lyophilization using
annealing. The PdI of nanoparticles were similar after lyophiliza-
3.4. Particle size, zeta potential and drug retention efficiency after tion, also demonstrating the low size heterogeneity and the
lyophilization absence of significant particle aggregation.
Regarding the zeta potential, it was observed a significant
After resuspension of lyophilizates, the nanoparticles were decrease (p < 0.05) of the surface charge of nanoparticles upon
characterized in terms of mean particle size, PdI and zeta potential lyophilization, leading to a better colloidal stability of nano-
(Table 2). The overall mean particle size of nanoparticles after particles. The drug leakage during lyophilization may occur due to
lyophilization were similar to that obtained after nanoparticles possible modifications of nanoparticles integrity. Therefore, the
production, with the exception for insulin-PLGA nanoparticles insulin retention efficiency in nanoparticles after lyophilization
lyophilized with annealing that presented a significant size was evaluated (Table 2). The leakage of insulin from nanoparticles
modification (p < 0.05). This result showed the importance of during lyophilization was negligent, demonstrating the integrity of
presence of co-encapsulated trehalose, acting as cryoprotectant, nanoparticles was preserved upon lyophilization. These results
when annealing was used. The potential residual leakage of also showed the annealing did not induce additional stresses that
trehalose from nanoparticles may protect them in a localized could damage the nanoparticles and lead to insulin leakage.
manner, by forming a protective matrix around nanoparticles
(Fonte et al., 2016; Tang and Pikal, 2004). The lyophilization ratio is 3.5. Morphology of nanoparticles
also shown in Table 2. This ratio was obtained by dividing the mean
particle size of nanoparticles after with that before lyophilization. 3.5.1. Prior lyophilization
Thus, a value around 1 demonstrates the mean particle size of The morphology of nanoparticles and lyophilizates were
nanoparticles was maintained after lyophilization. The lyophiliza- assessed by SEM to assess the shape, surface and size of
tion ratio of nanoparticles was around 1, demonstrating the nanoparticles. These features are important to infer the stability
lyophilization process used did not lead to significant particle of nanoparticles and the loaded protein. Fig. 1 shows the insulin-
aggregation or fusion. When using annealing in lyophilization of loaded PLGA nanoparticles after production. The nanoparticles of
nanoparticle suspensions, the main problem is the mechanical both formulations were similar, presenting a smooth surface and
stress of ice crystals that may lead to nanoparticle aggregation, spherical shape, which are characteristic of the used polymer and
since this effect in nanoparticle stability is more accentuated for encapsulation method. Microscopy studies by SEM also confirmed
larger ice crystals than for smaller ones (Abdelwahed et al., 2006a; the particle size distributions previously established from the
Fonte et al., 2016; Searles et al., 2001b). Therefore, the mean dynamic light scattering experiments, described in Section 3.1.

Fig. 1. SEM microphotographs of insulin-loaded PLGA nanoparticles after production. Scale bar: 1 mm.
168 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173

Fig. 2. SEM microphotographs of the lyophilizates of insulin-loaded PLGA nanoparticles. Scale bar: 10 mm.

3.5.2. After lyophilization similarity of the second derivative spectra of the native protein
Fig. 2 shows the SEM microphotographs of lyophilizates with that of the loaded protein. Therefore, the highest the
obtained with and without annealing. The lyophilizates presented similarity between those spectra, represents the preservation of
a porous structure, which were formed during water sublimation the secondary structure of protein. This protein structure may be
and are necessary to the adequate resuspension of nanoparticles. It assessed quantitatively and qualitatively by analyzing the AO and
was also possible to visualize the arrangement of nanoparticles the area-normalized second derivative amide I spectra, respec-
that maintained their spherical shape upon lyophilization. tively.
However, lyophilizates obtained using annealing showed a more
homogenous structure, compared to those obtained without 3.6.1.1. Area of overlap. The AO measures the degree of similarity
annealing. This occurred mainly due to the decrease of heteroge- between the area-normalized second derivative spectra of native
neity of the sublimation rate caused by annealing, obtaining a more insulin, and the insulin loaded into PLGA nanoparticles. The
homogeneous cake structure (Nail et al., 2002; Tang and Pikal, spectrum of native insulin was obtained by FTIR analysis of an
2004). insulin solution 30 mg/ml in HCl 0.1 M. The AO results for
After lyophilizate resuspension, nanoparticles were again lyophilized insulin-loaded PLGA nanoparticles are depicted in
observed by SEM (Fig. 3). The nanoparticles maintained their Fig. 4.
spherical shape and smooth surface throughout the lyophilization It was observed that either for lyophilization with and without
procedure. The size range observed in the microphotographs was annealing, nanoparticles co-encapsulated with trehalose had a
also in accordance with the mean particle size described in significantly higher AO of insulin (p < 0.05), compared to
Section 3.4. In addition, the microphotographs clearly demon- formulation containing no cryoprotectant. The AO of insulin-
strated that the lyophilization process with and without annealing trehalose 10%-PLGA nanoparticles was 85.3  0.7% and 86.0  1.0%
was able to attain its main objective, i.e. to preserve the integrity of for annealing and no annealing conditions, respectively. These
nanoparticles. results demonstrated that the presence of trehalose as cryopro-
tectant improved the preservation of the structure of insulin
3.6. Insulin structural integrity loaded into PLGA nanoparticles upon lyophilization, both with
annealing and no annealing conditions. This may be explained by
3.6.1. Fourier-transform infrared spectroscopy analysis the ability of cryoprotectants, to stabilize proteins by a preferential
FTIR spectroscopy is a powerful technique in the assessment of exclusion during freezing (Carpenter et al., 1992). The cryoprotec-
the secondary structure of proteins loaded into nanoparticles, in a tant co-encapsulated with insulin into PLGA nanoparticles has a
non-invasive manner (Sarmento et al., 2007). In a protein spectra, tendency to be excluded from the protein surface, originating a
the amide I region at 1710–1590 cm
1 is used to evaluate the preferential hydration of insulin and increasing the
 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173 169

Fig. 3. SEM microphotographs of lyophilized insulin-loaded PLGA nanoparticles after resuspension. Scale bar: 1 mm.

thermodynamic stability of its native state. Additionally, the annealing conditions was even almost identical. This clearly
cryoprotectant may also preserve insulin structure by reducing its demonstrated that annealing allowed the optimization of the
adsorption on the ice surface during freezing, and also by lyophilization cycle by decreasing its duration time, and simulta-
increasing the freeze-concentrate viscosity (Searles, 2010). neously ensuring the structural stability of the loaded protein. The
Regarding the comparison of lyophilization with and without possible explanation for this phenomenon is that the stability of
annealing, no differences were observed in the AO of both insulin- proteins loaded into nanoparticles may be more related with the
PLGA nanoparticles and insulin-trehalose 10%-PLGA nanoparticles. freezing method, rather than the annealing conditions during
The insulin AO of those formulations, for annealing and no freezing (Fonte et al., 2016).

Fig. 4. AO percentages of insulin loaded into PLGA nanoparticle formulations after lyophilization with and without annealing (n = 3, mean  SD). Np stands for nanoparticles.
Results are significantly different (p < 0.05) from formulation with no cryoprotectant co-encapsulated, insulin-PLGA nanoparticles, when marked with *.
170 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173

3.6.1.2. Visual comparison of area-normalized second derivative amide containing no cryoprotectant showed a more pronounced decrease
I spectra. The AO is a quantitative indicator of the preservation of of the a-helix band and its shift into 1653 cm
1, a modification that
insulin native structure, whereas the analysis of the area- was similar to that of the respective band position of lyophilized
normalized second-derivative amide I spectra allows the insulin. These structural features demonstrated the co-encapsula-
evaluation of the qualitative changes of insulin structure. This tion of the cryoprotectant was crucial, to better preserve the
data is depicted in Fig. 5. structure of insulin loaded into PLGA nanoparticles upon
Generally, upon lyophilization, the protein structure suffers a lyophilization with and without annealing.
decrease in a-helix, with a concomitant increase in the b-sheet
content, and it is usually accepted that the a-helix content is the 3.6.2. Circular dichroism analysis
best indicator of the integrity of protein structure (Griebenow and The CD spectroscopy was also used to evaluate the secondary
Klibanov, 1996). The area-normalized second-derivative FTIR structure of insulin. Prior to analysis, insulin was extracted from
spectrum of native insulin was obtained by analysis of an insulin PLGA nanoparticles to avoid spectral artifacts caused by light
solution 30 mg/ml. The spectrum showed the insulin structure was scattering. During the extraction process, some minor modifica-
dominated by a-helix band at 1657 cm
1, including also b-sheets tions of insulin structure may occur, nevertheless CD is still a
with bands at 1640 cm
1 and 1690 cm
1, which is in agreement powerful tool to corroborate the results obtained by FTIR
with the findings reported in previous works (Fonte et al., 2016; spectroscopy. The far-UV CD spectra of insulin are depicted in
Jørgensen et al., 2003; Sarmento et al., 2007). The changes of such Fig. 6. To obtain the spectrum of native insulin, a solution of insulin
bands occurring during lyophilization, represent the changes in the 0.2 mg/ml in HCl 0.01 M was analyzed, resulting in a spectrum with
secondary structure of insulin that may lead to its denaturation or two ellipticity minima, characteristic of the predominant a-helical
aggregation, hampering insulin bioactivity or even causing structure of the protein, at 208 nm and 222 nm (Kelly et al., 2005).
possible adverse effects after administration (Van de Weert The spectrum of native insulin was similar to those described in
et al., 2005). previous works (Sarmento et al., 2007; Yong et al., 2009).
Overall, no denaturation or aggregation of insulin was noticed Overall, the spectra of insulin did not show relevant shifts of the
upon lyophilization of formulations, since these phenomena, when characteristic minima, demonstrating the prevalent a-helical
occurring, are evidenced by a clear decrease in a-helix content and structure of insulin. The structural modification that could lead
a concomitant increase in b-sheet. This result was a good indicator to denaturation, aggregation or fibrillation of insulin is evidenced
that insulin could maintain its bioactivity upon lyophilization with by prevalent b-sheet content, with an ellipticity minimum around
and without annealing. The in vivo bioactivity of insulin loaded into 216 nm (Ahmad et al., 2005; Bouchard et al., 2000). Since this
the PLGA nanoparticles used in the present work, upon lyophili- detrimental modification did not occur, the CD results were in
zation without annealing, has been previously demonstrated agreement with those of FTIR on demonstrating the ability of
(Fonte et al., 2016). The insulin contained in formulations lyophilization cycles with and without annealing, to preserve
presented distinct structural rearrangements upon lyophilization. insulin structure in such a way that it could maintain its bioactivity.
Interestingly, the structural features of insulin in the different The variability in the rearrangement of insulin structure upon
formulations lyophilized both with and without annealing were lyophilization was demonstrated by the decrease of the negative
practically identical, demonstrating the robustness of the lyophi- ellipticity signal. Despite the possible influence of the extraction
lization cycles. Additionally, the distinct morphology of ice crystals process of insulin, the CD data were similar to those obtained by
and liquid/water interfaces formed during annealing, in which FTIR. For instance, the CD spectra of insulin from formulations
surface-induced denaturation of insulin may occur (Wang, 2000), lyophilized with no annealing were practically identical, to those
did not yield a detrimental effect in the structural stability of obtained after lyophilization with annealing. Furthermore, the
insulin. insulin from formulations with co-encapsulated trehalose showed
The insulin-trehalose 10%-PLGA nanoparticles presented the a closer similarity with native insulin, compared to insulin from
closest similarity with native insulin, with just a slight decrease PLGA nanoparticles containing no cryoprotectant co-encapsulated,
and shift of a-helix band into 1659 cm
1, and a decrease of the since the latter evidenced a more pronounced decrease of the
high-wavenumber b-sheet content. In turn, the nanoparticles negative ellipticity signal. These results confirmed both that

Fig. 5. Area-normalized second-derivative amide I FTIR spectra of insulin loaded into PLGA nanoparticles lyophilized using no annealing (A), and annealing (B). Np stands for
nanoparticles.
 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173 171

Fig. 6. Far-UV CD spectra of insulin extracted from insulin-loaded PLGA nanoparticles lyophilized using no annealing (A), and annealing (B). Np stands for nanoparticles.

annealing did not present a detrimental effect in insulin structure, stability of insulin in order to assure its bioactivity. No differences
and that PLGA nanoparticles co-encapsulated with trehalose were found between the insulin spectra from nanoparticles
presented a superior structural stability of insulin, compared to lyophilized using annealing, with those lyophilized with no
nanoparticles without cryoprotectant co-encapsulated. annealing.

3.6.3. Fluorescence spectroscopy analysis 3.7. Thioflavin T experiment

The fluorescence spectroscopy is another technique used in the
evaluation of proteins, particularly their tertiary structure (Lako- The thioflavin T experiment is a useful tool to assess the
wicz, 2006). Prior to analysis, insulin was also extracted from PLGA fibrillation of insulin, which may occur during nanoparticle
nanoparticles to avoid potential spectral artifacts. Insulin lacks processing and lyophilization. The amyloid fibrils are protein
tryptophan, so the protein fluorescence is mostly dependent in the aggregates that indicate the protein structure and its bioactivity
four tyrosine residues (Bekard and Dunstan, 2009). Therefore, the are decisively compromised. Thioflavin T is a dye able to bind to
modification of the emission spectra of insulin fluorescence is an amyloid fibrils with crossed b-sheet structures, leading to a shift of
indicator of possible changes in protein structure. Fig. 7 shows the the fluorescence excitation maximum from 340 nm to 450 nm, and
fluorescence spectra of insulin from nanoparticles, in which the an increase of fluorescence emission at 485 nm (LeVine, 1995;
spectrum of native insulin (insulin solution 0.2 mg/ml) evidenced Maskevich et al., 2007). Thus, the enhancement of the thioflavin T
maximum fluorescence intensity at 304 nm. The spectrum of fluorescence intensity is indicative of insulin aggregation/fibrilla-
native insulin was in agreement with those previously reported tion (Malik and Roy, 2011).
(Amidi et al., 2008; Falconi et al., 2001; Fonte et al., 2016; Malik and The insulin from PLGA nanoparticles did not evidence the
Roy, 2011). presence of amyloid fibrils, since no fluorescence emission at
The structural modifications related to insulin denaturation, 485 nm was detected (data not shown). In turn, the positive control
aggregation or fibrillation are characterized by a decrease in the of fibrillated insulin showed a clear fluorescence emission. These
intensity of fluorescence emission and the shift of its maxima results were in agreement with the aforementioned structural
(Bekard and Dunstan, 2009). Since all the insulin spectra were characterization of insulin, and were elucidative on demonstrating
practically identical, it was possible to conclude that those that both the nanoparticle production and lyophilization cycles
structural modifications did not occur, and the lyophilization with and without annealing, did not act as stress factors that could
cycles both with and without annealing were able to preserve the originate insulin fibrillation.

Fig. 7. Fluorescence spectra of insulin extracted from insulin-loaded PLGA nanoparticles lyophilized using no annealing (A), and annealing (B). Np stands for nanoparticles.
172 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173

4. Conclusion Abdelwahed, W., Degobert, G., Stainmesse, S., Fessi, H., 2006d. Freeze-drying of
nanoparticles: formulation, process and storage considerations. Adv. Drug
Deliv. Rev. 58, 1688–1713.
In this research work, the main objective was to develop an Ahmad, A., Uversky, V.N., Hong, D., Fink, A.L., 2005. Early events in the fibrillation of
optimized lyophilization cycle using annealing to decrease the monomeric insulin. J. Biol. Chem. 280, 42669–42675.
duration time of the process, and simultaneously ensure the Amidi, M., Pellikaan, H.C., de Boer, A.H., Crommelin, D.J., Hennink, W.E., Jiskoot, W.,
2008. Preparation and physicochemical characterization of supercritically dried
stability of nanoparticles and with upmost importance the insulin-loaded microparticles for pulmonary delivery. Eur. J. Pharm. Biopharm.
maintenance of the loaded protein stability. Additionally, the 68, 191–200.
effect of the cryoprotectant in this stabilization process was duly Bekard, I.B., Dunstan, D.E., 2009. Tyrosine autofluorescence as a measure of bovine
insulin fibrillation. Biophys. J. 97, 2521–2531.
assessed. Insulin and PLGA nanoparticles were used as models in Bouchard, M., Zurdo, J., Nettleton, E.J., Dobson, C.M., Robinson, C.V., 2000. Formation
this study. of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron
Annealing allowed to obtain lyophilizates with a more microscopy. Protein Sci. 9, 1960–1967.
Bozdag, S., Dillen, K., Vandervoort, J., Ludwig, A., 2005. The effect of freeze-drying
homogenous structure and faster reconstitution, than those
with different cryoprotectants and gamma-irradiation sterilization on the
obtained without annealing. The decrease of lyophilization time characteristics of ciprofloxacin HCl-loaded poly(D,L-lactide-glycolide)
using annealing did not increase the residual moisture content in nanoparticles. J. Pharm. Pharmacol. 57, 699–707.
the lyophilizates, which was a good achievement. The presence of Carpenter, J.F., Arakawa, T., Crowe, J.H., 1992. Interactions of stabilizing additives
with proteins during freeze-thawing and freeze-drying. Dev. Biol. Stand. 74
cryoprotectant co-encapsulated was found important to better (225–238), 229–238 (discussion).
stabilize the nanoparticles size during lyophilization with anneal- Danhier, F., Ansorena, E., Silva, J., Coco, R., Le Breton, A., Preat, V., 2012. PLGA-based
ing, mitigating the stresses caused by ice crystals growth. nanoparticles: an overview of biomedical applications. J. Control. Release 161,
505–522.
Regarding the maintenance of protein structure upon lyophili- Dong, A., Huang, P., Caughey, W., 1990. Protein secondary structures in water from
zation, no insulin denaturation, aggregation or fibrillation was second-derivative amide i infrared spectra. Biochemistry 29, 3303–3308.
observed, being a good indicator that the lyophilization process Falconi, M., Bozzi, M., Paci, M., Raudino, A., Purrello, R., Cambria, A., Sette, M.,
Cambria, M.T., 2001. Spectroscopic and molecular dynamics simulation studies
was able to preserve the bioactivity of insulin. It was also of the interaction of insulin with glucose. Int. J. Biol. Macromol. 29, 161–168.
demonstrated that nanoparticle formulations with co-encapsulat- Fonte, P., Araújo, F., Seabra, V., Reis, S., van de Weert, M., Sarmento, B., 2015. Co-
ed cryoprotectant, improved the preservation of the structure of encapsulation of lyoprotectants improves the stability of protein-loaded PLGA
nanoparticles upon lyophilization. Int. J. Pharm. 496 (2), 850–862.
insulin upon lyophilization, compared to nanoparticles without
Fonte, P., Andrade, F., Pinto, J., Seabra, V., van de Weert, M., Reis, S., Sarmento, B.,
cryoprotectant. Indeed, the structural features of insulin loaded Effect of the freezing step in the stability and bioactivity of protein-loaded PLGA
into nanoparticles co-encapsulated with trehalose presented the nanoparticles upon lyophilization.
Griebenow, K., Klibanov, A., 1996. On protein denaturation in aqueous-organic
closest similarity with those of native insulin. The level of insulin
mixtures but not in pure organic solvents. J. Am. Chem. Soc. 118, 11695–11700.
structural maintenance of nanoparticles lyophilized using anneal- Jørgensen, L., Vermehren, C., Bjerregaard, S., Froekjaer, S., 2003. Secondary structure
ing was similar to that obtained without annealing, demonstrating alterations in insulin and growth hormone water-in-oil emulsions. Int. J. Pharm.
that annealing allowed the optimization of the lyophilization cycle 254, 7–10.
Kelly, S., Jess, T., Price, N., 2005. How to study proteins by circular dichroism.
by decreasing its duration time with a simultaneous preservation Biochim. Biophys. Acta 1751, 119–139.
of protein structure. Kendrick, B., Dong, A., Allison, S., Manning, M., Carpenter, J., 1996. Quantitation of
Overall, this work emphasized the importance of the optimiza- the area of overlap between second-derivative amide I infrared spectra to
determine the structural similarity of a protein in different states. J. Pharm. Sci.
tion of lyophilization cycle of polymeric nanoparticles loaded with 85, 155–158.
proteins, and particularly recognizing annealing as a tool to Lakowicz, J.R., 2006. Principles of Fluorescence Spectroscopy. Springer, US, New
decrease the duration of the process and simultaneously ensure York.
LeVine, H., 1995. Thioflavine T interaction with amyloid b-sheet structures. Amyloid
the stability of nanoparticles and the structural preservation of the 2, 1–6.
encapsulated protein. Malik, R., Roy, I., 2011. Probing the mechanism of insulin aggregation during
agitation. Int. J. Pharm. 413, 73–80.
Maltesen, M., Bjerregaard, S., Hovgaard, L., Havelund, S., Van De Weert, M., 2009.
Acknowledgments Analysis of insulin allostery in solution and solid state with FTIR. J. Pharm. Sci.
98, 3265–3277.
The authors would like to thank Fundação para a Ciência e a Maskevich, A.A., Stsiapura, V.I., Kuzmitsky, V.A., Kuznetsova, I.M., Povarova, O.I.,
Uversky, V.N., Turoverov, K.K., 2007. Spectral properties of thioflavin t in
Tecnologia (FCT), Portugal (PTDC/SAL-FCT/104492/2008) and
solvents with different dielectric properties and in a fibril-incorporated form. J.
(SFRH/BD/78127/2011) for financial support. This work was also Proteome Res. 6, 1392–1401.
financed by European Regional Development Fund (ERDF) through Nail, S.L., Jiang, S., Chongprasert, S., Knopp, S.A., 2002. Fundamentals of freeze-
the Programa Operacional Factores de Competitividade—COM- drying. Pharm. Biotechnol. 14, 281–360.
Nemati, F., Cavé, G.N., Couvreur, P., 1992. Lyophilization of substances with low
PETE, by Portuguese funds through FCT in the framework of the water permeability by a modification of crystallized structures during freezing,
project PEst-C/SAU/LA0002/2013, and cofinanced by North Assoc. Pharm. Galenique Ind. 3, 487–493.
Portugal Regional Operational Programme (ON.2
O Novo Norte) Patapoff, T.W., Overcashier, D.E., 2002. The importance of freezing on lyophilization
cycle development. BioPharm 15, 16–21.
in the framework of Project SAESCTN-PIIC&DT/2011 under the Pikal, M.J., Shah, S., Roy, M.L., Putman, R., 1990. The secondary drying stage of freeze
National Strategic Reference Framework (NSRF). It is also drying: drying kinetics as a function of temperature and chamber pressure. Int.
acknowledged the financial support from FCT/MEC through J. Pharm. 60, 203–207.
Sameti, M., Bohr, G., Ravi Kumar, M.N.V., Kneuer, C., Bakowsky, U., Nacken, M.,
national funds and co-financed by FEDER, under the Partnership Schmidt, H., Lehr, C.-M., 2003. Stabilisation by freeze-drying of cationically
Agreement PT2020 (UID/MULTI/04378/2013>—POCI/01/0145/ modified silica nanoparticles for gene delivery. Int. J. Pharm. 266, 51–60.
FERDER/007728). Sarmento, B., Ribeiro, A., Veiga, F., Ferreira, D., 2006. Development and validation of
a rapid reversed-phase HPLC method for the determination of insulin from
nanoparticulate systems. Biomed. Chromatogr. 20, 898–903.
References Sarmento, B., Ferreira, D., Jorgensen, L., van de Weert, M., 2007. Probing insulin’s
secondary structure after entrapment into alginate/chitosan nanoparticles. Eur.
Abdelwahed, W., Degobert, G., Fessi, H., 2006a. Freeze-drying of nanocapsules: J. Pharm. Biopharm. 65, 10–17.
impact of annealing on the drying process. Int. J. Pharm. 324, 74–82. Searles, J.A., Carpenter, J.F., Randolph, T.W., 2001a. Annealing to optimize the
Abdelwahed, W., Degobert, G., Fessi, H., 2006b. Investigation of nanocapsules primary drying rate, reduce freezing-induced drying rate heterogeneity, and
stabilization by amorphous excipients during freeze-drying and storage. Eur. J. determine T(g)0 in pharmaceutical lyophilization. J. Pharm. Sci. 90, 872–887.
Pharm. Biopharm. 63, 87–94. Searles, J.A., Carpenter, J.F., Randolph, T.W., 2001b. The ice nucleation temperature
Abdelwahed, W., Degobert, G., Fessi, H., 2006c. A pilot study of freeze drying of poly determines the primary drying rate of lyophilization for samples frozen on a
(epsilon-caprolactone) nanocapsules stabilized by poly(vinyl alcohol): temperature-controlled shelf. J. Pharm. Sci. 90, 860–871.
formulation and process optimization. Int. J. Pharm. 309, 178–188.
 P. Fonte et al. / International Journal of Pharmaceutics 503 (2016) 163–173 173

Searles, J., 2010. Freezing and annealing phenomena in lyophilization. In: Rey, L., Wang, W., 2000. Lyophilization and development of solid protein pharmaceuticals.
May, J.C. (Eds.), Freeze Drying/Lyophilization of Pharmaceutical and Biological Int. J. Pharm. 203, 1–60.
Products. Informa Healthcare, pp. 52–81. Webb, S.D., Cleland, J.L., Carpenter, J.F., Randolph, T.W., 2003. Effects of annealing
Shi, A.-M., Wang, L.-J., Li, D., Adhikari, B., 2012. The effect of annealing and lyophilized and spray-lyophilized formulations of recombinant human
cryoprotectants on the properties of vacuum-freeze dried starch nanoparticles. interferon-gamma. J. Pharm. Sci. 92, 715–729.
Carbohydr. Polym. 88, 1334–1341. Williams, N.A., Polli, G.P., 1984. The lyophilization of pharmaceuticals: a literature
Tang, X., Pikal, M., 2004. Design of freeze-drying processes for pharmaceuticals: review. J. Parenter. Sci. Technol. 38, 48–59.
practical advice. Pharm. Res. 21, 191–200. Yong, Z., Yingjie, D., Xueli, W., Jinghua, X., Zhengqiang, L., 2009. Conformational and
Van de Weert, M., Hering, J., Haris, P., 2005. Fourier transform infrared spectroscopy. bioactivity analysis of insulin: freeze-drying TBA/water co-solvent system in the
In: Jiskoot, W., Crommelin, D. (Eds.), Methods for Structural Analysis of Protein presence of surfactant and sugar. Int. J. Pharm. 371, 71–81.
Pharmaceuticals. AAPS Press, pp. 131–166.