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Journal ArticleDOI

PEGylated and poloxamer-modified chitosan nanoparticles incorporating a lysine-based surfactant for pH-triggered doxorubicin release.

TL;DR: The overall results suggest that these pH-responsive CS-NPs are highly potent delivery systems to target tumor and intracellular environments, rendering them promising DOX carrier systems for cancer therapy.
About: This article is published in Colloids and Surfaces B: Biointerfaces.The article was published on 2016-02-01 and is currently open access. It has received 53 citations till now. The article focuses on the topics: Poloxamer & Nanocarriers.

Summary (4 min read)

1. Introduction

  • The NPs were well characterized and the mathematical modeling of pH-triggered DOX release profiles was discussed.
  • NP suspensions and lyophilized samples were analyzed regarding their stability at low temperature and under UVA radiation.
  • Finally, in order to gain preliminary insights into the role of the modifiers on the antitumor activity of NPs, the cytotoxicity of free and entrapped drug was assessed by an in vitro cell-based assay.

2.1. Materials

  • Chitosan (CS) of low molecular weight (deacetylation degree, 75-85%; viscosity, 20-300 cP according to the data sheet of the manufacturer), pentasodium tripolyphosphate (TPP), polyethylene glycol methyl ether (mPEG, Mn = 5,000), poloxamer 188 solution (10%, w/v) and 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl) and tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • Reagents for cell culture were from Vitrocell (Campinas, SP, Brazil).
  • Doxorubicin (DOX, state purity 98.32%) was obtained from Zibo Ocean International Trade (Zibo, Shangdong, P.R., China).
  • Acetonitrile and glacial acetic acid were purchased from Tedia (Fairfield, USA).
  • All other solvents and reagents were of analytical grade.

2.2. Surfactant included in the nanoparticles

  • The surfactant chemical structure is formed by two alkyl chains (each with eight carbon atoms) bound to the amino acid lysine.
  • This surfactant was synthesized as described elsewhere [29] .

2.3. Preparation of nanoparticles

  • Unloaded NPs were prepared similarly for each formulation, thus omitting the drug.
  • All procedures involving DOX were conducted in a low incidence of light.
  • The resulting DOXloaded NPs were purified by dialysis for 1 h in distilled water (dialysis bag -Sigma-Aldrich, 14,000 MWCO), in order to remove the non-encapsulated drug and non-incorporated constituents.

2.4. Characterization of nanoparticles

  • Each measurement was performed using at least three sets of ten runs at 25°C.
  • The pH measurements were verified directly in the NP suspensions, using a calibrated potentiometer (UB-10; Denver Instrument, Bohemia, NY, USA), at room temperature.
  • Finally, the spectral properties of the drug were assessed before its encapsulation and also after extraction from the NP structure.
  • This assay was performed in order to verify the stability of DOX after entrapment into the NP matrix.
  • The diluent optimized was water pH 3.0, acidified with glacial acetic acid.

2.5. Drug encapsulation efficiency

  • The quantitative analyses were performed by a reversed-phase liquid chromatography (RP-LC) method that was previously validated according to international guidelines and proved to be specific, linear, precise, accurate and robust (unpublished data).
  • Chromatographic analyses were carried out on a LC 1260 Agilent Technologies system (Agilent Technologies, Santa Clara, CA, USA), using a Waters XBridgeTM C18 column (250 mm x 4.6 mm I.D., 5μm), with a mobile phase consisting of 90% (v/v) acetonitrile in water and water pH 3.0, acidified with glacial acetic acid (33:67, v/v) and UV detection set at 254 nm.
  • Data analysis was performed with EZChrom software program (version A.01.05).
  • An amount of the non-purified NP suspension was placed into this device and submitted to 10,000 rpm for 20 min in a Sigma 2-16P Centrifuge (Sigma, Germany).
  • The encapsulation efficiency (EE%) was calculated as the difference between total and free DOX concentrations determined in the NP suspension (total drug content) and in the ultrafiltrate, respectively, using the mentioned analytical method.

2.6. pH-dependent in vitro DOX release

  • For understanding the pH-sensitivity behavior of NPs, swelling studies were performed by soaking lyophilized NPs into PBS pH 7.4, 6.6 and 5.4 at room temperature and under gentle shake.
  • Hydrodynamic diameter was measured after 3 h incubation.

2.11. Cytotoxicity assays

  • The in vitro antitumor activity of unloaded-CS-NPs, DOX-loaded CS-NPs and free DOX was determined against HeLa cell line (human epithelial cervical cancer), which was cultured in DMEM medium (4.5 g/l glucose) supplemented with 10% (v/v) FBS, at 37ºC in a 5% CO2 atmosphere.
  • Free DOX as well as DOXloaded CS-NPs were assayed at 1 and 10 μg/ml DOX concentration, while unloaded CS-NPs were assessed at 50 and 200 μg/ml.
  • Finally, the MTT containing medium was removed and 100 µl of DMSO was added to each well in order to dissolve the purple formazan product.
  • After shaking, the absorbance of the resulting solution was measured using a SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA) microplate reader at 550 nm.
  • The untreated cell control (cells with medium only) was taken as 100% viability.

3. Results and discussion

  • Different concentrations of poloxamer were tested (0.2%, 0.5% and 1%, w/v), and the intermediate one (0.5%) was chose with acceptable physicochemical characteristics.
  • It was previously reported that micelles containing block copolymers at 0.25 and 2% (w/v), in which DOX is also non-covalently incorporated, exhibited greater efficacy than free DOX in in vitro and in vivo tumor models [50] .

3.1. Characterization and EE% of nanoparticles

  • (with only one negatively charged group) binds to CS, no free negative charge remains available to interact with DOX, therefore leading to diminished EE%.
  • It is important to highlight that when 77KS was incorporated, the authors achieved higher EE% values than previous studies that reported DOX EE% values in the order of 47% for PLGA NPs and 20% for CSbased NPs [5, 53] .

3.2. In vitro DOX release

  • Moreover, the low DOX release at normal physiological conditions may reduce the side effects that can occur during cancer treatment.
  • Altogether, these results support the idea that these nanocarriers are a potential design to be used as a pH-sensitive system to improve the drug availability on tumor microenvironment and intracellular compartments.

3.3. Mathematical modeling

  • Finally, n = 0.2276 was obtained for non-encapsulated DOX, indicating that its release profile is diffusion-controlled.
  • Altogether, their results demonstrated the remarkable contribution of the relaxational process of the polymeric matrix for DOX release at pH 7.4, which may justify the slower drug release under physiological conditions.

3.4. Lyophilization of nanoparticles

  • When mannitol and glycerol were tested as protectants, the obtained result was not satisfactory since the redispersion procedure showed a strong tendency to form aggregates.
  • For the sake of choosing between 1, 5 and 10% lactose, the major criteria evaluated were the yield, drug content and redispersibility index (ratio between the size after lyophilization and before lyophilization).
  • Moreover, only 10% lactose was able to produce a clear suspension, without any visible precipitates (Table 1 ).
  • Sugars are suitable protective agents, acting by hydrogen bonding and maintaining the solute in a pseudo hydrated state during the dehydration step, which thus protects the NP structure from damage in dehydration and rehydration process [63] .

3.5. FT-IR analysis

  • This shifting was confirmed by analyzing the spectrum of unloaded CS-NPs (data not showed).
  • Another peak that can be observed in CS-NPs spectra (Fig. 4C results indicate that DOX was loaded into CS-NPs [18] .
  • The same strong peak appears for pure poloxamer, which represents the stretching vibrational band of methylene group [49, 65] .

3.6. Stability studies of nanoparticles

  • With the aim to study the ability of the nanosystems to protect the encapsulated drug from photodegradation, DOX water solution, as well as DOX-CS-NPs and PEG-DOX-CS-NPs in both suspension and lyophilized states were exposed to UVA radiation.
  • These findings of t1/2, therefore, revealed that the nanostructured systems were not able to protect DOX from the UVA radiation during the entire study period.
  • In contrast, the lyophilized samples L-DOX-CS-NPs and L-PEG-DOX-CS-NPs followed a second kinetic degradation order (r = 0.9975 and 0.9950, respectively) and presented encouraging results about t1/2. L-DOX-CS-NPs and L-PEG-DOX-CS-NPs demonstrated t1/2 values 15-and 7.5-fold greater (62.5 h and 41.67 h) compared to their suspension forms, respectively, suggesting an improvement on photostability of dry solid forms.

3.7. Cytotoxicity assays

  • In vitro assays are very attractive due to ethical aspects and for being a rapid and effective pathway to assess toxicological responses of new nanotechnologies before going to in vivo studies.
  • Therefore, here the authors performed a preliminary study on the potential antitumor activity of the pH-responsive DOX-loaded NPs using an in vitro cell model.
  • The cytotoxic responses of unloaded CS-NPs, DOX-loaded CS-NPs and free DOX were evaluated against HeLa tumor cells using MTT viability assay.
  • The results obtained with DOX-loaded NPs were compared to those with free DOX in order to ensure that the drug encapsulation improves or at least maintains the cytotoxic effects of DOX.
  • Finally, the cell viability was higher than 85% at both tested concentrations of unloaded CS-NPs, indicating that the surfactant 77KS did not promote significant cytotoxic effects [12] .

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PEGylated and poloxamer-modified chitosan nanoparticles incorporating a
1
lysine-based surfactant for pH-triggered doxorubicin release
2
3
4
5
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Laís E. Scheeren
1,2
, Daniele R. Nogueira
1,2,*
, Letícia B. Macedo
1,2
, M. Pilar Vinardell
3
,
8
Montserrat Mitjans
3
, M. Rosa Infante
4
, Clarice M. B. Rolim
1,2
9
10
11
12
13
1
Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Av. Roraima
14
1000, 97105-900, Santa Maria, RS, Brazil
15
2
Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa
16
Maria, Av. Roraima 1000, 97105-900, Santa Maria, RS, Brazil
17
3
Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII
18
s/n, 08028, Barcelona, Spain
19
4
Departamento de Tecnología Química y de Tensioactivos, IQAC, CSIC, C/ Jordi Girona 18-
20
26, 08034, Barcelona, Spain
21
22
23
24
25
* Corresponding author: Phone: +55 55 3220 9548; Fax: +55 55 3220 8248
26
E-mail address: daniele.rubert@gmail.com (Daniele Rubert Nogueira).
27

2
ABSTRACT
28
The growing demand for efficient chemotherapy in many cancers requires novel approaches in
29
target-delivery technologies. Nanomaterials with pH-responsive behavior appear to have
30
potential ability to selectively release the encapsulated molecules by sensing the acidic tumor
31
microenvironment or the low pH found in endosomes. Likewise, polyethylene glycol (PEG)-
32
and poloxamer-modified nanocarriers have been gaining attention regarding their potential to
33
improve the effectiveness of cancer therapy. In this context, DOX-loaded pH-responsive
34
nanoparticles (NPs) modified with PEG or poloxamer were prepared and the effects of these
35
modifiers were evaluated on the overall characteristics of these nanostructures. Chitosan and
36
tripolyphosphate were selected to form NPs by the interaction of oppositely charged
37
compounds. A pH-sensitive lysine-based amphiphile (77KS) was used as a bioactive adjuvant.
38
The strong dependence of 77KS ionization with pH makes this compound an interesting
39
candidate to be used for the design of pH-sensitive devices. The physicochemical
40
characterization of all NPs has been performed, and it was shown that the presence of 77KS
41
clearly promotes a pH-triggered DOX release. Accelerated and continuous release patterns of
42
DOX from CS-NPs under acidic conditions were observed regardless of the presence of PEG
43
or poloxamer. Moreover, photodegradation studies have indicated that the lyophilization of NPs
44
improved DOX stability under UVA radiation. Finally, cytotoxicity experiments have shown
45
the ability of DOX-loaded CS-NPs to kill HeLa tumor cells. Hence, the overall results suggest
46
that these pH-responsive CS-NPs are highly potent delivery systems to target tumor and
47
intracellular environments, rendering them promising DOX carrier systems for cancer therapy.
48
Keywords: chitosan nanoparticles; doxorubicin; in vitro release; in vitro cytotoxicity; lysine-
49
based surfactant; pH-sensitivity
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1. Introduction
51

3
Doxorubicin (DOX) is an anthracycline antibiotic commonly used as a chemotherapeutic agent
52
[1]. Due to its broad-spectrum of antitumor activity, it has been incorporated into several nano-
53
sized materials, including pH-responsive microgels [2], temperature-responsive micelles [3],
54
liposomes [4] and polymeric nanoparticles (NPs) [5,6]. DOX antineoplastic effects can occur
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by different mechanisms, such as free radical generation, which is well associated with the
56
cardiotoxicity of anthracyclines [7]. Drug delivery systems have been gaining attention in recent
57
years as a promising approach to improve cancer treatment and prevent toxicity in healthy
58
tissues. It is noteworthy that by adding different modifiers, these systems can be designed for
59
cancer cell-specific targeting as well as for biological, chemical, or physical stimulus response
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[8,9].
61
Considering that endosomal pH (~ 6.5 to 5.5) [10] and the tumor extracellular pH (pH
e
62
~ 6.6) are notably lower than those of normal tissue (pH ~ 7.4) [11], pH-sensitive devices have
63
been widely researched as drug delivery strategies for cancerous diseases [9]. In this context,
64
our group has paid special attention to a bioactive amino acid-based surfactant derived from
65
N
α
,N
ε
-dioctanoyl lysine with an inorganic sodium counterion (77KS), which in previous studies
66
shown pH-responsive properties and low cytotoxicity [12-14]. Therefore, here we selected
67
77KS as an adjuvant with potential ability to promote the pH-triggered DOX release in the
68
tumor microenvironment and endosomal compartments (Fig. 1).
69
Chitosan (CS) is a nontoxic, biocompatible and biodegradable polymer that has been
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emerging as one of the most promising delivery vehicles for cancer chemotherapy [15].
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Chitosan has been widely used for the development of DOX-loaded NPs by simple and mild
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preparation techniques [5,16-18]. CS-NPs modified by polyethylene glycol (PEG) are explored
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due to the ability of this hydrophilic polymer to prolong the circulation time of nanocarriers in
74
the blood stream. This mechanism allows NP accumulation in the tumor region via enhanced
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permeability and retention (EPR) effect, which, in turn, increases tumor exposure to the
76

4
encapsulated drug [19-22]. Likewise, Pluronic block copolymers (or non-proprietary name
77
“poloxamer”) have been studied as biological response modifiers. They are amphiphilic
78
synthetic polymers with tumor-sensitizing activity in multidrug resistant (MDR) cells, which is
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especially attributed to the inhibition of P-glycoprotein [23]. For this reason, it has been reported
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that the association of DOX to poloxamer-based formulations potentiates the drug activity
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against non-MDR and, especially, MDR tumor cells [24-26].
82
Therefore, the aim of the present study was to prepare PEGylated and poloxamer-
83
modified CS-NPs incorporating a lysine-based surfactant as a pH-responsive bioactive adjuvant.
84
The NPs were well characterized and the mathematical modeling of pH-triggered DOX release
85
profiles was discussed. NP suspensions and lyophilized samples were analyzed regarding their
86
stability at low temperature and under UVA radiation. Finally, in order to gain preliminary
87
insights into the role of the modifiers on the antitumor activity of NPs, the cytotoxicity of free
88
and entrapped drug was assessed by an in vitro cell-based assay.
89
2. Materials and methods
90
2.1. Materials
91
Chitosan (CS) of low molecular weight (deacetylation degree, 75-85%; viscosity, 20-300 cP
92
according to the data sheet of the manufacturer), pentasodium tripolyphosphate (TPP),
93
polyethylene glycol methyl ether (mPEG, M
n
= 5,000), poloxamer 188 solution (10%, w/v) and
94
2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl) and tetrazolium bromide (MTT) were purchased
95
from Sigma-Aldrich (St. Louis, MO, USA). Reagents for cell culture were from Vitrocell
96
(Campinas, SP, Brazil). Doxorubicin (DOX, state purity 98.32%) was obtained from Zibo
97
Ocean International Trade (Zibo, Shangdong, P.R., China). Acetonitrile and glacial acetic acid
98
were purchased from Tedia (Fairfield, USA). All other solvents and reagents were of analytical
99
grade.
100

5
2.2. Surfactant included in the nanoparticles
101
An anionic amino acid-based surfactant derived from N
α
,N
ε
-dioctanoyl lysine and with an
102
inorganic sodium counterion (77KS) was included in the NP formulation. The surfactant
103
chemical structure is formed by two alkyl chains (each with eight carbon atoms) bound to the
104
amino acid lysine. It has a molecular weight of 421.5 g/mol and a critical micellar concentration
105
(CMC) of 3 x 10
3
µg/ml [27,28]. This surfactant was synthesized as described elsewhere [29].
106
2.3. Preparation of nanoparticles
107
CS-NPs were spontaneously formed by ionotropic gelation process, according to the
108
methodology first described by Calvo et al. [30].
DOX stock solution was prepared in ultrapure
109
water in order to give a final concentration of 2.0 mg/ml. Chitosan at 1.0 mg/ml was dissolved
110
in a 1.0% (v/v) acetic acid aqueous solution under magnetic stirring for 2 h, and pH was adjusted
111
to 5.5 with 10 M NaOH [31]. A mixed solution of the cross-linker TPP and the surfactant 77KS
112
was prepared in ultra-pure water at 2.0 mg/ml and 0.5 mg/ml, respectively. Initially, DOX stock
113
solution was added to 5 ml of CS solution (CS:DOX ratio 5:0.5, w/w) and maintained under
114
magnetic stirring (1000 rpm) for 10 min. Then, 1 ml of a premixed TPP:77KS solution (ratio
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equal 2:0.5, w/w) was added drop-wise into the CS:DOX solution. NPs (DOX-CS-NPs) were
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formed spontaneously and the gelation process was carried out under constant magnetic stirring
117
for 20 min at room temperature.
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In order to obtain PEGylated DOX-CS-NPs (PEG-DOX-CS-NPs), a mixed solution of
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CS and PEG (at 1 mg/ml and 10 mg/ml, respectively) was prepared in 1.0% (v/v) acetic acid.
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To 5 ml of this solution, DOX stock solution was added and stirred for 10 min (CS:PEG:DOX
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ratio 5:50:0.5, w/w/w). Afterwards, 1 ml of TPP:77KS (2:0.5, w/w) was added drop-wise and
122
stirred for 20 min.
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