Preclinical report 323
Chitosan nanoparticles for dermaseptin peptide delivery toward tumor cells in vitro Kelliane A. Medeirosa,b, Graziella A. Joanittia,b,c and Luciano P. Silvaa,b The present study aimed to entrap and characterize the morphology and antitumor effects of a dermaseptin (DStomo01) peptide in chitosan nanoparticles, in vitro. DStomo01 nanoparticles showed moderate polydispersivity, excellent colloidal stability, and slow release. It was noted that free DStomo01 induced DNA fragmentation and mitochondrial hyperpolarization in HeLa cells. However, when entrapped in chitosan nanoparticles, DStomo01 was slightly more active against HeLa cells than the free peptide. In conclusion, the present sustained release system was efficient in entrapping the peptide and reducing tumor cell viability, which are promising steps for future studies involving specific targeting of nanoparticles
c and in-vivo treatments. Anti-Cancer Drugs 25:323–331 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
the spontaneous reaction between protonated chitosan molecules and the polyanion sodium tripolyphosphate [4].
Peptides found in amphibians belonging to the Phyllomedusinae subfamily, in particular peptides classified as dermaseptins, have shown potent cytolytic activity on cell membranes including in cancer cells [1]. Dermaseptin peptides present cationic and amphiphilic features that are related to their interaction with cell membranes; however, their precise mechanism of action remains unknown [1]. Despite showing cytolytic effects that could be explored in anticancer treatments, these peptides are not selective against tumors and could therefore damage healthy cells. In addition, peptides are bioactive molecules that could be hydrolyzed by peptidases, or, if aggregated, easily removed from the body by macrophages present in the blood stream and in the tumor microenvironment [1]. An alternative to overcome both nonspecific cytolytic effects and loss of function is to entrap the peptides in nanoparticulated systems, enabling their protection from enzymatic degradation and macrophages clearance, and even directing them to targeted cells [2]. Among the organic polymers used for nanoparticles preparation, chitosan has been widely explored. This polymer is biocompatible, biodegradable, and facilitates the penetration of drugs across epithelial barriers because of its mucoadhesive properties [3–5]. The use of copolymers, such as polyethylene glycol (PEG), in the production of chitosan nanoparticles make them even more biocompatible and more stable in biological fluids by reducing their natural tendency to aggregate [6]. Among the methods used for peptide entrapment in chitosan nanoparticles [7], ionic gelation has been indicated as it does not include toxic components during the preparation steps and is a simple procedure based on c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:323–331 Keywords: antitumor, cancer, chitosan, dermaseptin, nanoparticle, sustained release a
Embrapa Genetic Resources and Biotechnology, bInstitute of Biology and Ceilandia Faculty, University of Brasilia, Brasilia, DF, Brazil
c
Correspondence to Luciano P. Silva, PhD, Laboratory of Mass Spectrometry, Embrapa Genetic Resources and Biotechnology, Pq. Est. Biol. Final W5, Brası´lia 70770-917, DF, Brazil Tel: + 55 61 3448 4794; fax: + 55 61 34403658; e-mails: [email protected], [email protected] Received 15 August 2013 Revised form accepted 20 October 2013
In this study, we used the ionic gelation method to prepare PEG–chitosan nanoparticles entrapping the chemically synthesized frog peptide dermaseptin (DStomo01). The peptide cytotoxic effects, in its free or entrapped forms, were evaluated on cancer cells, in vitro. Our results showed that the entrapped peptide was stable and more effective in decreasing cell viability than the free peptide.
Materials and methods Materials
Fmoc-amino acids and resin for peptide synthesis were purchased from Peptides International (Louisville, Kentucky, USA). Chitosan, pentasodium [oxido(phosphonatooxy)phosphoryl] phosphate (TPP), and PEG 5000 monomethyl ether were Sigma–Aldrich products (St Louis, Missouri, USA). N,N-Dimethylformamide was acquired from Vetec Quimica (Rio de Janeiro, Brazil) and was treated with KOH for 12 h and distilled before use. 2-(3,5-Diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole bromide (MTT) was an Invitrogen product (Carlsbad, California, USA). Type 1 water (Milli-Q, Bedford, Massachusetts, USA) was employed in all the experiments. Rhodamine 123 and propidium iodide were purchased from Molecular Probes (Eugene, Oregon, USA). Atomic force microscopy (AFM) silicon tips were purchased from Nanosensors (Neuchatel, Switzerland). Synthesis of dermaseptin peptide
The dermaseptin peptide DStomo01 (UniProt database under accession code P85523) was naturally found in the amphibian Phyllomedusa tomopterna, and further synthesized DOI: 10.1097/CAD.0000000000000052
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324 Anti-Cancer Drugs 2014, Vol 25 No 3
using the Fmoc strategy/t-butyl manual synthesis on solid support [Fmoc-Gln (Trt)-Wang Resin, 0.50 mmol/g; Peptides International]. The synthesized crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC; Shimadzu, Kyoto, Japan) using a semipreparative column (Vydac 218TP510, 10 250 mm, ˚; Separations Group, Hesperia, California, USA) and 300 A the sequence identity was confirmed by MALDI-TOF/TOF mass spectrometry (UltraFlex III; Bruker Daltonics, Billerica, Massachusetts, USA) using a-ciano-4-hydroxycinnamic acid as matrix. Preparation of dermaseptin–chitosan nanoparticles
Chitosan nanoparticles were prepared according to the ionic gelation technique as described by Batista et al. [8]. Thus, 1.5 mg/ml of chitosan (75% deacetylated; Sigma–Aldrich) was dissolved in 0.1 mol/l acetic acid under mild magnetic stirring for 1.5 h, and the DStomo01 peptide (100 mg/ml) was added, followed by the addition of TPP aqueous solution (2 mg/ml) dropwise over 30 s. Finally, PEG 5000 monomethyl ether (4.16 mg/ml) was added, while maintaining magnetic stirring for 5 min at 251C. Empty nanoparticles were named CH (formed by the addition of chitosan and TPP) or PEG (obtained with chitosan, TPP, and PEG) and nanoparticles containing the peptide were named CH DStomo01 (formed by the addition of chitosan, TPP, and DStomo01) or PEG DStomo01 (obtained with chitosan, TPP, PEG, and DStomo01).
371C, and after 0, 1, 3, 5, 24, and 48 h in water an aliquot of each sample was collected and centrifuged at 20 000g for 1 h at room temperature. The supernatant was transferred to a new vial and the free DStomo01 peptide was quantified by RP-HPLC as previously described. Cell culture
Mouse melanoma (B16F10) and nontumorigenic mouse fibroblast (NIH/3T3) cell lines were purchased from the cell bank of the Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil); human breast (MCF-7) and cervical (HeLa) cancer cells were purchased from the American Type Culture Collection (http://www.atcc.org/). The HeLa-derivative cell line HSG was donated by Dr B.J. Baum (National Institute of Health, Bethesda, Maryland, USA). The cells were routinely maintained in culture flasks (TPP; Sigma Chemical Co., St Louis, Missouri, USA), at 371C in 5% CO2, in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated FBS (Invitrogen) and 100 IU/ml penicillin and 100 mg/ml streptomycin. The cells were seeded on 96-well plates at a density of 8 103 cells per well at 371C in 5% CO2 overnight. The medium was changed and cells were incubated with different concentrations of DStomo01 (0, 2, 4, 8, 16, 32, 64, 128, and 256 mg/ml) at 371C in 5% CO2. The control group corresponded to cells receiving ultrapure water at the same volume of the peptide. After 24 h treatment, cell viability was assessed as described below.
Characterization of nanoparticles
To determine the nanoparticle loading efficiency, free DStomo01 peptides were removed from the nanoparticle suspension by ultrafiltration through a microcon device (YM-100; Millipore, Bedford, Massachusetts, USA) under centrifugation (3040g, 5 min). The concentration of free peptide was determined using RP-HPLC based on the integrated peak area compared with a standard curve of known concentrations (0.25–4 mg/ml). The nanoparticles were filtered through a 0.22 mm membrane and characterized according to hydrodynamic diameter and polydispersivity by means of dynamic light scattering at an angle of 1731 at 251C. The surface charge of the nanoparticles was determined by zeta potential measurements acquired using ZetaSizer equipment (Malvern Instruments, Malvern, UK). The nanoparticle morphology, diameter, and height were determined by AFM (SPM-9600; Shimadzu) under dynamic mode, using a silicon conical tip attached to a rectangular cantilever with spring constant of 42 N/m and operating at a resonance frequency of about 250 kHz. For these measurements, the samples were diluted at 1 : 1000 (v/v), deposited onto a thin layer of freshly cleaved mica, and mounted on a sample holder. In-vitro release studies
To analyze the DStomo01 release profile, six aliquots (1 ml) from each nanoparticle sample were incubated at
Cell viability assay
Cell viability was determined by a 3,4,5-dimethylthiazol2,5-biphenyl tetrazolium bromide (MTT; Invitrogen) assay. After the cell treatment, the culture media was removed and 150 ml of MTT solution (0.5 mg/ml MTT in culture media) was added to each microplate well. After 3 h of incubation at 371C in 5% CO2, the culture media was aspirated and 100 ml of dimethyl sulfoxide was added. The absorbance was monitored using a spectrophotometer with a microplate reader at a wavelength of 595 nm (SpectraMax; Molecular Devices, Sunnyvale, California, USA). DNA fragmentation, cell cycle, and mitochondrial membrane potential
HeLa cells were seeded on 12-well plates at a density of 5 104 cells in culture medium and incubated with DStomo01 as previously described. After 24 h treatment, the cells were harvested by trypsinization, centrifuged, and prepared for the assays described below. To evaluate DNA fragmentation and cell cycle, the cells were resuspended in 200 ml of 0.1% sodium citrate, 0.1% Triton-X 100, 20 mg/ml propidium iodide, and PBS at pH 7.4 (Invitrogen) and incubated for 30 min at room temperature. For mitochondrial membrane potential analysis, the cells were washed twice with 500 ml of PBS. Then, 0.5 ml
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Chitosan nanoparticles for drug delivery Medeiros et al. 325
of rhodamine 123 solution (5 mg/ml diluted in ethanol; Sigma–Aldrich) was added to each cell group and incubated for 15 min at room temperature. The cells were washed twice with PBS. Finally, the cells were analyzed using a FACSCalibur flow cytometer (Becton & Dickenson, Aalst, Belgium). A total of 10 000 events were collected per sample. Annexin-V/propidium iodide staining
HeLa cells were seeded and incubated with DStomo01. After 24 h, the cells were harvested, washed with PBS, and resuspended in 100 ml binding buffer [10 mmol/l HEPES/NaOH (pH 7.4), 140 mmol/l NaCl, and 2.5 mmol/l CaCl2]. Five microliters of annexin-V FITC (Biosource, Camarillo, California, USA) was added and incubated for 15 min in the dark at room temperature. At this step, propidium iodide at a concentration of 5 mg/ml was added. The cells were analyzed using a FACSCalibur flow cytometer (Becton & Dickenson) and a total of 10 000 events were collected per sample. Morphology of HeLa cells treated with DStomo01
Cell morphology was analyzed by contrast phase microscopy (Zeiss, Oberkochen, Germany) and AFM (Shimadzu). AFM analysis was carried out under contact mode using a silicon nitride tip and a triangular integrated cantilever with spring constant of 0.15 N/m. The cells adhered on coverslips present in the bottom of the culture plate after treatment with 64 mg/ml DStomo01 for 24 h, which decreased the cell viability by about 40%. After removing the culture media, the cells were washed with PBS and fixed with methanol before AFM analysis.
effective against HeLa cells, decreasing their viability by 70 and 100% at the concentrations of 128 and 256 mg/ml, respectively, whereas in the case of HSG and NIH/3T3 cells, the viability decreased by about 90% at the concentration of 256 mg/ml (Fig. 1). On the basis of the MTT assay data, the treatment with 64 mg/ml DStomo01, which decreased about 35% of HeLa cell viability, was further investigated to evaluate specific cytotoxic effects, without leading to pronounced cell death. Investigation of DStomo01 cytotoxic effects
HeLa cells treated with DStomo01 showed apoptosis markers such as annexin-V + staining (Fig. 2a) and DNA fragmentation (Fig. 2b) of 56 and 14%, respectively, both evaluated by flow cytometry, and these values were significantly different from control (P < 0.05). DStomo01 treatment also induced mitochondrial membrane hyperpolarization as indicated by a 48% increase in cells labelled with rhodamine 123 (Fig. 2c). In addition, no significant effects were observed in the cell cycle phase analysis (Fig. 2d). Cell morphology
The HeLa cell morphology treated or not with DStomo01 was analyzed under a phase-contrast inverted microscope (Fig. 3a and b) and the following differences were observed: control cells were adhered to the plate, had colony-forming extensions between them, and presented well-defined nucleus and nucleolus, whereas cells treated with DStomo01 were not adhered to the plate, lost the characteristic cell format, and showed no extensions and no defined nucleus or nucleolus. When the cells were
Effect of DStomo01 chitosan nanoparticles on cell viability
Statistical analysis
All values were expressed as mean±SEM. Each value is the mean of at least three different experiments in each group. The differences in the effects of the treatments compared with control values were analyzed using analysis of variance and Tukey’s post-hoc test. Values significantly different from the control at P value less than 0.05 are indicated in the figures by an asterisk.
Fig. 1
DStomo01 HeLa MCF-7 B16F10
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HSG NIH/3T3
140 Cell viability (%)
To check the possible effects of DStom01 nanoparticles on cell viability, they were tested at peptide concentrations of 0, 2, 4, 8, 16, 32, and 64 mg/ml on HeLa and 32 and 64 mg/ml on NIH/3T3 cell lines, respectively. The effect on cell viability was tested after 24 h treatment as previously described.
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Results DStomo01 treatment moderately impairs cancer cell viability
All the cell lines showed significant decrease in cell viability after 24 h incubation with DStomo01 concentrations above 128 mg/ml (Fig. 1), but their susceptibilities were clearly variable. The treatment was overall more
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Cell viability of tumoral (HeLa, MCF-7, B16F10) and nontumoral (HSG and NIH/3T3) cell lines after DStomo01 treatment for 24 h. The results were obtained using MTT assay. Data are expressed as mean±SEM. *P < 0.05 was considered to be statistically significant compared with the control group using Tukey’s post-hoc test.
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326 Anti-Cancer Drugs 2014, Vol 25 No 3
Fig. 2
(a) Control
Dermaseptin
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Analysis of the mechanism of action of dermaseptin (DStomo01) on the HeLa strain. Treatment was done with 64 mg/ml for 24 h. (a) Alteration of plasma membrane by exposure of phosphatidylserine labeled with annexin-V + and propidium iodide (PI); (b) DNA fragmentation (M1) by PI labeling; (c) marking the change of mitochondrial membrane potential by rhodamine 123 labeling; and (d) phases of the cell cycle by PI labeling. Readings were performed by flow cytometry. Data are expressed as mean±SEM. *P < 0.05 was considered to be statistically significant compared with the control group using Tukey’s post-hoc test.
analyzed under an AFM, it was observed that the cells without treatment had a sharp contrast when viewed twodimensionally and that the treatment with DStomo01 caused profound morphological changes in the cells because the central region showed depth areas (Fig. 3c – untreated and three-dimensionally treated). Nanoparticle characterization
CH DStomo01 and PEG DStomo01 nanoparticles showed an entrapment efficiency of 73 and 80%,
respectively (Table 1). Both nanoparticles showed average hydrodynamic diameters ranging from 100 to 200 nm (Table 1). The empty CH and CH DStomo01 had bimodal and trimodal distribution and positive zeta potential with colloidal stability considered good and excellent (Table 1), respectively, according to American Society for Testing and Materials (ASTM) standardization (1985). The empty nanoparticles of PEG and PEG DStomo01 had unimodal and bimodal distribution, respectively, whereas colloidal stability was considered
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Chitosan nanoparticles for drug delivery Medeiros et al. 327
Fig. 3
(c)
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951.93 (nm)
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Morphology of HeLa cells after treatment with DStomo01 for 24 h. Phase-contrast microscopy (a, b) and atomic force microscopy operated in contact mode (c, d). (a) and (c) without treatment and (b) and (d) treated at a concentration of 64 mg/ml, respectively.
Table 1
Physicochemical properties of chitosan nanoparticles with or without DStomo01 (n = 3 independent measurements)
Nanoparticles CH CH DStomo01 PEG PEG DStomo01
Hydrodynamic diameter average (nm)
PdI
Zeta potential (mV)
EE (%)
1281 105.7 220.2 164.2
0.69 0.17 0.41 0.48
+ 42 + 67 + 39 + 30
– 73 – 80
CH, nanoparticles with chitosan and TPP (CH); CH DStomo01, chitosan, TPP, and peptide DStomo01; EE, entrapment efficiency; PdI, polydispersivity index; PEG, chitosan and TPP coated with polyethylene glycol (PEG); PEG DStomo01, chitosan, TPP, DStomo01 reversed peptide with polyethylene glycol.
moderate for both (Table 1). These same characteristics were maintained after storage at 41C for 1 month. There was reproducibility of measured parameters among several distinct nanoparticle preparations. AFM analyses of the nanoparticles showed overall spherical shape. CH DStomo01 nanoparticles presented polydisperse unimodal distribution with average diameters ranging from 100 to 650 nm and bimodal distribution of average heights ranging from 18 to 24 nm (Fig. 4a, c and d). PEG DStomo01 nanoparticles were also polydisperse and showed spherical shape but with size distribution reaching larger diameters ranging from 100 to 1500 nm. The distribution was bimodal, whereas the height distribution was unimodal and 70 nm on average (Fig. 4b, e and f).
Release of DStomo01 from the nanoparticles
After CH DStomo01 and PEG DStomo01 nanoparticle incubation in water at 371C for different periods of time, it was observed that there was no significant release of the peptide at any of the times tested (Fig. 5). Effect of entrapped DStomo01 on HeLa and NIH/3T3 cell viability
The nanoparticles containing entrapped DStomo01 significantly decreased the viability of HeLa and NIH/3T3 cells at the concentrations of 64 and 32 mg/ml, respectively (Fig. 6a and b). The nanoparticles with PEG DStomo01 were more effective than the CH DStomo01 or the free peptide. Empty nanoparticles with PEG and CH had no effect on cell viability when compared with control cells.
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328 Anti-Cancer Drugs 2014, Vol 25 No 3
Fig. 4
(a)
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Topographic images of DStomo01 nanoparticles acquired by atomic force microscopy operated in dynamic mode (a and b). Histograms of class distributions with varied values of height and diameter of the nanoparticles: chitosan DStomo01 (c and d); PEG chitosan DStomo01 (e and f).
Fig. 5
amphibian), and have 86% primary structure identity to dermadistinctin L isolated from Phyllomedusa distincta. Batista et al. [8] analyzed the hemolytic effect of some dermaseptins, and the dermadistinctin L was the only one that showed significant lytic activity toward erythrocytes. Thus, the similar peptide DStomo01 was selected for assays on eukaryote cells, especially for tumor cell lines in culture.
Peptide entrapped (%)
100 80 CH DStomo01 PEG DStomo01
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Release profile of DStomo01 entrapped in chitosan nanoparticles coated or not with polyethylene glycol in water at 371C.
Discussion The peptide DStomo01 showed significant but moderate cytotoxic activity against all tumor cells tested. This peptide completely abolished HeLa cell viability at the highest concentration assayed. Dstomo01 belongs to the class of dermaseptin antimicrobial peptides, present in amphibians of the genus Phyllomedusa (an anuran
According to Morton et al. [9], cationic peptides with ahelical structure, such as expected in DStomo01s, induce programmed cell death by triggering processes such as apoptosis. This is a useful effect when considering the use of such peptides to treat tumors. Other antimicrobial cationic peptides, such as magainin, cecropin, and melittin, exhibit activity on cancer cells in vitro, and although their mechanisms of action are not well known, they are known to induce apoptosis as indicated by phosphatidylserine exposure, DNA fragmentation, and mitochondrial membrane depolarization [10,11]. In the present study, DStomo01 induced hyperpolarization of the mitochondrial membrane and DNA fragmentation on HeLa cells, without affecting their cell cycle. The combined data show that DStomo01 induced cytotoxic effects on cancer cells without altering the cell cycle.
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Chitosan nanoparticles for drug delivery Medeiros et al. 329
Fig. 6
(a)
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Effects of DStomo01 entrapped in chitosan nanoparticles on cell viability for 24 h. The results were obtained by MTT assay. Empty chitosan nanoparticles (CH), empty chitosan with PEG (PEG), and free peptide (DStomo01) were used as controls. (a) Tumoral cell line (HeLa); (b) nontumoral cell line (NIH/3T3). The symbols %, #, and ? represent significant differences from CH, CH DStomo01, and PEG DStomo01, respectively; and the letters b, and d refer to the difference in the corresponding concentration compared with the control (0 mg/ml), and $ when the free peptide was compared with the control (0 mg/ml). Data are expressed as mean±SEM. *P < 0.05 was considered to be statistically significant compared with the control group using Tukey’s post-hoc test.
The effects observed indicated that DStomo01 cytotoxicity is more closely related to apoptosis cell death. Therefore, the DStomo01 mechanism of action may differ from other peptides that act exclusively on membranes. The hyperpolarization may be because of the entry of this peptide by interaction with the cell membrane and then
with the outer mitochondrial membrane. This would prevent the transport of ATP (biosynthesized in the oxidative phosphorylation process) from the inner mitochondrial membrane to the cytosol, leading to hyperpolarization, which precedes mitochondrial disruption with the release of apoptosis-promoting factors, DNA fragmentation, and externalization of phosphatidylserine [12]. This result was similar to that found by Sa´nchez-Alca´zar et al. [13], who investigated camptothecin and T lymphocytes and observed that initially hyperpolarization of the mitochondrial membrane causes the release of cytochrome c from the surface of the inner mitochondrial membrane to cytosol, starting the process of apoptosis. Thus, it can be suggested that in the present study we observed the effects that precede DNA fragmentation: the mitochondrial membrane hyperpolarization with a possible phosphatidylserine externalization in the cell membrane. After investigating the effects of DStomo01 in the free form on cells, it was entrapped in chitosan nanoparticles with average hydrodynamic diameters ranging from 100 to 200 nm. Douglas et al. [14] and Pan et al. [15] obtained chitosan nanoparticles with sizes similar to those observed in the present study, showing an average hydrodynamic diameter of 157 and 250–400 nm, respectively. According to Hughes [16], the nanoparticles size is very important for drug delivery systems, and particles smaller than 100 nm are required to cross the blood–brain barrier, ramifications of the pulmonary system, and joints of the skin, among other tissue and organ barriers. Likewise, the nanoparticle size must also be smaller than 100 nm for them to penetrate tumors and also not to be removed from the body by the immune system. Thus, the nanoparticles developed in the present study are suitable to penetrate tumors, but their in-vivo biodistribution needs to be further investigated. Throughout nanoparticles characterization the high level of entrapment efficiency shown in this study was similar to those described in literature, in which the ionic gelation method was also used. Pan et al. [15] obtained 80% encapsulation yield of insulin in chitosan nanoparticles. Wu et al. [17] entrapped ammonium glycyrrhizinate with an efficiency of 83% for particles of chitosan and 90% for a composite of chitosan and PEG. Garcia-Fuentes et al. [18] also showed that the yield of encapsulation of calcitonin in chitosan nanoparticles was 90%. Therefore, the entrapment efficiency obtained in this study by the ionic gelation technique makes this procedure feasible from the standpoint of obtaining nanosystems for the sustained release of peptides. In addition to showing high entrapment rates, zeta potential data suggested that the CH DStomo01 nanoparticles obtained had an excellent colloidal stability. This result may have been favored by the increase in chitosan solubility [18] or even by the presence of cationic peptide. In contrast, the colloidal stability
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of PEG DStomo01 nanoparticles could be classified only as moderate because of the fact that this polymer has the characteristic of decreasing the nanoparticle surface charge since it is a negatively charged molecule. During the optimization steps of nanoparticle production, it was observed that PEG increased the size of the nanoparticles. This effect is caused by the weak bonding between the oxygen atoms of PEG and the amino group of chitosan, which make the structure more flexible and thus increased in size [18]. De Jintapattanakit et al. [19] also observed that chitosan nanoparticles entrapping insulin and produced by ionic gelation were larger and had a spherical shape when coated by PEG. The present study showed that the nanoparticles had a homogeneous surface, which may suggest that the peptide and other nanoparticle components could be homogeneously dispersed therein. The opposite was found by Simon et al. [20], who visualized insulin nanoparticles by AFM operating in a dynamic phase acquisition, and observed a heterogeneous distribution of components on the surface of the rough and porous nanoparticles, probably with a heterogeneous distribution of insulin. The slow initial DStomo01 release from the nanoparticles in the present developed system follows a different pattern from those described in the literature, in which the bioactive compound is rapidly dissociated at first and then the release continues slowly and steadily with a lower apparent rate [17,21]. This slow release could be related to nanoparticle components and peptide affinity and/ or to morphological features such as low surface roughness of the nanoparticles. In addition, this low release rate can still be related to the deacetylation degree of chitosan used in this study, which was greater than 75%. This fact was verified previously by Xu and Du [21] after observing that the greater the degree of chitosan deacetylation, the slower the release of BSA, since the cross-link with the TPP grew stronger causing decreased permeability of the nanoparticles. In addition, according to Leffler and Muller [22], the acid type in which chitosan is first dissolved can also change the form of drug release. In this study, chitosan used for the preparation of the nanoparticles was initially dissolved in acetic acid, which may also have contributed to the release profile of DStomo01 observed as the nanoparticles became more compact. After nanoparticle characterization, they were used in cell viability tests. Despite nearly no release of DStomo01 when the nanoparticles were suspended in water, a decrease in cell viability was observed after nanoparticles treatment, suggesting that DStomo01 is released when the nanoparticles are suspended in culture medium, upon contact with the cells, and/or that the nanoparticles were internalized by the cells. The nanoparticles containing the peptide DStomo01 were slightly more effective when compared with the free peptide, whereas empty nanoparticles did not affect the cell viability of tumor (HeLa) or nontumoral (NIH/3T3) cell lines. These results are different from those from the current study. Garcia-Fuentes et al. [18] showed that chitosan
nanoparticles with PEG were not toxic to Caco-2 cells and Soman et al. [23] noted that melittin-loaded nanoparticles were less active in B16F10 than free peptide. To overcome the observed toxicity on nontumoral cells and minimize side effects, future studies related to the attachment of ligands (hormones, peptides, or antibodies) that specifically target the DStomo01 nanoparticles to tumor cells are under way. Conclusion
The peptide DStomo01 was active against several cancer cell lines, showing a more pronounced effect on HeLa cells. DStomo01 interacts with membranes, leading to mitochondrial membrane hyperpolarization, DNA fragmentation, and phosphatidylserine externalization. DStomo01 was efficiently entrapped in chitosan nanoparticles with spherical shape, average diameter between 100 and 200 nm, high colloidal stability, and slow release profile. The addition of PEG to the nanoparticle formulation resulted in a decreased colloidal stability and greatly increased the hydrodynamic diameter. It is noteworthy that the nanoparticles developed in this study are promising for future studies as they showed high entrapping rates and were more active against tumor cells than the DStomo01 in the free form.
Acknowledgements The authors are grateful for the financial support of the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), and Fundac¸˜ao de Apoio a Pesquisa no Distrito Federal (FAP-DF) that enabled the development of this study. Conflicts of interest
There are no conflicts of interest.
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Chitosan nanoparticles for dermaseptin peptide delivery toward tumor cells in vitro Kelliane A. Medeirosa,b, Graziella A. Joanittia,b,c and Luciano P. Silvaa,b The present study aimed to entrap and characterize the morphology and antitumor effects of a dermaseptin (DStomo01) peptide in chitosan nanoparticles, in vitro. DStomo01 nanoparticles showed moderate polydispersivity, excellent colloidal stability, and slow release. It was noted that free DStomo01 induced DNA fragmentation and mitochondrial hyperpolarization in HeLa cells. However, when entrapped in chitosan nanoparticles, DStomo01 was slightly more active against HeLa cells than the free peptide. In conclusion, the present sustained release system was efficient in entrapping the peptide and reducing tumor cell viability, which are promising steps for future studies involving specific targeting of nanoparticles
c and in-vivo treatments. Anti-Cancer Drugs 25:323–331 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
the spontaneous reaction between protonated chitosan molecules and the polyanion sodium tripolyphosphate [4].
Peptides found in amphibians belonging to the Phyllomedusinae subfamily, in particular peptides classified as dermaseptins, have shown potent cytolytic activity on cell membranes including in cancer cells [1]. Dermaseptin peptides present cationic and amphiphilic features that are related to their interaction with cell membranes; however, their precise mechanism of action remains unknown [1]. Despite showing cytolytic effects that could be explored in anticancer treatments, these peptides are not selective against tumors and could therefore damage healthy cells. In addition, peptides are bioactive molecules that could be hydrolyzed by peptidases, or, if aggregated, easily removed from the body by macrophages present in the blood stream and in the tumor microenvironment [1]. An alternative to overcome both nonspecific cytolytic effects and loss of function is to entrap the peptides in nanoparticulated systems, enabling their protection from enzymatic degradation and macrophages clearance, and even directing them to targeted cells [2]. Among the organic polymers used for nanoparticles preparation, chitosan has been widely explored. This polymer is biocompatible, biodegradable, and facilitates the penetration of drugs across epithelial barriers because of its mucoadhesive properties [3–5]. The use of copolymers, such as polyethylene glycol (PEG), in the production of chitosan nanoparticles make them even more biocompatible and more stable in biological fluids by reducing their natural tendency to aggregate [6]. Among the methods used for peptide entrapment in chitosan nanoparticles [7], ionic gelation has been indicated as it does not include toxic components during the preparation steps and is a simple procedure based on c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:323–331 Keywords: antitumor, cancer, chitosan, dermaseptin, nanoparticle, sustained release a
Embrapa Genetic Resources and Biotechnology, bInstitute of Biology and Ceilandia Faculty, University of Brasilia, Brasilia, DF, Brazil
c
Correspondence to Luciano P. Silva, PhD, Laboratory of Mass Spectrometry, Embrapa Genetic Resources and Biotechnology, Pq. Est. Biol. Final W5, Brası´lia 70770-917, DF, Brazil Tel: + 55 61 3448 4794; fax: + 55 61 34403658; e-mails: [email protected], [email protected] Received 15 August 2013 Revised form accepted 20 October 2013
In this study, we used the ionic gelation method to prepare PEG–chitosan nanoparticles entrapping the chemically synthesized frog peptide dermaseptin (DStomo01). The peptide cytotoxic effects, in its free or entrapped forms, were evaluated on cancer cells, in vitro. Our results showed that the entrapped peptide was stable and more effective in decreasing cell viability than the free peptide.
Materials and methods Materials
Fmoc-amino acids and resin for peptide synthesis were purchased from Peptides International (Louisville, Kentucky, USA). Chitosan, pentasodium [oxido(phosphonatooxy)phosphoryl] phosphate (TPP), and PEG 5000 monomethyl ether were Sigma–Aldrich products (St Louis, Missouri, USA). N,N-Dimethylformamide was acquired from Vetec Quimica (Rio de Janeiro, Brazil) and was treated with KOH for 12 h and distilled before use. 2-(3,5-Diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole bromide (MTT) was an Invitrogen product (Carlsbad, California, USA). Type 1 water (Milli-Q, Bedford, Massachusetts, USA) was employed in all the experiments. Rhodamine 123 and propidium iodide were purchased from Molecular Probes (Eugene, Oregon, USA). Atomic force microscopy (AFM) silicon tips were purchased from Nanosensors (Neuchatel, Switzerland). Synthesis of dermaseptin peptide
The dermaseptin peptide DStomo01 (UniProt database under accession code P85523) was naturally found in the amphibian Phyllomedusa tomopterna, and further synthesized DOI: 10.1097/CAD.0000000000000052
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324 Anti-Cancer Drugs 2014, Vol 25 No 3
using the Fmoc strategy/t-butyl manual synthesis on solid support [Fmoc-Gln (Trt)-Wang Resin, 0.50 mmol/g; Peptides International]. The synthesized crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC; Shimadzu, Kyoto, Japan) using a semipreparative column (Vydac 218TP510, 10 250 mm, ˚; Separations Group, Hesperia, California, USA) and 300 A the sequence identity was confirmed by MALDI-TOF/TOF mass spectrometry (UltraFlex III; Bruker Daltonics, Billerica, Massachusetts, USA) using a-ciano-4-hydroxycinnamic acid as matrix. Preparation of dermaseptin–chitosan nanoparticles
Chitosan nanoparticles were prepared according to the ionic gelation technique as described by Batista et al. [8]. Thus, 1.5 mg/ml of chitosan (75% deacetylated; Sigma–Aldrich) was dissolved in 0.1 mol/l acetic acid under mild magnetic stirring for 1.5 h, and the DStomo01 peptide (100 mg/ml) was added, followed by the addition of TPP aqueous solution (2 mg/ml) dropwise over 30 s. Finally, PEG 5000 monomethyl ether (4.16 mg/ml) was added, while maintaining magnetic stirring for 5 min at 251C. Empty nanoparticles were named CH (formed by the addition of chitosan and TPP) or PEG (obtained with chitosan, TPP, and PEG) and nanoparticles containing the peptide were named CH DStomo01 (formed by the addition of chitosan, TPP, and DStomo01) or PEG DStomo01 (obtained with chitosan, TPP, PEG, and DStomo01).
371C, and after 0, 1, 3, 5, 24, and 48 h in water an aliquot of each sample was collected and centrifuged at 20 000g for 1 h at room temperature. The supernatant was transferred to a new vial and the free DStomo01 peptide was quantified by RP-HPLC as previously described. Cell culture
Mouse melanoma (B16F10) and nontumorigenic mouse fibroblast (NIH/3T3) cell lines were purchased from the cell bank of the Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil); human breast (MCF-7) and cervical (HeLa) cancer cells were purchased from the American Type Culture Collection (http://www.atcc.org/). The HeLa-derivative cell line HSG was donated by Dr B.J. Baum (National Institute of Health, Bethesda, Maryland, USA). The cells were routinely maintained in culture flasks (TPP; Sigma Chemical Co., St Louis, Missouri, USA), at 371C in 5% CO2, in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated FBS (Invitrogen) and 100 IU/ml penicillin and 100 mg/ml streptomycin. The cells were seeded on 96-well plates at a density of 8 103 cells per well at 371C in 5% CO2 overnight. The medium was changed and cells were incubated with different concentrations of DStomo01 (0, 2, 4, 8, 16, 32, 64, 128, and 256 mg/ml) at 371C in 5% CO2. The control group corresponded to cells receiving ultrapure water at the same volume of the peptide. After 24 h treatment, cell viability was assessed as described below.
Characterization of nanoparticles
To determine the nanoparticle loading efficiency, free DStomo01 peptides were removed from the nanoparticle suspension by ultrafiltration through a microcon device (YM-100; Millipore, Bedford, Massachusetts, USA) under centrifugation (3040g, 5 min). The concentration of free peptide was determined using RP-HPLC based on the integrated peak area compared with a standard curve of known concentrations (0.25–4 mg/ml). The nanoparticles were filtered through a 0.22 mm membrane and characterized according to hydrodynamic diameter and polydispersivity by means of dynamic light scattering at an angle of 1731 at 251C. The surface charge of the nanoparticles was determined by zeta potential measurements acquired using ZetaSizer equipment (Malvern Instruments, Malvern, UK). The nanoparticle morphology, diameter, and height were determined by AFM (SPM-9600; Shimadzu) under dynamic mode, using a silicon conical tip attached to a rectangular cantilever with spring constant of 42 N/m and operating at a resonance frequency of about 250 kHz. For these measurements, the samples were diluted at 1 : 1000 (v/v), deposited onto a thin layer of freshly cleaved mica, and mounted on a sample holder. In-vitro release studies
To analyze the DStomo01 release profile, six aliquots (1 ml) from each nanoparticle sample were incubated at
Cell viability assay
Cell viability was determined by a 3,4,5-dimethylthiazol2,5-biphenyl tetrazolium bromide (MTT; Invitrogen) assay. After the cell treatment, the culture media was removed and 150 ml of MTT solution (0.5 mg/ml MTT in culture media) was added to each microplate well. After 3 h of incubation at 371C in 5% CO2, the culture media was aspirated and 100 ml of dimethyl sulfoxide was added. The absorbance was monitored using a spectrophotometer with a microplate reader at a wavelength of 595 nm (SpectraMax; Molecular Devices, Sunnyvale, California, USA). DNA fragmentation, cell cycle, and mitochondrial membrane potential
HeLa cells were seeded on 12-well plates at a density of 5 104 cells in culture medium and incubated with DStomo01 as previously described. After 24 h treatment, the cells were harvested by trypsinization, centrifuged, and prepared for the assays described below. To evaluate DNA fragmentation and cell cycle, the cells were resuspended in 200 ml of 0.1% sodium citrate, 0.1% Triton-X 100, 20 mg/ml propidium iodide, and PBS at pH 7.4 (Invitrogen) and incubated for 30 min at room temperature. For mitochondrial membrane potential analysis, the cells were washed twice with 500 ml of PBS. Then, 0.5 ml
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Chitosan nanoparticles for drug delivery Medeiros et al. 325
of rhodamine 123 solution (5 mg/ml diluted in ethanol; Sigma–Aldrich) was added to each cell group and incubated for 15 min at room temperature. The cells were washed twice with PBS. Finally, the cells were analyzed using a FACSCalibur flow cytometer (Becton & Dickenson, Aalst, Belgium). A total of 10 000 events were collected per sample. Annexin-V/propidium iodide staining
HeLa cells were seeded and incubated with DStomo01. After 24 h, the cells were harvested, washed with PBS, and resuspended in 100 ml binding buffer [10 mmol/l HEPES/NaOH (pH 7.4), 140 mmol/l NaCl, and 2.5 mmol/l CaCl2]. Five microliters of annexin-V FITC (Biosource, Camarillo, California, USA) was added and incubated for 15 min in the dark at room temperature. At this step, propidium iodide at a concentration of 5 mg/ml was added. The cells were analyzed using a FACSCalibur flow cytometer (Becton & Dickenson) and a total of 10 000 events were collected per sample. Morphology of HeLa cells treated with DStomo01
Cell morphology was analyzed by contrast phase microscopy (Zeiss, Oberkochen, Germany) and AFM (Shimadzu). AFM analysis was carried out under contact mode using a silicon nitride tip and a triangular integrated cantilever with spring constant of 0.15 N/m. The cells adhered on coverslips present in the bottom of the culture plate after treatment with 64 mg/ml DStomo01 for 24 h, which decreased the cell viability by about 40%. After removing the culture media, the cells were washed with PBS and fixed with methanol before AFM analysis.
effective against HeLa cells, decreasing their viability by 70 and 100% at the concentrations of 128 and 256 mg/ml, respectively, whereas in the case of HSG and NIH/3T3 cells, the viability decreased by about 90% at the concentration of 256 mg/ml (Fig. 1). On the basis of the MTT assay data, the treatment with 64 mg/ml DStomo01, which decreased about 35% of HeLa cell viability, was further investigated to evaluate specific cytotoxic effects, without leading to pronounced cell death. Investigation of DStomo01 cytotoxic effects
HeLa cells treated with DStomo01 showed apoptosis markers such as annexin-V + staining (Fig. 2a) and DNA fragmentation (Fig. 2b) of 56 and 14%, respectively, both evaluated by flow cytometry, and these values were significantly different from control (P < 0.05). DStomo01 treatment also induced mitochondrial membrane hyperpolarization as indicated by a 48% increase in cells labelled with rhodamine 123 (Fig. 2c). In addition, no significant effects were observed in the cell cycle phase analysis (Fig. 2d). Cell morphology
The HeLa cell morphology treated or not with DStomo01 was analyzed under a phase-contrast inverted microscope (Fig. 3a and b) and the following differences were observed: control cells were adhered to the plate, had colony-forming extensions between them, and presented well-defined nucleus and nucleolus, whereas cells treated with DStomo01 were not adhered to the plate, lost the characteristic cell format, and showed no extensions and no defined nucleus or nucleolus. When the cells were
Effect of DStomo01 chitosan nanoparticles on cell viability
Statistical analysis
All values were expressed as mean±SEM. Each value is the mean of at least three different experiments in each group. The differences in the effects of the treatments compared with control values were analyzed using analysis of variance and Tukey’s post-hoc test. Values significantly different from the control at P value less than 0.05 are indicated in the figures by an asterisk.
Fig. 1
DStomo01 HeLa MCF-7 B16F10
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HSG NIH/3T3
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To check the possible effects of DStom01 nanoparticles on cell viability, they were tested at peptide concentrations of 0, 2, 4, 8, 16, 32, and 64 mg/ml on HeLa and 32 and 64 mg/ml on NIH/3T3 cell lines, respectively. The effect on cell viability was tested after 24 h treatment as previously described.
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Results DStomo01 treatment moderately impairs cancer cell viability
All the cell lines showed significant decrease in cell viability after 24 h incubation with DStomo01 concentrations above 128 mg/ml (Fig. 1), but their susceptibilities were clearly variable. The treatment was overall more
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Cell viability of tumoral (HeLa, MCF-7, B16F10) and nontumoral (HSG and NIH/3T3) cell lines after DStomo01 treatment for 24 h. The results were obtained using MTT assay. Data are expressed as mean±SEM. *P < 0.05 was considered to be statistically significant compared with the control group using Tukey’s post-hoc test.
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326 Anti-Cancer Drugs 2014, Vol 25 No 3
Fig. 2
(a) Control
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Analysis of the mechanism of action of dermaseptin (DStomo01) on the HeLa strain. Treatment was done with 64 mg/ml for 24 h. (a) Alteration of plasma membrane by exposure of phosphatidylserine labeled with annexin-V + and propidium iodide (PI); (b) DNA fragmentation (M1) by PI labeling; (c) marking the change of mitochondrial membrane potential by rhodamine 123 labeling; and (d) phases of the cell cycle by PI labeling. Readings were performed by flow cytometry. Data are expressed as mean±SEM. *P < 0.05 was considered to be statistically significant compared with the control group using Tukey’s post-hoc test.
analyzed under an AFM, it was observed that the cells without treatment had a sharp contrast when viewed twodimensionally and that the treatment with DStomo01 caused profound morphological changes in the cells because the central region showed depth areas (Fig. 3c – untreated and three-dimensionally treated). Nanoparticle characterization
CH DStomo01 and PEG DStomo01 nanoparticles showed an entrapment efficiency of 73 and 80%,
respectively (Table 1). Both nanoparticles showed average hydrodynamic diameters ranging from 100 to 200 nm (Table 1). The empty CH and CH DStomo01 had bimodal and trimodal distribution and positive zeta potential with colloidal stability considered good and excellent (Table 1), respectively, according to American Society for Testing and Materials (ASTM) standardization (1985). The empty nanoparticles of PEG and PEG DStomo01 had unimodal and bimodal distribution, respectively, whereas colloidal stability was considered
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Chitosan nanoparticles for drug delivery Medeiros et al. 327
Fig. 3
(c)
(a)
951.93 (nm)
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Morphology of HeLa cells after treatment with DStomo01 for 24 h. Phase-contrast microscopy (a, b) and atomic force microscopy operated in contact mode (c, d). (a) and (c) without treatment and (b) and (d) treated at a concentration of 64 mg/ml, respectively.
Table 1
Physicochemical properties of chitosan nanoparticles with or without DStomo01 (n = 3 independent measurements)
Nanoparticles CH CH DStomo01 PEG PEG DStomo01
Hydrodynamic diameter average (nm)
PdI
Zeta potential (mV)
EE (%)
1281 105.7 220.2 164.2
0.69 0.17 0.41 0.48
+ 42 + 67 + 39 + 30
– 73 – 80
CH, nanoparticles with chitosan and TPP (CH); CH DStomo01, chitosan, TPP, and peptide DStomo01; EE, entrapment efficiency; PdI, polydispersivity index; PEG, chitosan and TPP coated with polyethylene glycol (PEG); PEG DStomo01, chitosan, TPP, DStomo01 reversed peptide with polyethylene glycol.
moderate for both (Table 1). These same characteristics were maintained after storage at 41C for 1 month. There was reproducibility of measured parameters among several distinct nanoparticle preparations. AFM analyses of the nanoparticles showed overall spherical shape. CH DStomo01 nanoparticles presented polydisperse unimodal distribution with average diameters ranging from 100 to 650 nm and bimodal distribution of average heights ranging from 18 to 24 nm (Fig. 4a, c and d). PEG DStomo01 nanoparticles were also polydisperse and showed spherical shape but with size distribution reaching larger diameters ranging from 100 to 1500 nm. The distribution was bimodal, whereas the height distribution was unimodal and 70 nm on average (Fig. 4b, e and f).
Release of DStomo01 from the nanoparticles
After CH DStomo01 and PEG DStomo01 nanoparticle incubation in water at 371C for different periods of time, it was observed that there was no significant release of the peptide at any of the times tested (Fig. 5). Effect of entrapped DStomo01 on HeLa and NIH/3T3 cell viability
The nanoparticles containing entrapped DStomo01 significantly decreased the viability of HeLa and NIH/3T3 cells at the concentrations of 64 and 32 mg/ml, respectively (Fig. 6a and b). The nanoparticles with PEG DStomo01 were more effective than the CH DStomo01 or the free peptide. Empty nanoparticles with PEG and CH had no effect on cell viability when compared with control cells.
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328 Anti-Cancer Drugs 2014, Vol 25 No 3
Fig. 4
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Topographic images of DStomo01 nanoparticles acquired by atomic force microscopy operated in dynamic mode (a and b). Histograms of class distributions with varied values of height and diameter of the nanoparticles: chitosan DStomo01 (c and d); PEG chitosan DStomo01 (e and f).
Fig. 5
amphibian), and have 86% primary structure identity to dermadistinctin L isolated from Phyllomedusa distincta. Batista et al. [8] analyzed the hemolytic effect of some dermaseptins, and the dermadistinctin L was the only one that showed significant lytic activity toward erythrocytes. Thus, the similar peptide DStomo01 was selected for assays on eukaryote cells, especially for tumor cell lines in culture.
Peptide entrapped (%)
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Release profile of DStomo01 entrapped in chitosan nanoparticles coated or not with polyethylene glycol in water at 371C.
Discussion The peptide DStomo01 showed significant but moderate cytotoxic activity against all tumor cells tested. This peptide completely abolished HeLa cell viability at the highest concentration assayed. Dstomo01 belongs to the class of dermaseptin antimicrobial peptides, present in amphibians of the genus Phyllomedusa (an anuran
According to Morton et al. [9], cationic peptides with ahelical structure, such as expected in DStomo01s, induce programmed cell death by triggering processes such as apoptosis. This is a useful effect when considering the use of such peptides to treat tumors. Other antimicrobial cationic peptides, such as magainin, cecropin, and melittin, exhibit activity on cancer cells in vitro, and although their mechanisms of action are not well known, they are known to induce apoptosis as indicated by phosphatidylserine exposure, DNA fragmentation, and mitochondrial membrane depolarization [10,11]. In the present study, DStomo01 induced hyperpolarization of the mitochondrial membrane and DNA fragmentation on HeLa cells, without affecting their cell cycle. The combined data show that DStomo01 induced cytotoxic effects on cancer cells without altering the cell cycle.
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Chitosan nanoparticles for drug delivery Medeiros et al. 329
Fig. 6
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Effects of DStomo01 entrapped in chitosan nanoparticles on cell viability for 24 h. The results were obtained by MTT assay. Empty chitosan nanoparticles (CH), empty chitosan with PEG (PEG), and free peptide (DStomo01) were used as controls. (a) Tumoral cell line (HeLa); (b) nontumoral cell line (NIH/3T3). The symbols %, #, and ? represent significant differences from CH, CH DStomo01, and PEG DStomo01, respectively; and the letters b, and d refer to the difference in the corresponding concentration compared with the control (0 mg/ml), and $ when the free peptide was compared with the control (0 mg/ml). Data are expressed as mean±SEM. *P < 0.05 was considered to be statistically significant compared with the control group using Tukey’s post-hoc test.
The effects observed indicated that DStomo01 cytotoxicity is more closely related to apoptosis cell death. Therefore, the DStomo01 mechanism of action may differ from other peptides that act exclusively on membranes. The hyperpolarization may be because of the entry of this peptide by interaction with the cell membrane and then
with the outer mitochondrial membrane. This would prevent the transport of ATP (biosynthesized in the oxidative phosphorylation process) from the inner mitochondrial membrane to the cytosol, leading to hyperpolarization, which precedes mitochondrial disruption with the release of apoptosis-promoting factors, DNA fragmentation, and externalization of phosphatidylserine [12]. This result was similar to that found by Sa´nchez-Alca´zar et al. [13], who investigated camptothecin and T lymphocytes and observed that initially hyperpolarization of the mitochondrial membrane causes the release of cytochrome c from the surface of the inner mitochondrial membrane to cytosol, starting the process of apoptosis. Thus, it can be suggested that in the present study we observed the effects that precede DNA fragmentation: the mitochondrial membrane hyperpolarization with a possible phosphatidylserine externalization in the cell membrane. After investigating the effects of DStomo01 in the free form on cells, it was entrapped in chitosan nanoparticles with average hydrodynamic diameters ranging from 100 to 200 nm. Douglas et al. [14] and Pan et al. [15] obtained chitosan nanoparticles with sizes similar to those observed in the present study, showing an average hydrodynamic diameter of 157 and 250–400 nm, respectively. According to Hughes [16], the nanoparticles size is very important for drug delivery systems, and particles smaller than 100 nm are required to cross the blood–brain barrier, ramifications of the pulmonary system, and joints of the skin, among other tissue and organ barriers. Likewise, the nanoparticle size must also be smaller than 100 nm for them to penetrate tumors and also not to be removed from the body by the immune system. Thus, the nanoparticles developed in the present study are suitable to penetrate tumors, but their in-vivo biodistribution needs to be further investigated. Throughout nanoparticles characterization the high level of entrapment efficiency shown in this study was similar to those described in literature, in which the ionic gelation method was also used. Pan et al. [15] obtained 80% encapsulation yield of insulin in chitosan nanoparticles. Wu et al. [17] entrapped ammonium glycyrrhizinate with an efficiency of 83% for particles of chitosan and 90% for a composite of chitosan and PEG. Garcia-Fuentes et al. [18] also showed that the yield of encapsulation of calcitonin in chitosan nanoparticles was 90%. Therefore, the entrapment efficiency obtained in this study by the ionic gelation technique makes this procedure feasible from the standpoint of obtaining nanosystems for the sustained release of peptides. In addition to showing high entrapment rates, zeta potential data suggested that the CH DStomo01 nanoparticles obtained had an excellent colloidal stability. This result may have been favored by the increase in chitosan solubility [18] or even by the presence of cationic peptide. In contrast, the colloidal stability
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of PEG DStomo01 nanoparticles could be classified only as moderate because of the fact that this polymer has the characteristic of decreasing the nanoparticle surface charge since it is a negatively charged molecule. During the optimization steps of nanoparticle production, it was observed that PEG increased the size of the nanoparticles. This effect is caused by the weak bonding between the oxygen atoms of PEG and the amino group of chitosan, which make the structure more flexible and thus increased in size [18]. De Jintapattanakit et al. [19] also observed that chitosan nanoparticles entrapping insulin and produced by ionic gelation were larger and had a spherical shape when coated by PEG. The present study showed that the nanoparticles had a homogeneous surface, which may suggest that the peptide and other nanoparticle components could be homogeneously dispersed therein. The opposite was found by Simon et al. [20], who visualized insulin nanoparticles by AFM operating in a dynamic phase acquisition, and observed a heterogeneous distribution of components on the surface of the rough and porous nanoparticles, probably with a heterogeneous distribution of insulin. The slow initial DStomo01 release from the nanoparticles in the present developed system follows a different pattern from those described in the literature, in which the bioactive compound is rapidly dissociated at first and then the release continues slowly and steadily with a lower apparent rate [17,21]. This slow release could be related to nanoparticle components and peptide affinity and/ or to morphological features such as low surface roughness of the nanoparticles. In addition, this low release rate can still be related to the deacetylation degree of chitosan used in this study, which was greater than 75%. This fact was verified previously by Xu and Du [21] after observing that the greater the degree of chitosan deacetylation, the slower the release of BSA, since the cross-link with the TPP grew stronger causing decreased permeability of the nanoparticles. In addition, according to Leffler and Muller [22], the acid type in which chitosan is first dissolved can also change the form of drug release. In this study, chitosan used for the preparation of the nanoparticles was initially dissolved in acetic acid, which may also have contributed to the release profile of DStomo01 observed as the nanoparticles became more compact. After nanoparticle characterization, they were used in cell viability tests. Despite nearly no release of DStomo01 when the nanoparticles were suspended in water, a decrease in cell viability was observed after nanoparticles treatment, suggesting that DStomo01 is released when the nanoparticles are suspended in culture medium, upon contact with the cells, and/or that the nanoparticles were internalized by the cells. The nanoparticles containing the peptide DStomo01 were slightly more effective when compared with the free peptide, whereas empty nanoparticles did not affect the cell viability of tumor (HeLa) or nontumoral (NIH/3T3) cell lines. These results are different from those from the current study. Garcia-Fuentes et al. [18] showed that chitosan
nanoparticles with PEG were not toxic to Caco-2 cells and Soman et al. [23] noted that melittin-loaded nanoparticles were less active in B16F10 than free peptide. To overcome the observed toxicity on nontumoral cells and minimize side effects, future studies related to the attachment of ligands (hormones, peptides, or antibodies) that specifically target the DStomo01 nanoparticles to tumor cells are under way. Conclusion
The peptide DStomo01 was active against several cancer cell lines, showing a more pronounced effect on HeLa cells. DStomo01 interacts with membranes, leading to mitochondrial membrane hyperpolarization, DNA fragmentation, and phosphatidylserine externalization. DStomo01 was efficiently entrapped in chitosan nanoparticles with spherical shape, average diameter between 100 and 200 nm, high colloidal stability, and slow release profile. The addition of PEG to the nanoparticle formulation resulted in a decreased colloidal stability and greatly increased the hydrodynamic diameter. It is noteworthy that the nanoparticles developed in this study are promising for future studies as they showed high entrapping rates and were more active against tumor cells than the DStomo01 in the free form.
Acknowledgements The authors are grateful for the financial support of the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), and Fundac¸˜ao de Apoio a Pesquisa no Distrito Federal (FAP-DF) that enabled the development of this study. Conflicts of interest
There are no conflicts of interest.
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