1
CAPES Foundation, Ministry of Education of Brazil,
2
Centre for Textile Science and Technology, University of Minho,
3
Department of Mechanical Engineering, Center for Nanofibers & Nanotechnology, National University of Singapore, Singapore
4
Anti-Infectives Research Group, Singapore Eye Research Institute, Singapore
5
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
6
Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan
University, China
Corresponding author details:
Luis Almeida
Centre for Textile Science and Technology
University of Minho
China
Copyright:
© 2018 Naves LB, et al. This is
an open-access article distributed under the
terms of the Creative Commons Attribution 4.0
international License, which permits unrestricted
use, distribution, and reproduction in any
medium, provided the original author and source
are credited.
In this study, we present a potential alternate approach for the treatment of melanoma
skin cancer and skin tissue regeneration, a comparison of polycaprolactone (PCL) and
polycaprolactone blended with linear (LPEI) and branched polyethylenimine (BPEI). This
research presents the biocompatibility and feasibility of PCL, PCL loaded with LPEI and PCL
loaded with BPEI in different concentrations, producing electrospun scaffolds. SEM images
show that the nanofibers developed between 74 ± 419 nm. Contact angle assay demonstrated
high hydrophobicity for all mats, which could be overcome by surface modification, namely,
plasma treatment, ameliorating the hydrophilicity of the mats, providing excellent cells
adhesion to the scaffolds surface. We demonstrate the biocompatibility of the scaffolds
developed by electrospinning techniques, followed by in vitro tests with Human Dermal
Fibroblasts (HDFs) and murine melanoma cells (B16), by using MTT assay to determinate
the biocompatibility with all cells, and confocal images to give as insights of cell morphology
(nucleus and cellular membrane). Sirius red collagen assay was performed for HDFs to give
the collagen release profile after 6 days of incubation, and the possibility of the mats to
help in skin regeneration process by forming extra cellular matrix (ECM). The CFMDA dye
suggested no cytotoxicity for HDFs to monitor the morphology of live cells. The results
have shown that all the scaffolds developed have good cell adhesion and proliferation
properties. We could also observe high cytotoxicity for B16 melanoma cells for PCL_BPEI
nanofibers. This primary in vitro study suggests that the mats developed may increase
the skin regeneration process and at the same time promote apoptosis of melanoma cells,
therefore it can be an emerging technology for skin regeneration, wound healing process
and treatment of melanoma through electrospun drugs delivery system.
Melanoma; Scaffolds; Electrospinning; Drug Delivery; Tissue Engineering
Nanofibers are widely used in various medical biomedical application such as tissue engineering, cell therapy, regenerative medicine, drug delivery and cancer therapy. These fibers have diameter range of 1 to 100 nanometers, when the diameter is higher than 100 nanometers is called either electrospun mats or scaffolds developed by electrospinning technique. Thus, the scaffolds properties, they have been proven to be much more efficient than any other system for molecular and cellular applications, as compared to their micro- macro scale, we can distinguish some functional properties, such as: high aspect ratio, quantum confinement effects, large surface area, fast- absorbing biomolecules which provides abundant binding and adhesion to cell receptors. The strong cell- matrix interaction allows to develop cell engineering, organs and tissues [1]. Electrospun scaffolds possess among all their characteristics surface morphology, mechanical strength, structural integrity, chemical functionalities and porosity, these characteristics can be tailored, accordingly to their employed application by modulating the fiber orientation, material composition, dimension and alignment. Monophasic electrospun mats have some numerous advantages, however, for some biomedical application is required composites scaffolds owing superior functional and structural properties [2].
Several studies have been carried out on nanofibers composites, demonstrating that for biomedical and biological application, nanofibers composites are better option than their monophasic nanofibers [3].
The interaction within the surrounding matrix and the nanofibers is one of the important factors that determine the composite properties interactions. These interface properties have been demonstrated to have the capability to control broadly properties, such as bonding strengths, bonding types and dislocation densities. The surface structure of each nanofiber will determine each nanofiber composites interface, therefore, every single electrospun mats surface will have an unique properties [2].
In the 21st century, drugs microencapsulation technology has been applied for drugs deliveries. It possesses a significant potential for therapeutic and pharmaceutical fields, as it provides controlled and sustained release of pharmaceutical agents for various medical proposes. It is necessary to have some technical consideration regarding the futures and requirements when designing new pharmaceutical agents; these agents must be identified and specified before the drug design. Ethical approval of pharmaceutical products should be obtained to demonstrate use of the drugs, as being supported by results of clinical trials and animals experiments. Lucas et. al. [4], mentioned that according to the EU 7th amendment of the Cosmetic Directive, the studies in vivo should only be performed when it is not possible to achieve reliable and better scientific outputs in vitro, therefore, in vivo studies should be replaced for in vitro models and procedures. Economic, quality and technical issue should be considered when the pharmaceutical agents microencapsulated technology is employed for mass production. When these drugs are designed for therapeutic application, is very important to identify and examine careful the [4] chemical, physical and therapeutic properties of microcapsules. To avoid an overdose administration of drug, the drug entrapment amount in microcapsules should be cautiously controlled. In addition to that, the particle size of microcapsules designed could alter the total surface area for the drug release. The quality control is necessary for producing microencapsulated medicine with an acceptable quality [5]. Drugs topical delivery is related to the application of drugs to the skin surface, aiming to delivery therapeutic agents trough skin into the systemic circulation or blood stream (transdermal delivery) or to pathological sites of the skin (dermal delivery) [6]. As example of skin disorder treated using transdermal delivery we can mention severe pain which is treated with fentanyl [7], while eczema and psoriasis are treated using dermal delivery [8]. The efficacy of topical drugs delivery is directly related to the diffusion of therapeutic agent through the skin and the drug formulation.
It is known that cancer cell lysosomes may play an important role in intrinsic multidrug resistance (MDR), which is an important hindrance to effective cancer therapy by accumulating chemotherapy drugs and deactivating their therapeutic action. The polymer studied in this manuscript, the cationic charged organic macromolecule which contains amino nitrogen as every third atom, polyethyleneimine (PEI), can disrupt the lysosomal/endosomal membrane via proton-sponge effect (PSE) to provide an effective buffering system for the sudden decrease in pH from the extracellular environment to the endolysosomal compartment [9,10]. Therefore (PEI) has been used to transfect a variety of cell-types, both in vitro and in vivo [11]. PEI is available in both branched (BPEI) and linear forms (LPEI), they belong to the class of synthetic cationic polymer with excellent transfection efficiency with antiviral properties and gene delivery [12-14]. A number of quaternized derivatives of PEIs, either in form of coatings or nanoparticles have shown to elicit potent antimicrobial activity against Gram-negative and Gram- positive pathogens. However, there exist only a limited number of studies that have evaluated the cytotoxicity of PEIs for melanoma cells. Linear or branched PEIs cytotoxicity is directly related to the polymer molecular weight and exposure time. The use of PEIs for biomedical applications are attractive because PEI-coated medical devices are under medical trials for extracorporeal blood purification therapies and PEI-based hydrogels have been approved by the US Food and Drug Administration (FDA) [12]. Scientists have done several approaches for melanoma cancer treatment, and gene therapy including both PEI polymers, either Linear or branched [15], given by their attractive properties for drugs delivery systems [16]. PEIs are promising synthetic vector for drug or gene delivery due to its high cationicity, allowing it to protect and condense small interfering RNA or nucleic acids (plasmid DNA), enabling proton sponge effect by endosomal scape and well nuclease digestion [17]. The kinetics of cells association of both, is very important, once that, there is substantial internalization of PEI bound to the plasma membrane of malignance cells. However, some non-specific binding could be expected due to the ionic interaction, the polycations interactions with the components of the cellular membranes, may regulate their cellular uptake route [18]. The toxicities and transfection efficiencies of PEIs differ in structure dependent or mass dependent manner, so far is very difficult to have any defined relationship between these parameters [19,20]. The use of PEIs might be limited by its proinflammatory effects and high toxicity effects [21].
Using electrospun technology possible to develop new methods in tissue engineering, as organs and tissue that are developed by using a combination of biocompatible polymer scaffolds and patient´s own cells.
Up to now, scientists have focused their studies on developing polymer scaffolds using biocompatible and biodegradable polymers, designing a scaffold that can mimic the extracellular matrix (ECM) proteins, regulating the cells activities and providing mechanical support [22]. Skin substitutes such as autografts, allografts and xenografts have been used for wound healing of patients with significant dermal injuries [23].
Some studies showed that the cell adhesion and proliferation can be enhanced by protein absorption on the surface of the scaffolds [24,25]. For this study we used as main polymer polycaprolactone (PCL) which is a synthetic polymer, semi- crystalline, extensively used for biomedical applications due to its properties like high tensile strength, low melting point, biodegradability and non-toxic nature [26]. Skin tissue engineering, PCL has been investigated as potential matrix for the regeneration of damaged tissues. However, PCL use is limited due to its lack of functional groups, high hydrophobicity, neutral charge and poor bioregulatory activity [27]. To overcome these limitations various post-processing and physico-chemical surface modification techniques have been reported in the literature [28]. It is possible to bring down the disadvantages of PCL by blending another polymer with desired characteristics [29].
Malignant melanoma cells have enhanced proliferation and survival abilities, the major reason for this behavior is their antiapoptosis capacity, which is a problem faced by clinical chemotherapy drug tolerance [30]. In addition to that, chemotherapy treatment is very invasive for patients having low self-immune defense. This is a complex disease that arises through multiple etiologic pathway. Limited number of treatments are available for melanoma medical application, mostly of patients with a more aggressive form of the disease with neither long-lasting nor effective treatment presently exists decline treatment [31]. Although, the major etiologic agent in the development of skin cancer is ultraviolet radiation (UVR) exposure, which is responsible for some gene mutation, such as MAPKERK, which includes the cascade of BRAF, NRAS, MEK1/2 and ERK 1/2proteins, involved in the control of cell proliferation, growth and migration [32]. Mutations in this pathway may play a major role in the progression and development of melanoma cancer [33]. The National Comprehensive Cancer Network (NCCN) guidelines metastatic melanoma can be treated with dacarbazine (DTIC) which is FDA drug approved [34], also with paclitaxel, temozolomide; these treatments are not specific MEK and BRAF inhibitors [35]. Several studies have been carried out on nanofibers composites, demonstrating that for biomedical and biological application, nanofibers composites a better option than their monophasic nanofibers. When compared chitosan nanofibers to blended polycaprolactone (PCL)/chitosan, the cell proliferation increased (> 50%) and obtained better mechanical properties to the composite nanofibers polycaprolactone (PCL)/ chitosan than the monophasic chitosan nanofibers system [3].
Therefore, in this article we investigated the cytotoxicity of
PEIs (LPEI/BPEI) electrospun mats blended with PCL against a
panel of cells, which includes melanoma (B16) and humam dermal
fibroblast (HDF). We developed five difeferent mats with differnte
LPEI and BPEI loads, namely PCL, blended PCL_2LPEI, PCL_5LPEI,
PCL_2BPEI and PCL_5BPEI. Using MTT assay we have shown the high cytotoxicity of PCL coated with PEIs for melanoma cells. Finally, we
demonstrate, using SEM microscopy, CMFDA, and Sirius red collagen,
that crosslink of LPEI/BPEI on to an (PCL) matrix can attenuate the
cytotoxicity for HDFs. It was noted that after 6 days, the collagen
release profile was increased. The present study suggests that the
mats developed may increase the skin regeneration process and at
the same time promote apoptosis of melanoma cells, therefore it can
be an emerging technology for skin regeneration, wound healing
process and treatment of melanoma through electrospun nanofibers
for drugs delivery applications.
Table 1 shows the diameters and the water contact angles of the five different types of nanofibers (Figure 2). Notably, we observed an increase in the fibre diameter in 10% of poly-(ε-caprolactone) blended with 5% of branched polyethylenimine scaffolds, which could be due to possible decrease in the conductivity of the polymer solution.
All the scaffolds prepared in this study showed hydrophobic behaviorwith a contact angle higher than 87°, as we can observe in Figure 3 for non-treated mats. To ameliorate the hydrophilic properties, the scaffolds kept for plasma treatment for 8s. The observed results after plasma treatment are shown in Figure 3. After plasma treatment, we could observe the improvementof hydrophilic properties. The hydrophilic properties of the nanofibrous scaffolds is directly proportional to the rate of water absorbance. This is showed in Table 1, where we can observe the water contact angle values dropped, ranging from 31° to 37°
In Figure 8, is possible to observe the MTT assay, which the results emphasize the good biocompatibility of all mats developed, PCL, PCL_2LPEI, PCL_5LPEI, PCL_2BPEI, PCL_5BPEI, and TCP that served as control. In general, MTT assy is based on the fact that metabolic active cells interact with a tetrazolium salt (in a MTT reagent), therefore, producing a soluble formazan dye. The absorbance intensity is an indication of viable cells present, the higher the absorbance, the higher the viability is [42]. To obtain better insight, data was expressed in cell proliferation ratio after dividing cell number at each time point with initial cell density, the assay was performed on day 1, 4, 7 and 10 after seeding the HDFs cells into the scaffolds. The relatively high optical densities after 4 days of incubation, indicated that PCL, LPEI and BPEI supported cell adhesion on nanofibrous matrices [39]. Results indicated that there was no cytotoxicity rate to all mats. The scaffolds volume ratio and surface area increased the nutrients and oxygen transport, therefore promoting good cell adhesion, growth and proliferation for all the scaffolds. It was not observed apoptotic behavior of human dermal fibroblast cells, on the contrary, over the course of 10 days, the proliferation rate increased meaning a good biocompatibility for HDF, which may be useful for wound healing.
We next determined cell viability using CMFDA live-dead staining. After four days post seeding (p.s.) of hDF cells on various scaffolds, the cells were stained with green CMFDA dye (5-Chloromethylfluorescein diacetate) to obtain semi qualitative information about living and dead cells. All the scaffolds studied displayed no discernible cytotoxicity to HDF cells, therefore, confirming the biocompatibility of all scaffolds for HDF (Figure 9).Next, we determined secretion of collagen using Picro-Sirius red (Figure 10). After 6 days, PCL_2BPEI, PCL_5BPEI and PCL_2LPEI showedhigher collagen release than that in control.
The metabolic assay for living cells MTT, was also performed to melanoma cells, as we can observe in Figure 12. The reading was taken after 24 hours of incubation time, data obtained suggested high toxicity for the mats PCL_2BPEI and PCL_5BPEI, doxorubicin is an anticancer drug and was used as positive control. TCP served as negative control.
To gain insights of cell morphology, confocal images of the melanoma cells cultured on various scaffolds are shown in Figure 13, 24 hours after seeding the cells. The cells were labeled with Hoechst and Phalloidin Rhodamine Actin, for better visualization of the nucleus and membrane of the cells respectively. By taking advantage of the photoluminescent properties of the scaffolds, we used confocal microscopy to confirm the integrity of the fiber mats after 24 hours in the cell culture. It is possible to observe that the multiple layers for TCP, the cells had a normal proliferation and growth, however, we had a different outcome for PCL_2LPEI, PCL_5LPEI, PCL_2BPEI and PCL_5BPEI, the images suggest cytotoxicity for the cells, the membrane becomes rounded, which indicates cell death. Interesting, in our study we did not use any green stain, which was observed on the layer of the scaffolds for PCL, PCL_2LPEI, PCL_5LPEI, PCL_2BPEI and PCL_5BPEI, this suggest that the melanin produced by melanoma cells give green fluorescent color, this is not observer in TCP, which served as control. In addition to that, the fluorescent signals from the cells, confirmed the stability of the mats in the cultured media.
Next we performed the same study for HDFs 24 hours after seeding the cells. The cells were analyzed with Hoechst and Phalloidin Rhodamine Actin, to have a better insight of the nucleus and membrane of the cells respectively. It is possible to observe that nontoxicity is presented in the mats, due to the normal proliferation rate and morphology of cells membrane and nucleus of HDFs seeded in PCL_2LPEI, PCL_5LPEI, PCL_2BPEI and PCL_5BPEI (Figure 14). As comparison on the right side, we have the melanoma confocal images, giving us a clear picture of the toxicity presented in the scaffolds as mentioned above for the control the proliferation of melanoma cells.
Protein kinase C (PKC) family of serine/threonine was one of the first protein kinase to be identified. It is a heterogeneous group of enzymes integrating and receiving signals in both normal melanocytes and melanoma pathology. The chromatographic purification of PKC shows that this activity was composed by at least three distinct species, designated as α, β and γ isotypes [43]. Nowadays, it is known that PKC isotypes of mammalians are superfamily comprising twelve distinct genes. The members of mammalian, PKC, superfamily play an important key on regulatory roles in cellular processes, it has a wide range, from fundamentals autonomous activities such proliferation to cellular memory. Comparative analysis over the last few years have provide insights and defining a number of regulatory elements in PKC which confer to each isotype specific activation signals and location [44].
The alteration of PKC enzyme activation and expression contribute directly to the malignant phenotype of melanoma in both tumour suppressive and oncogenic roles. These enzymes are involved in a varied array of biological process including, migration, cell polarity, proliferation, differentiation and apoptosis. PCK enzymes have profound influences on actin cytoskeleton organization and PCKα, which have been linked to melanoma invasion. Thus, activation and/or alteration in the expression of PCKα cooperate with β3 subunit signalling, this can disrupt stable cell-matrix adhesion and facilitate melanoma invasion. The changes in integrin expression and signalling are important to melanoma invasion and metastasis. The high expression of the subunit β3 is associated with melanoma invasion and progression [45].
PCK activation inhibits the growth of melanoma cells while stimulating proliferation of normal melanocytes cells. The inhibitory growth effect of PKC activation is due to some PKC enzymes possess tumour suppressive functions in melanoma cells [46].
Agonist is a chemical that binds to a receptor and activate the receptor to produce biological response. PCK agonist with differential desensitization/activation specificities holds a promise to the development of PKC based therapeutics for melanoma, by having multiple mechanisms of actions, such as inducing expression of their target PKC enzyme or disrupting tumour vasculature [45].
Multidrug resistance (MDR) is an important hindrance for cancer therapy. Polymeric gene carriers have attracted attention for potential application in the fled of gene therapy, especially for cancer therapy. One of the most important approach to avoid side effects is cancer- specific gene delivery. As we know, protein kinase (PKs) play an important role in regulating various cellular functions, so, dysregulation of specific PKs activity is closely associated with many disease, including cancer. As mentioned previously, there are various PKs, among them, the PCKα is reported as suitable marker to distinguish carcinogenic cell from healthy cells, once that in tumour cells the PCKα activity is much higher than in healthy cells. The buffering capacity of the polyethylenimine main chain, results in polyplexes efficient escape from endosome, showing a clear-cut response towards PKs activity. LPEI and BPEI based carries can be generally applicable to any kind of protein kinase, which are specifically activated in disease cells.
As LPEI and BPEI can target PKCα which has more intense activity
in malignance cells and less activity in healthy cells, we can observe
high cytotoxicity for B16 melanoma cells, and non-cytotoxicity to
human dermal fibroblast cells, as shown in confocal, Figure 14.
Table 1: Characterization of biocomposite nanofibrous scaffolds
Figure 2: Scanning Electron Microspe (SEM) images of all nanofibers (500x magnification). Scale bar = 1µm
Figure 3: Water contact angle for all mats, non- treated mats on the left and after 8s of plasma treatment on the right
Figure 4: Fourier transform infrared spectroscopy spectra for all
nanofibrous mats
Figure 5: Tensile stress- strain curves of PCL, PCL_2LPEI, PCL_5LPEI,
PCL_2BPEI and PCL_5BPEI
Figure 6: Scanning Electron Microspe ( SEM) images show the
nanofibers-cell interaction on the day 1.
PCL, 2LPEI, 5LPEI, 2BPEI, 5BPEI and TCP (500x magnification) on
day 7. Scale bar = 1µm
Figure 7: Scanning Electron Microspe ( SEM) images show the
nanofibers-cell interaction on the day 6. PCL, 2LPEI, 5LPEI, 2BPEI,
5BPEI and TCP (500x magnification) on day 7. Scale bar = 1µm.
Figure 8: MTS assay of electrospun mats after 1, 4, 7 and 4 days of
incubation time of human dermal fibroblast (HDF) cells. Absorbance
Index 490 nm.
Figure 10: Sirius red collagen assay, observation of collagen
release on the day 6 for cultured HDFs
Figure 11: Scanning Electron Microspe ( SEM) images show the
biomaterial-cell interaction with murine melanoma cells B16 after
24 hours of incubation time
Figure 12: MTS assay reading of electrospun mats after 24 hours
of incubation time of murine melanoma cells B16. (****, and ***
represents high cytotoxicity). Absorbance Index 490 nm
Mimicking the formation of ECM, which regulates and supports
cell activities, we developed nanofibrous scaffolds based on PCL
10%, PCL 10%_LPEI2%, PCL 10%_LPEI 5%, PCL 10%_ BPEI 2%,
PCL 10%- BPEI 5% for the use of human dermal fibroblast during
the early stage for skin tissue regeneration; this approach may be
useful for injured skin as burns, diabetic foot ulcers and chronic
wounds. Surface chemical characterization and morphological
analysis confirmed the successful formation of PCL and PCL blended
with PEIs composite structures. The surface wettability issue was
overcome by plasma treatment. Biological studies with HDFs shown
no toxicity for all mats, do not affect the surrounding tissue, good
Figure 13: Confocal images of electrospun mats seeded with murine melanoma cells B16 after 24 hours of incubation time.
Figure 14: Confocal images of electrospun mats seeded with human dermal fibroblasts (HDFs) and murine melanoma cells B16 (Mnoma)
after 24 hours of incubation time
Abbreviations: PCL, -poly-(ε-caprolactone); PCL_2LPEI, poly-(ε-caprolactone) blended with 2% of linear polyethylenimine; PCL_5LPEI,
poly-(ε-caprolactone) blended with 5% of linear polyethylenimine; PCL_2BPEI, poly- (ε-caprolactone) blended with 2% of branched
polyethylenimine; PCL_5BPEI, poly-(ε-caprolactone) blended with 5% of branched polyethylenimine; TCP, tissue culture plate
cell adhesion, cell proliferation and collagen release properties. The
porous nanofibrous scaffolds interconnection may provide more
structural space for HDFs adhesion and proliferation, permitting
efficient metabolic wastes and nutrients exchanges. Together these
data demonstrate that PCL blended with PEIs nanofibrous mats can
provide multifunctional scaffolds properties, ranging from collagen
secretion, cell adhesion and proliferation, acting as localized delivery
of biomolecules to the injured skin, possessing the possibility
to accelerate wound healing, useful for skin tissue engineering
applications. Regarding the B16 murine melanoma cells, we could
conclude that PCL_2BPEI and PCL_5BPEI had the higher anticancer
effect, it is important to note that PCL_5BPEI was toxic for HDFs cells.
Protein kinase (PKs) play an important role in regulating various
cellular functions, so, dysregulation of specific PKs activity is closely
associated with many disease, including cancer. As mentioned
previously, there are various PKs, among them, the PCKα is reported
as suitable marker to distinguish carcinogenic cell from healthy cells, once that in tumour cells the PCKα activity is much higher
than in healthy cells. The buffering capacity of the polyethylenimine
main chain, results in polyplexes efficient escape from endosome,
showing a clear-cut response towards PKs activity. Together, all
these data, suggested that PCL_2BPEI composite nanofibrous mats
would provide multifunctional scaffolds properties for possible skin
engineering, wound healing system and drug delivery application
(Figure 15).
Figure 15: Graphical representation of Protein kinase C, α subunit
Authors wish to acknowledge financial support from
CAPES foundation for the Ph.D. Grant with process Number
13543/13-0, Brazilian Ministry of Education-Brazil. Authors also
like to thank FEDER funds through the Competitivity Factors
Operational Programme—COMPETE and national funds through
FCT—Foundation for Science and Technology (POCI-01-0145-
FEDER-007136). RL thanks the funding support from Singapore
National Research Foundation under its Translational and Clinical
Research Flagship Programme (NMRC/TCR/008-SERI/2013)
and administered by the Singapore Ministry of Health’s National
Medical Research Council and Co-operative Basic Research Grant
from the Singapore National Medical Research Council (Project No.
NMRC/CBRG/0048/2013). NKV acknowledges Start-Up Grant, Lee
Kong Chian School of Medicine, Nanyang Technological University,
Singapore.
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