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Journal of Microencapsulation
Micro and Nano Carriers
ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: https://www.tandfonline.com/loi/imnc20
Designing siRNA-conjugated plant oil-based
nanoparticles for gene silencing and cancer
therapy
Nur Merve Anilmis, Goknur Kara, Ebru Kilicay, Baki Hazer & Emir Baki
Denkbas
To cite this article: Nur Merve Anilmis, Goknur Kara, Ebru Kilicay, Baki Hazer & Emir Baki Denkbas (2019) Designing siRNA-conjugated plant oil-based nanoparticles for gene silencing and cancer therapy, Journal of Microencapsulation, 36:7, 635-648, DOI: 10.1080/02652048.2019.1665117
To link to this article: https://doi.org/10.1080/02652048.2019.1665117
Accepted author version posted online: 11 Sep 2019.
Published online: 19 Sep 2019. Submit your article to this journal
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ORIGINAL ARTICLE
Designing siRNA-conjugated plant oil-based nanoparticles for gene silencing
and cancer therapy
Nur Merve Anilmisa, Goknur Karab, Ebru Kilicayc, Baki Hazerd,e,f,gand Emir Baki Denkbasb,g a
Nanotechnology Engineering Division, Institute of Science and Technology, Bulent Ecevit University, Zonguldak, Turkey;
b
Department of Chemistry, Biochemistry Division,Hacettepe University, Ankara, Turkey;cVocational School of Higher Education, Programme of Biomedical Device Technology, Bulent Ecevit University, Zonguldak, Turkey;dDepartment of Aircraft Mechanic-Engine Maintenance, Cappadocia University,Urgup, Nevsehir, Turkey;eDepartment of Chemistry, Bulent Ecevit University, Universite Caddes, Zonguldak, Turkey;fDepartment of Nanotechnology Engineering, Bulent Ecevit University, Zonguldak, Turkey;gDepartment of Biomedical Engineering, Baskent, University, Ankara, Turkey
ABSTRACT
In this study, the anticancer activities of two siRNA carriers were compared using a human lung adenocarcinoma epithelial cell line (A549). Firstly, poly(styrene)-graft-poly(linoleic acid) (PS-g-PLina) and poly(styrene)-graft-poly(linoleic acid)-graft-poly(ethylene glycol) (PS-g-PLina-g-PEG) graft copolymers were synthesized by free-radical polymerization. PS-PLina and PS-PLina-PEG nanoparticles (NPs) were prepared by solvent evaporation method and were then characterized. The size was found as 150 ± 10 nm for PS-PLina and 184 ± 6 nm for PS-PLina-PEG NPs. The NPs were functionalized with poly(l-lysine) (PLL) for c-myc siRNA conjugation. siRNA entrapment effi-ciencies were found in the range of 4–63% for PS-PLina-PLL and 6–42% for PS-PLina-PEG-PLL NPs. The short-term stability test was realised for 1 month. siRNA release profiles were also investigated. In vitro anticancer activity of siRNA-NPs was determined by MTT, flow cytometry, and fluorescence microscopy analyses. Obtained findings showed that both NPs systems were promising as siRNA delivery tool for lung cancer therapy.
ARTICLE HISTORY Received 11 May 2019 Accepted 4 September 2019 KEYWORDS siRNA; PLina; PS-g-PLina-PEG; nanoparticles; lung cancer Introduction
RNA interference (RNAi), a conserved self-defence mechanism, has gained great attention for the treat-ment of cancer in recent years (Xia et al. 2007, Kim et al. 2008, Tan et al. 2016). siRNA, that consists of 21–23 nucleotides, is the key molecule of RNAi owing to its ability on silencing target oncogene expression in cell cytoplasm (Lee et al. 2016). The penetration of siRNA into the cells results in specific inhibition of messenger RNA (mRNA) and protein of the target gene (Jere et al. 2009). siRNA-based cancer therapy plays a critical role by inhibiting the specific genes in tumour progression and treating cancer without affecting the genome. However, the naked siRNA has a poor bioavailability due to its rapid degradation, low intracellular uptake, inefficient intracellular release, and stability problem in the body. In addition, naked siRNA cannot properly define the target cells (Oh and Park
2009). Since siRNA is a large molecule (13 kDa) and contains negative charge, it is difficult to permeate through the negatively charged cellular membrane. Therefore, suitable carrier systems are needed to
overcome these limitations (Kachalaki et al. 2015). When siRNA is conjugated with proper nanoparti-cles,these systems can accumulate at tumour side, eas-ily transport and penetrate to the target cells in order to effectively inhibit the target gene (Wu et al.2016).
Nanocarriers based on non-viral vectors have been designed to efficiently deliver nucleic acids in many biomedical applications. Nano-carrier systems contrib-ute siRNA for its cellular uptake and endosomal escape, and also protect siRNA from RNase degrad-ation (Convertine et al.2009, Benoit and Boutin 2012, Malcolm et al. 2017, Wang et al. 2017, Malcolm et al.
2018). Cellular uptake of non-viral carriers realises by endocytosis. The fusogenic lipids facilitate the cellular uptake and endosomal release of the drug from the particles. The fusogenic systems are one of the ways that can enhance endosomal escape by damaging the endosomal membrane which causes the cytoplasmic translocation of the nucleic acid and contribute to cytoplasmic delivery of nucleic acids (Farhood et al.
1992, Legendre and Szoka 1992, Felgner et al. 1994, Zhou and Huang 1994). . Koltover et al. investigated
CONTACT Ebru Kilicay ebru.kilicay@gmail.com B€ulent Ecevit University, Zonguldak Vocational High School, Electronic and Automation
Department, Biomedical Device Technology Program, Zonguldak, Turkey ß 2019 Informa UK Limited, trading as Taylor & Francis Group
the interference of lipoplexes with giant anionic vesicles. It was seen that the H11 hexagonal phase
forming lipoplex fused with the surface of giant vesicles, a model for the endosomal membrane. Therefore these lipids, arranged in H11 phase, are
called fusogenic.
Other researchers used the stable nucleic acid lipid particles (SNALPs) 1,2-dioleyloxy-N,Ndimethyl-3-amino-propane (DODMA) (includes a single double bond/ lipid chain) to synthesize lipids with 0, 1, 2, or 3 dou-ble bonds. They reported that H11phase was affected
from the saturation degree of the hydrophobic lipid part. The increasing number of double bonds tended to form the non-bilayer phase. The SNALP bilayer showed propensity to form the fusogenic H11 due to
the decrease of saturation degree in the lipid hydro-phobic domain at the cationic lipid component (Heyes et al.2005).
Many studies were carried out to show that the fusogenicity is important forendosomal escape strat-egy. It was reported from the literature that linoleic acid (C18:2) demonstrated the highest fusogenic activ-ity. Hence, the various cationic lipids were constructed using the hydrophobic tails derived from linoleic acid (Sato et al.2019).
The biodegradable polymeric NPs were used as delivery agents in cancer treatment. The polymer-mediated delivery systems are divided into two groups: natural and synthetic polymers. Chitosan, gel-atin, albumin, and etc. are natural polymers. PEG, PLL, poly(Ɛ-caprolactone) (PCL), and poly (D,L-lactide) (PLA) are the most used synthetic polymers (Lee et al.2016). Among the biodegradable polymers, natural oils, and derivates based on unsaturated oils have a significant impact on the renewable resources. In addition, plant oil-based polymers are promising natural materials to be used as nano-delivery systems. Polyunsaturated plant oils obtained from renewable resources have attracted considerable attention as monomers for gen-erating biodegradable polymers due to their low tox-icity, low price, and universal feasibility.
The auto-oxidation of polyunsaturated oil/oily acids is the available way to synthesize oil-based polymers for obtaining macroperoxy initiators (Cakmakli et al.
2013; Alli et al. 2014; _Ince et al. 2016). The autoxida-tion is carried out under oxygen and sunlight by per-oxidation, epper-oxidation, and so on. The macroperoxides can initiate easily the free radical polymerization of vinyl monomers to obtain graft copolymers (Cakmakli et al.2004,2005,2007, Alli and Hazer2008,2011).
PEG, a hydrophilic, biodegradable, and uncharged polymer, is widely used in drug delivery systems. It
can be prepared as a block copolymer by using proper polymers or coating materials to provide long blood circulation (Guo et al. 2010; Nag and Awasthi 2013). Synthesis and characterization of PS-g-PLina-g-PEG amphiphilic rod-coil tadpole containing a hydrophobic head and a hydrophilic tail and PS-g-PLina-g-poly(di-methyl siloxane) (PDMS) double hydrophobic rod-coil tadpole graft copolymers were reported in 2016 (_Ince et al. 2016). Kilicay et al. synthesized poly(methyl methacrylate) (PMMA) and PMMA copolymers derived from plant oils; polylinseed oil-g-PMMA (PLO-g-PMMA), polysoybean oil-g-PMMA (PSB-g-PMMA), polylinoleic acid-g-PMMA (PLina-g-PMMA) and polyhydroxy alka-noate-sy-g-polylinoleic acid-g-PMMA (PHA-g-PLina-g-PMMA). Micro/nanospheres were prepared from the synthesized graft copolymers. A model drug, acetylsali-cylic acid, was used for loading into microspheres. Different experimental parameters were evaluated to obtain ideal micro/nanosphere formulations (Kilicay et al. 2011). In another study, PLO-g-PMMA, PLO-g-PS, PSB-g-PMMA, PLina-g-PMMA, PLina-g-PS, PLina-g-poly-n-butyl methacrylate (PnBMA), PHA-g-PSB-g-PMMA, and PHA-g-PLina-g-PMMA were synthesized. Dynamic mechanical properties of the homopolymers and copolymers were investigated via tensile test. The copolymers including PLO, PSB, and PLina demon-strated the transition from glassy to rubbery behaviour due to the presence of the triglyceride/oil acid mole-cules plasticiser effect. DNA adsorption of the copoly-mers was also evaluated. The result showed that DNA adsorption and hydrophilicity of the copolymers con-taining PLO, PSB, and PLina blocks were higher than those of the homopolymers (Cakmakli et al.2013).
PLL has been widely studied for nucleic acid-based nanocarrier systems due to its biodegradable, biocom-patible nature, and its intense cationic charge (Kano et al. 2011). Therefore, some cationic polymers like PLL, polyethylene imine (PEI), and histone (Bertrand et al. 2002) can form complexes with siRNA by the electrostatic interactions between the positively charged polymers and the negatively charged phos-phate groups of siRNA (Leng et al. 2007). This can improve the transfection efficiency of siRNA (Reischl and Zimmer 2009). However, the cationic nature of PLL can lead to non- specific interaction with cells. Therefore, the attachment of PLL with amphiphilic or hydrophilic molecules like PEG to avoid non-specific interactions has been extensively searched. Kano et al. (2011) prepared PLL-g-PEG conjugated with siRNA and proved the increasing life time of siRNA in the blood circulation is more than that of the naked siRNA (Sato et al.2007).
The aim of this study was to investigate the effect of PEG grafting to PS-PLina NPs on their in vitro siRNA delivery. For this purpose, at first step, PLina were pre-pared by the autoxidation of linoleic acid under sun-light and then the obtained macroperoxy initiator, PLina, was used to initiate graft copolymers of the styrene via free radical polymerization. PS-g-PLina and PS-g-PLina-g-PEG amphiphilic graft copolymers were attained via the polymerization reaction between the amine terminated PEG and the carboxylic ends of PS-g-PLina. Thereafter, we prepared a series of PS-PLina NPs and PS-PLina-PEG NPs with a diameter of about 150 ± 10 and 184 ± 6 nm, respectively. After functional-izing the NPs surface with PLL, siRNA conjugated com-plexes were designed using c-myc silencing siRNA. To the best of our knowledge, there are no studies on siRNA conjugated PS-PLina and PS-PLina-PEG NPs for cancer treatment applications. The in vitro effect of transfecting A549 cells with siRNA-NPs platforms and subsequently silencing c-myc oncogene were investi-gated. Compared to siRNA-PS-PLina NPs, the low PEGylation rate did not alter the ability to inhibit the target oncogene expression. Therefore, the findings demonstrated that both plant oil-based NPs exhibited a promising siRNA carrying and delivery performance resulting in a significant anticancer activity in cells, with a slight difference between each other.
Experimental
Materials
Linoleic acid (cis-cis-9–12-octadecadienoic acid) was supplied from Fluka (USA) and used as received. Polyethylene glycol-bis-(2-aminopropyl ether) (Jeffamine D-2000; PEG-NH2) (Mw: 2.000 g/mol) was
obtained from Huntsman Co. (Basel, Switzerland). Styrene was supplied by Sigma-Aldrich (Germany) and it was purified by the following steps: washing with 10% (wt) aqueous NaOH solution, drying with anhyd-rous CaCl2 overnight, and distilling with CaH2 under
reduced pressure. PLina-ox was obtained via autoxida-tion of linoleic acid as previously reported by Alli and Hazer (2011). Dichloromethane (DCM) was obtained from Merck (Germany) and chloroform (99% GC grade), stannous octanoate (Snoct2), sodium dodecyl sulphate (SDS), PLL solution (P4707, Mw: 70.000–150.000), and other chemicals were purchased from Sigma-Aldrich. The surfactant, Polyvinyl alcohol (PVA, Mw: 72.000) was purchased from Fluka. All other chemicals were of analytical grade and used without further purification.
C-myc-specific siRNA (Sense strain:
50CCGUGGAUCUGAAUUACAATT-30; antisense strain: 50UUGUAAUUCAGAUCCACGGAA-30) was supplied by Qiagen (Hilden, Germany). A549 and mouse fibroblast cells, L929 cells, were obtained from American Type Culture Collection (ATCC) (Rockwille, MD). Cell culture media, Rosewell Park Memorial Institute-1640 (RPMI-1640), fetal bovine serum (FBS), penicillin–streptomycin, trypsin-ethylenediaminetetraacetic acid (EDTA), and phosphate-buffered saline (PBS) were purchased from Pan-Biotech (Germany). Cytotoxicity agent, 3 –(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was supplied from Glentham Life Sciences (UK). For flow cytometry and fluorescence microscopy analyses, Alexa FluorVR
488 Annexin V/Dead Cell Apoptosis Kit was pur-chased from Thermo Fisher Scientific (Germany).
Autoxidation polymerization of linoleic acid
Autoxidation of linoleic acid was carried out via expose to sunlight under air oxygen at room tempera-ture soto get PLina as macroperoxide initiator. According to the procedure previously cited in the lit-erature (Cakmakli et al. 2007), 5 g of linoleic acid spread out in a petri dish (ؼ 5 cm) was subjected to sunlight at room temperature. About 2 months later, a viscous pale, yellow liquid linoleic peroxide was attained. Approximately, 9% of partitions of the obtained viscous liquid were isolated through the sol–gel analysis with using chloroform (Alli and Hazer2011).
Synthesis of PS-Lina and PS-PLina-PEG graft copolymers
PLina, themacroperoxide initiator, was used to initiate the free radical polymerization of styrene to obtain PS-g-PLina copolymers having carboxylic acid terminal groups. Briefly, 8 g of styrene and 3 g of PLina were dissolved in 10 ml of pure toluene under argon atmos-phere (about 3 min) and then this solution was mixed in oil bath at 95C for 5 h. After that, the raw polymer was dissolved in 20 ml of chloroform and next it was precipitated in 200 ml of methanol. Finally, the raw polymer was filtered and dried at room temperature for 24 h and then dried in vacuum oven at 40C for 24 h.
PS-g-PLina-g-PEG amphiphilic copolymers were obtained between the reaction of the primary amine group of PEG and the carboxylic acid groups of PS-g-PLina copolymer. Briefly, 0.5 g of PS-g-PS-g-PLina and 0.25 g of Jeffamine ED-2003 (amine terminated PEG) were
dissolved in 10 ml of pure toluene under argon atmos-phere (about 3 min) and then this solution was mixed in oil bath at 95C for 5 h. The reaction was stopped, the half of the solvent was evaporated and the same procedure as mentioned above was applied for the raw polymer. In addition to that, the amphiphilic graft copolymer was immersed into distilled water for 24 h so as to remove the unreacted PEG residues and the purified polymer was filtered and dried at room tem-perature for 24 h. In the final step, it was dried under vacuum oven at 40C for 24 h (_Ince et al.2016).
Polymer characterization
FTIR was carried out by using a Perkin-Elmer 1600 Series FTIR Spectrometer. 1H NMR was recorded by using a Varian/Mercury-200 NMR Spectrometer, in deuterochloroform (CDCl3) as solvent and
tetramethyl-silane (TMS or Si(CH3)4) as internal standard.
Synthesis of PS-PLina and PS-PLina-PEG NPs
PS-PLina and PS-PLina-PEG NPs were prepared via double emulsion solvent evaporation method (Perez et al. 2001, Song et al. 2006, Cohen-Sela et al. 2009). Briefly, 10 mg of PS-g-PLina and 10 mg of PS-g-PLina-g-PEG copolymers were dissolved in 10 ml of DCM for forming the organic phase. 50 ml of PVA (0.5 mg/ml) was used as a dispersion medium. The organic phase was added dropwise to dispersion medium by means of an ultrasonic probe (IKA T 125 Digital Ultra Turrax Homogeniser, Germany) in an ice bath at 90% ampli-tude. After that, the same volume of PVA solution was added again into the final emulsion (w/o). To remove the organic solvent from the medium, the mechanical stirrer (Heidolph RZR 2012) and the ultrasonic bath (Alex, Germany) were used at the same time. The sus-pensions were centrifuged at 12000 rpm for 30 min and obtained NPs were washed with deionized water to remove the unreacted residues. Finally, the NPs were dried using a freeze-dryer (Martin Christ GmbH, Germany) and stored until further experiments.
Functionalizing the NPs with PLL
For electrostatically attachment of the siRNA mole-cules to the surface of the NPs, the negatively charged PS-PLina and PS-PLina-PEG NPs were functionalized with PLL (Chevalier et al. 2017). Lysine is a polar basic amino acid that carries proton acceptor molecules in its side chain at physiological pH 7.4. Lyophilised PS-PLina and PS-PS-PLina-PEG NPs were poured in distilled
water at a concentration of 40lg/ml and left in a magnetic stirrer for approximately 1 h to disperse in water well. 250, 500, and 1000ml of PLL solution (0.1% w/v in water) were added to the NP solutions, separ-ately. The resulting solutions were left to stir in the magnetic stirrer for 24 h to ensure that the PLL was attached to the NPs. The mixtures were then centri-fuged at 12000 rpm for 15 min and supernatants were discarded to remove the unbound PLL.
Characterization of the NPs
The size–size distributions and zeta potential values of the NPs prepared with different parameters were ana-lysed using Zeta Sizer (Malvern Instruments, 3000 HSA, England). All measurements were carried out in tripli-cate and the mean ± standard deviation (SD) was recorded for each sample. SEM (FEI Quanta 450 FEG, Germany) was used for morphological evaluation of the NPs. The structural characterization of the NPs was determined by FTIR (Perkin Elmer SpectrumOne, Nicolet 520, USA).
Conjugation of PS-PLina-PLL and PS-PLina-PEG-PLL NPs with c-myc siRNA
The stock siRNA solution silencing c-myc gene was prepared following the manufacturer’s instructions. The lyophilised siRNA purchased from supplier was diluted with RNase-free water reaching a final concen-tration of 20mM. To generate siRNA-attached NPs, dif-ferent siRNA concentrations were interacted with PLL modified NPs that prepared as above (Sato et al.
2007). For PS-PLina-PLL NPs, stock siRNA solutions at the volumes of 3, 7, and 10ml were added to the NP solutions having a final siRNA concentration of 100, 280, and 400 nM, respectively. Likewise, 3, 10, and 15ml of siRNA stock solutions were added to PS-PLina-PEG-PLL NP solutions reaching a final siRNA concentration of 100, 400, and 600 nM, respectively. The resulting solutions in mini centrifuge tubes were placed on a rotor at room temperature and left for complexation reaction for 1 h. The obtained siRNA-NPs formulations were centrifuged at 12000 rpm for 15 min and washed with distilled water. The size and zeta potential of siRNA-attached particles were meas-ured. All measurements were carried out in triplicate and the mean ± SD was recorded for each sample.
Determination of siRNA entrapment efficiency (%)
c-myc siRNA conjugated NPs were produced as men-tioned above. At the centrifuge step, the supernatants of each suspensions for PLL and PS-PLina-PEG-PLL NPs were collected. The amount of unconju-gated siRNAs remaining in the supernatants was determined at 260 nm using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific) (Pittella et al. 2011). The supernatants obtained from siRNA-free NPs were used as a blank. siRNA entrapment effi-ciencies (%) for each used siRNA concentration were calculated according to the following formula:
The amount of conjugated siRNA with nanoparticle ¼ The amount of added siRNA
The amount of unconjugated siRNA remaining in the supernatant
siRNA entrapment efficiency ð%Þ
¼ ðThe amount of conjugated siRNA with nanoparticle =The amount of added siRNAÞ 100
Short-term stability studies of the NPs
PS-PLina and PS-PLina-PEG NPs were freeze-dried prior to determination of their physicochemical stability. The NPs were stored in mini centrifuge tubes at þ4C in aqueous media for one month time period. Aliquots were taken at certain times. The particle size, polydis-persity index (PDI), and zeta potential values of the NPs were recorded via Zeta-sizer for 30 days. All meas-urements were carried out in triplicate and the mean ± SD was recorded for each sample.
In vitro release kinetics
After successfully attachment of c-myc siRNA to the surface of the NPs, the release profiles of the particles at physiological pH were investigated. siRNA conju-gated PS-PLina-PLL and PS-PLina-PEG-PLL NPs were diluted with PBS (pH 7.4) in mini centrifuge tubes and incubated in a water bath at 60 rpm at 37C for 8 days. 100ll of each sample was taken and centrifuged at 12000 rpm for 15 min at different time intervals (0, 2, 4, 24, 48, 72, 90, 144, 168, 216, 240 and 264 h). The same amount of fresh PBS was added into tubes, instead. After centrifugation, the concentration of siRNA released from NPs in supernatant was deter-mined spectrophotometrically at 260 nm. The cumula-tive siRNA release graph against time was drawn using the obtained values (Liu et al. 2012). All
measurements were carried out in triplicate and the mean ± SD was recorded for each sample.
Cell culture and treatment
A549 and L929 cells were growth and maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 at 37C. The cells were cultured in
75 cm3 culture flasks and split upon reaching 80% confluency. The culture medium was changed every 2 days. Before transfecting the cells, the experimental groups were prepared properly. Bare NPs (40, 80, and 120mg/ml) were diluted with completed RPMI-1640 and directly added onto L929 cells. However, the siRNA-conjugated samples and naked siRNAs were diluted with serum and penicillin/streptomycin free RPMI-1640. Then, A549 cells and siRNA containing samples were interacted for 4 h in incubator, after that, 10% FBS was added onto cells and incubation was proceeded until various assays were performed.
MTT cytotoxicity assay
Cytotoxicity of bare NPs againts L929 cells,and c-myc siRNA-conjugated NPs and naked c-myc siRNAs against A549 cells was evaluated by MTT colorometric test based on mitochondrial activity of living cells. A cell density of 5 103 cells per well were seeded in 96-well plates. Next day, the cells were treated with the experimental groups as above and incubated for 24, 48, and 72 h. At the end of the treatments, the content of each well was replaced with the mixture of MTT reagent (5 mg/ml in PBS) and RPMI-1640. The plates were covered with aluminium foil and incu-bated for 4 h at 37C. Then, remaining solution in each well was aspirated, 100ml of isopropanol solution (containing 0.04 N HCl) was added to dissolve the for-mazan crystals and the plates were incubated in dark for a further 30 min. The plates were read at 570 nm using a microplate reader (SpectroStar Nano, BMG Labtech). Cells incubated only with RPMI-1640 were used as control. Each experimental groups were repeated in octuplicate. The cell viability (%) was cal-culated by defining the viability of control cells as 100%. The results were given as mean ± SD.
Apoptosis analysis
The apoptotic effects of c-myc siRNA conjugated NPs on A549 cells were determined using Alexa FluorVR488 Annexin V/Dead Cell Apoptosis Kit according to the
manufacturer’s protocol. The cells were seeded in six-well plates at a density of 3 105 cells/well and cul-tured for 24 h. Various experimental groups were added onto cells and incubated for 48 h. The cells were then washed with PBS and harvested by trypsin addition. After washing with ice-cold PBS and centri-fuge step, the cell pellets were suspended in annexin-binding buffer. Next, annexin V and propidium iodide (PI) (100mg/ml) were added and the suspensions were incubated for 15 min in the dark at room temperature. Samples were analysed using flow cytometer (BD FACSAria II, Biosciences, USA).
Fluorescence microscopy analysis
Fluorescence microscopy analysis was carried out to demonstrate the live and dead cells after treating A549 cells with PS-PLina-PLL-siRNA and PS-PLina-PEG-PLL-siRNA NPs. For this purpose, the cells were seeded in 24-well plates at a density of 2x104 cells/well and cultured for 24 h. The following day, all prepared c-myc siRNA containing samples were added onto the cells and the plate was incubated for 48 h. After that, the cell medium was removed in each well and the cells were washed with 500ml of PBS and 500 ml of annexin-binding buffer, respectively. Subsequently, 100ml of annexin-binding buffer, 5 ml of annexin V and 1ml of PI were added onto the cells. After the wells were incubated in the dark for 15 min, the microscopic analysis was carried out.
Statistical analysis
Results were analysed via using standard analysis of variance (ANOVA) method and presented as the mean values ± SD of triplicate experiments.
Results and discussion
The physicochemical characterizations of synthesized graft copolymers and NPs
InFigure 1(a), the FTIR analysis results were shown for PS-g-PLina, PS-g-PLina-g-PEG copolymers and PS-PLina, PS-PLina-PEG NPs. The bands at 1705 and 1600 cm1 indicate the characteristic carbonyl and phenyl groups of polystyrene of PS-g-PLina and PS-PLina NPs, respectively. The bands at 1610 and 3029 cm1 repre-sent the presence of C¼O and amide CO-NH groups of PS-g-PLina-g-PEG and PS-PLina-PEG NPs, respect-ively. FTIR spectrums of obtained PLina-PLL and PS-PLina-PEG-PLL NPs were also given inFigure 1(a). As it is seen, PLL has characteristic signals at 2070 cm1 for
CON-H and 2860 cm1 for CH2 groups (Clifford
et al.2019).
The 1HNMR spectrums of PLina and PS-g-PLina-g-PEG copolymers were demonstrated in Figure 1(b). The PS-g-PLina copolymer included characteristic peaks of the related segments: 0.8 ppm for CH3group
of the fatty acid macroperoxides; 4.1 ppm for–CH–O– group belongs to PLina and 6–7.2 ppm for phenil pro-ton of polystyrene. The characteristic signals coming from PS-g-PLina-g-PEG were observed at 6.7–7.4 ppm for phenyl group of polystyrene; 3.8 ppm for PEG and 3.6 ppm for–CH–O– group of PLina, respectively (_Ince et al.2016).
Size–size distributions of the NPs
Particle size and size distribution have great import-ance on efficiency of most NPs considering them as in vitro and in vivo delivery systems. These parameters not only have an impact on stability and drug load-ing/release but also on toxicity, targeting capability and biodistribution of NPs (Singh and Lillard 2009). In addition, the biological fate of NPs also depends on their size. It is known that NPs 200 nm tend to be removed more from circulation whereas at size of 100 nm, NPs can escape immediate clearance by the lymphatic system, be able to cross the blood–brain barrier (BBB) and enable to deliver sufficient amount of drug due to their large surface area (Rizvi and Saleh
2018). Numerous studies have shown that NP size also affects cellular uptake level. In a work conducted by Desai et al. (1997), with 100 nm NPs, 2.5 and 6 fold higher Caco-2 cells uptake rates were observed com-paring to the ones with 1 and 100lm microparticles, respectively.
In this study, the amount of polymer and surfactant and the stirring rate were changed in order to realise the size–size distribution and surface charge of the obtained NPs. The other parameters were kept con-stant in all conditions. The results were shown in
Table 1. As seen, NPs were obtained in various sizes with varying experimental parameters. For PS-PLina, NPs between 150 and 512 nm were formed, while for PS-PLina-PEG, NPs ranging from 184 to 665 nm were formed. The repelling force between the NPs increased with the increase in surfactant amount and also the dynamic interfacial tension significantly decreased. Therefore, an increase in the amount of surfactant caused a decrease in the NP size and that way, the NPs became more stable. More NPs were pre-cipitated by increasing the amount of polymers in the preparation stage of the particles and thus, the size of
the NPs increased. The amount of energy transmitted to the suspension medium increased as the homojeni-zation rate increased and this effect decreased the NPs size. The experimental conditions for producing both NPs with the smallest diameter were determined
as: 1 mg/ml polymer concentration, 0.5 mg/ml PVA concentration, and 90 homogenization rate (ampli-tude %).
Khoee et al. synthesized triblock copolymer; amphiphilic poly(ethylene glycol)-poly(butylene
Figure 1. (A) FTIR spectrums of (a) PS-g-PLina, PS-PLina NPs, PS-g-PLina-g-PEG and PEG NPs, PLL and PS-PLina-PEG-PLL NPs; (B) (b) The1HNMR spectrums of PS-g-PLina and PS-g-PLina-g-PEG copolymers; (C) SEM images of (c) PS-PLina NPs, (d) PS-PLina-PEG NPs, (e) PS-PLina-PLL NPs and (f) PS-PLina-PEG-PLL NPs.
Table 1. The effect of polymer and PVA concentrations and homogenization rate on the size–size distribution and zeta potential of PS-PLina and PS-PLina-PEG NPs.
Samples Polymer conc. (mg/ml DCM) PVA conc. (mg/ml) Homogenization rate (amplitude %) Size (nm) PDI Zeta Potential (mV) PS-PLina NPs 5 0.5 90 411 ± 5 0.313 ± 0.042 20.3 ± 0.10 3 0.5 90 339 ± 7 0.216 ± 0.021 27.2 ± 0.31 1 0.5 90 150 ± 10 0.080 ± 0.009 32.8 ± 0.25 1 0.25 90 235 ± 16 0.158 ± 0.018 30.5 ± 0.02 1 0.125 90 328 ± 18 0.378 ± 0.024 19.8 ± 0.30 1 0.5 70 342 ± 17 0.285 ± 0.043 18.0 ± 0.33 1 0.5 50 512 ± 28 0.398 ± 0.075 12.6 ± 0.42 PS-PLina-PEG NPs 5 0.5 90 568 ± 14 0.495 ± 0.093 9.2 ± 0.78 3 0.5 90 499 ± 11 0.307 ± 0.078 10.1 ± 0.22 1 0.5 90 184 ± 6 0.113 ± 0.017 14.6 ± 0.19 1 2 90 265 ± 13 0.277 ± 0.021 13.3 ± 0.27 1 1 90 380 ± 11 0.348 ± 0.008 11.0 ± 0.16 1 0.5 70 402 ± 13 0.318 ± 0.060 12.1 ± 0.23 1 0.5 50 665 ± 17 0.523 ± 0.077 6.5 ± 0.11
adipate)-poly(ethylene glycol) (PEG-PBA-PEG) by poly-condensation reaction and prepared PEG-PBA-PEG NPs based on modified oil in water (O/W) emulsion method and then obtained O/S/W nanocapsules. They used 17b-estradiol valerate (17b-EV) as drug and inter-acted it with the NPs. The effect of polymer concentra-tion, drug content, polymer/oil weight ratio, and surfactant (Tween 80) concentration on nancapsules size were investigated. They demonstrated that the NP size increased as the amount of polymer increased and bimodal size distribution occured. Unimodal size distribution was observed with the small amount of polymer. As the drug loading increased, the particle size and the burst release of the NPs also increased. In addition, the encapsulation efficiency, drug content and the yield of nanocapsule were higher without using any surfactant. Since the PEG acted as a stabil-izer, the size of nanocapsules decreased (Khoee and Hossainzadeh2010).
Functionalization of the NPs with PLL
Another important feature for a delivery NP is its sur-face charge which determines the interaction between the NP and its environment such as target cell or other physiological molecules. Having a positive charge not only provides NPs to easily pass though the negatively charged cell membrane but also enhan-ces complex formation with polyanionic siRNA. However, in the systemic circulation, negatively charged serum proteins will bind to positively charged nanostructure resulting in a decrease in its activity. This can be eliminated by decorating the nanostruc-tures with PEG or other hydrophilic strucnanostruc-tures and forming a barrier that protects them from physio-logical environment, activation of immune responses, and phagocytes (Whitehead et al.2009).
Surface charges of produced PS-PLina and PS-PLina-PEG NPs were also investigated by measuring their zeta potential values that represent surface charge of
NPs in colloidal dispersion. Zeta potential values of PS-PLina NPs varied from32.8 to 12.6 mV while for PS-PLina-PEG NPs it varied between 14.6 and 6.5 mV (Table 1). The surface charge of the NPs was found as negative due to presence of carboxyl groups in the structures. After the characterization studies at the smallest NP size values, zeta potential for PS-PLina, and PS-PLina-PEG NPs were found as32.8 mV and as 14.6 mV, respectively. In addition, PDI values demon-strated that the NP formulations had a homogenous distribution (Table 1). After functionalizing the NPs with PLL, the obtained zeta potential values of NPs were given in Table 2. Various concentrations (250, 500, and 1000ml) of PLL were used. The NP surface charges changed from negative to positive in a con-centration-dependent manner. However, the excess addition of PLL may cause a toxic effect on the cells, therefore, the determination of the optimum amount of PLL is important. When 500ml PLL was used, þ14.9 mV and þ15.8 mV zeta potential values were obtained for PS-PLina-PLL and PS-PLina-PEG-PLL NPs, respectively (Table 2).
PLL modified poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared in a work conducted by Yuan et al. The microspheres were treated with NaOH solution in order to form carboxyl groups on the microsphere surface then they were immerged in PLL solution. PLGA-PLL microspheres were generated via electrostatic interaction between cationic PLL and anionic PLGA microspheres. After PLL modification, the viability of MG63 cells was higher due to the low cytotoxicity of the microspheres. Besides, the prolifer-ation and attachment of the cells with PLGA-PLL microspheres were more comparing to PLGA micro-spheres (Yuan et al. 2018). In another study, PLL-coated tamoxifen-loaded PLGA NPs (PLL-NP-TMX) were prepared to improve NP-cell interaction and hence the therapeutic effect of tamoxifen. PLL coating was carried out by the electrostatic interaction of posi-tively charged PLL (50ml, 100 ml, 200 ml) and negatively
Table 2. The zeta potential values of PS-PLina after conjugation with 250, 500, 1000ml of PLL and 100, 280, 400 nM of siRNA concentrations, respectively; PS-PLina-PEG NPs after conjugation with 250, 500, 1000ml of PLL and 100, 280, and 600 nM of siRNA concentrations, respectively.
Samples 0ml PLL 250ml PLL 500ml PLL 1000ml PLL
PS-PLina NPs 32.8 ± 0.25 þ4.14 mV ± 0.08 þ14.9 mV ± 0.06 þ17.13 mV ± 0.08
PS-PLina-PEG NPs 14.6 ± 0.19 þ9.14 mV ± 0.1 þ15.8 mV ± 0.05 þ20.77 mV ± 0.12
100 nM siRNA 280 nM siRNA 400 nM siRNA 600 nM siRNA
PS-PLina-PLL NPs (250ml) 9.67 mV ± 0.03 10.43mV ± 0.83 10.77 mV ± 0.34 – PS-PLina-PLL NPs (500ml) 5.13 mV ± 0.17 6.27mV ± 0.97 7.87 mV ± 0.56 – PS-PLina-PLL NPs (1000ml) 2.81 mV ± 0.29 4.6 mV ± 0.19 7.6 mV ± 0.01 – PS-PLina-PEG-PLL NPs (250ml) 6.70mV ± 0.94 8.65mV ± 0.92 – 10.12mV ± 0.29 PS-PLina-PEG-PLL NPs (500ml) 13.37 mV ± 1.74 13.67mV ± 1.97 – 14.87mV ± 1.56 PS-PLina-PEG-PLL NPs (1000ml) 11.07 mV ± 0.53 14.43 mV ± 1.2 – 15.83mV ± 0.61 Data represent mean ± SD, n¼ 3.
charged NP-TMX (500ml). In the study, it was demon-strated that these obtained NP formulations had a valuable antiproliferative effect against human breast adenocarcinoma cells (Chevalier et al.2017).
The surface morphology of the NPs
The surface morphology of the NPs was evaluated by SEM analysis and the results were given inFigure 1(c). Prior to the assay, the optimized experimental param-eters were used for preparing bare PLina and PS-PLina-PEG NPs. In addition, 500ml PLL was used to produce PLL-functionalized NPs. In Figure 1(c), it was seen that all NPs were in a well-shaped and properly spherical form with a smooth surface. For each sam-ple, smaller NPs were obtained as in the DLS analysis while larger ones were also observed because of the aggregation. This may be due to too much dilution of the samples when performing DLS measurement.
The conjugation of c-myc siRNA with PS-PLina-PLL and PS-PLina-PEG-PLL NPs
The conjugation reaction occurred via the electrostatic interaction between the positive charges of PS-PLina-PLL and PS-PLina-PEG-PS-PLina-PLL NPs and the negative charges of c-myc siRNA (Meyer et al. 2009, Liu et al.
2012). The zeta potential values obtained after the conjugation of all NP formulations having different amount of PLL with siRNAs at various concentrations were shown in Table 2. According to the results, after interaction with siRNA, the positive charges of all NPs shifted to negative charges. These gained negative charges demonstrated that c-myc siRNA was success-fully attached to the surfaces of all NPs. The negative phosphate groups present in the siRNA neutralize the NH3þ groups in the NPs, resulting in a reduction in
the zeta potential of the cationic NPs (Raja et al.
2015). In addition, a slight different was also observed between the surface charges of siRNA-PS-PLina-PLL and siRNA-PS-PLina-PEG-PLL NPs.
Evaluation of siRNA entrapment efficiency (%)
Figure 2 (a,b)shows the siRNA entrapment efficiencies of PLL functionalized PS-PLina and PS-PLina-PEG NPs evaluated spectrophotometrically. The obtained results revealed that using various amounts of siRNA and PLL, siRNA entrapment efficiencies in the range of 4–61% and 6–42% were achieved for PLina-PLL and PS-PLina-PEG-PLL NPs, respectively. Furthermore, it was observed that the entrapment efficiency decreased as
the amount of siRNA increased for both NPs. This can be explained by the fact that as the concentration of drug loaded into the NPs increases, they tend to pro-trude the drug outside (Anitha et al. 2014). The rela-tively higher entrapment efficiency was achieved through the interaction of NPs modified by 500ml PLL with different amounts of siRNA.
The increasing amount of PEG in the PS-g-PLina graft copolymers during the graft copolymers prepar-ation step caused the decreased solubility of the graft copolymers in organic solvent due to the cross linking of polymers. In this way, the PEG content in the graft copolymers was substantially decreased (Alli et al.
2012). Thus, the entrapment efficiency of PS-PLina-PEG-PLL with siRNA was not importantly increased as compared with PS-PLina-PLL ones.
The short-term stability test of NPs
The freeze-dried NPs were used for this study. All the prepared samples in aqueous medium were stored both at 4C refrigerator and 25C room temperature. In terms of the stability test results, all samples at 25C showed aggregation and instability after the first week. Thus, the stability studies were continued under 4C refrigerator condition. The results were demon-strated in Table 3. The NPs indicated mostly narrow size distrubution and the PDI values were good enough (<0.3) for stabilization. There was a slight increase but no significant change in the mean size, PDI and zeta potential of the particles stored for 30 days than those of day 1 due to small aggregation. As a result, all the lyophilized samples stored at 4C were more stable and they also kept their self-integrity during the 30 days period.
In vitro siRNA release
In vitro c-myc siRNA release profiles were shown in
Figure 2 (c,d). For PS-PLina-PLL NPs, an initial burst effect was observed within 4 h and cumulative siRNA release values were obtained as 37, 27.6, and 17% for 100, 280, and 400 nM siRNA concentrations, respect-ively. At the end of 240, 216, and 240 h, the cumula-tive release values slowed down and reached to a plateau, which were as 82% siRNA release for 100 nM; 62% siRNA release for 280 nM and 55% siRNA release for 400 nM siRNA concentrations, respectively. For PS-PLina-PEG-PLL NPs, an initial burst effect was observed within 4 h and 42, 24, and 15% of cumulative siRNA release values were obtained for 100, 400, and 600 nM siRNA concentrations, respectively. 90% of siRNA for
100 nM, 72% of siRNA for 400 nM, and 65% of siRNA for 600 nM siRNA concentration were released and all values reached to a plateau at the end of 240 h.
After the end of day 10, the sustained and con-trolled release profiles were observed. When we look at all formulations for both NPs, it was obtained that the percentage of first burst release at 100 nM siRNA concentration was more than the other ones. This is because, the small amount of siRNA bonded to the NP surface was released easier and faster than that of the higher amounts and this effect gradually decreased as siRNA concentration increased (Patil and Panyam
2009). In addition, when comparing both NPs loaded with the same concentration of siRNA (100 nM), the results demonstrated that total siRNA release from PEG-PLL NPs was slightly higher than PS-PLina-PLL NPs at the end of the experiment. The reason of this is, PEG content in PS-PLina-PEG-PLL NPs makes the structure hydrophilic and as a result of the inter-action between the NPs and the physiological
medium, the surface degradation rate of PEG-PLL NPs is faster than hydrophobic PS-PLina-PLL NPs.
Figure 2. siRNA (100, 280, and 400 nM concentrations) binding efficiencies (%) of (a) PS-PLina-PLL NPs with 250, 500, and 1000ml PLL concentrations; siRNA (100, 400, and 600 nM concentrations) binding efficiencies (%) of (b) PS-PLina-PEG-PLL NPs with 250, 500, and 1000ml PLL concentrations; In vitro c-myc siRNA release profile of (a) 100, 280, and 400 nM siRNA concentrations from PS-PLina-PLL NPs with 500ml PLL concentration; (b) 100, 400, and 600 nM siRNA concentrations from PS-Plina-PEG-PLL NPs with 500ml PLL concentration in PBS solution (pH ¼ 7.4) at 37C. Note: SD shown as error bars, n¼ 3.
Table 3. After 30 days period of storage at þ4C, the
changes in the mean diameter, PDI and zeta potential values of the PS-PLina NPs, PS-PLina-PEG NPs, PS-PLina-PLL NPs, and PS-PLina-PEG-PLL NPs. Data represent mean ± SD, n¼ 3.
þ4C
Samples Size (nm) PDI Zeta potential (mV) PS-PLina NPs Day 1 150 ± 10 0.080 ± 0.009 32.8 ± 0.25 Day 30 156 ± 2 0.090 ± 0.005 30.1 ± 0.12 PS-PLina-PEG NPs Day 1 184 ± 6 0.113 ± 0.017 14.6 ± 0.19 Day 30 189 ± 1 0.118 ± 0.004 13.1 ± 0.33 PS-PLina-PLL NPs Day 1 168 ± 3 0.120 ± 0.012 37.2 ± 0.11 Day 30 171 ± 4 0.132 ± 0.006 34.7 ± 0.20 PS-PLina-PEG-PLL NPs Day 1 200 ± 4 0.104 ± 0.035 20 ± 0.04 Day 30 204 ± 7 0.098 ± 0.049 14.1 ± 0.17
Xu et al. prepared three different siRNA-NP formu-lations which were: siRNA encapsulated PEG-Dlinkm
-PLGA NPs containing acid degradable amide bond (Dlinkm), PLA/Cy5-siRNA NPs, and PLGA/Cy5-siRNA
NPs. PEG-Dlinkm-PLGA NPs could reach easily to
tumour side and accumulate there and PEG facili-tated cellular uptake of NPs and it showed slightly fast release of siRNA than PLGA/Cy5-siRNA NPs due to experience of its surface to the corrosion more easily into the physiological medium. Nevertheless, the release percentage of siRNA from PLA/Cy5-siRNA NPs was considerably lower than both of them. This is because PLGA was more hydrophilic than PLA (Xu et al.2016).
In vitro cytotoxicity study
Figure 3 demonstrates the results of cytotoxicity of NPs against non-cancerous and cancer cells. After interacting L929 cells with various amounts of bare NPs for 72 h, it was observed that increase in NP con-centration resulted in more toxic effect on cells. 120mg/ml PS-PLina-PEG-PLL NPs had almost 15% of toxic effect while 40mg/ml of all NPs exhibited the lowest toxicity (Figure 3(a)). Because of this, NPs were prepared at this concentration for further experiments.
Figure 3(b–d) shows the viability (%) of A549 cells after transfecting them with PS-PLina-PLL-siRNA NPs, PS-PLina-PEG-PLL-siRNA NPS, and naked siRNAs for 24, 48, and 72 h, respectively. According to the results, the toxic effect of naked siRNAs on the cells was found to
be less than those of the same concentrations of siRNA conjugated PS-PLina-PLL NPs and PS-PLina-PEG NPs. Free siRNA is unstable under the physiological conditions. When siRNA was normally involved in blood circulation, it was easily digested by the nucle-ases in the serum, exposed to rapid renal clearance/ hepatic separation, and due to its large particle size and excessive negative charge, the cellular membrane may be very difficult to pass. Free siRNA cannot accur-ately identify the target cells in many human cells, and therefore, siRNA’s activity alone is low when observed in vitro (Kachalaki et al.2015).
Obtained results also revealed that the cytotoxicity of siRNA attached NPs on A549 cells increased as incu-bation time increased. The toxic effect of PS-PLina-PLL NPs attached with 400 nM c-myc-siRNA was found to be 57.24% while 59.35% of toxicity was observed for PS-PLina-PEG-PLL NPs attacted with 600 nM c-myc siRNA after 72 h treatment (Figure 3(d)). In addition, the cytotoxicity of siRNA-NPs formulations on A549 cells increased in a siRNA concentration-dependent manner. (Figure 3).
Flow cytometry analysis with annexin V-PI
The apoptotic and necrotic effects of silencing c-myc gene using PS-PLina-PLL and PS-PLina-PEG-PLL NPs on A549 cells were determined via Annexin V-PI staining. According to the results given inFigure 4, the percant-age of viability of control group cells, not treated with any reagent, was founded as 99.6%. When the cells
Figure 3. Cytotoxic effects of (a) bare PS-PLina NPs, PS-PLina-PLL NPs, PS-PLina-PEG NPs and PS-PLina-PEG-PLL NPs (40, 80, and 120mg/ml) on L929 cells for 72 h treatment. Cytotoxicity of PS-PLina-PLL-siRNA NPs with 100, 280, 400 nM siRNA concentrations, PS-PLina-PEG-PLL-siRNA NPs with 100, 400, and 600 nM siRNA concentrations and naked siRNA with 100, 280, 400, and 600 nM siRNA concentrations on A549 cells after (a) 24 h, (b) 48 h, and (c) 72 h treatments. Data represent mean ± SD, n¼ 8.
were treated with the increased amount of siRNA and NPs formulations, the number of necrotic and apoptotic cells increased. For both NPs, inhibition of c-myc gene
resulted in more than 25% apoptotic rate in cells. The highest apoptotic levels were obtained as 32.6% for 400 nM siRNA-PS-PLina-PLL NPs and 32.9% for 600 nM siRNA-PS-PLina-PEG-PLL NPs. Consequently, no
Figure 4. The results of flow cytometry analysis of A549 cells transfected with PS-PLina-PLL- siRNA NPs including 100, 280, 400 nM of siRNA and PS-PLina-PEG-PLL-siRNA NPs including 100, 400, and 600 nM of siRNA concentrations, respectively, for 48 h.
Figure 5. Fluorescence microscopy images of A549 cells after 48 h of incubation with various samples (a) non-treated cells, (b) naked 400 nM siRNA, (c) PS-PLina-PLL-400 nM siRNA, and (d) PS-PLina-PEG-PLL-400 nM siRNA. Scale bars are 100lm.
significant difference between the NPs were observed for causing apoptotis after c-myc inhibition via siRNA.
Fluoresence microscope assay
Figure 5 demonstrates the fluorescence microscopy images of A549 cells after 48 h of incubation with the experimental samples. Living cells show annexin V staining (only green) while dead cells show both annexin V and PI staining (red). Since live cell mem-branes are not damaged these cells can not be stained with PI. However, PI can pass through the damaged membrane of dead cells and dyes the nucleus to red. It can be seen from the results that when the cells were not treated with any agents, they were stained green (Figure 5(a)). For the same siRNA concentration (400 nM), the number of red stained cells (dead cells) increased after transfecting the cells with the siRNA-NPs formulations. Similar results were obtained for both for PS-PLina-PLL and PS-PLina-PEG-PLL NPs. (Figure 5(b–d)). These results also confirmed the flow cytometry assay.
Conclusions
The graft copolymers and NPs were successfully syn-thesized. Both PLL based NPs were considerably stable at 4C. The NPs were loaded with c-myc siRNA and showed high siRNA entrapment efficiency. It was the first time that PS-PLina-PLL and PS-PLina-PEG-PLL NPs were used as a siRNA carriers to inhibit human lung cancer progression. The promising anticancer activities of the siRNA-loaded NPs were proven by cytotoxicity and apoptosis results. The findings showed that tboth unsaturated fatty acid based nanoparticles conjugated c-myc siRNA have a great potential for suppressing tumour growth significantly than the naked siRNA.
Acknowledgments
The authors thank Faruk Bahadır for helping us to the prep-aration step of polymer.
Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding
This work was financially supported by Bulent Ecevit University Research Fund (Grant no. BEU-2016–33496813-01) and Kapadokya University #K€UN(0).2018-BAGP-001.
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