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Development and Evaluation of Solid Witepsol Nanoparticles for Gene Delivery

Objectives: Gene therapy approaches have become increasingly attractive in the medical, pharmaceutical, and biotechnological industries due to their applicability in the treatment of diseases with no effective conventional therapy. Non-viral delivery using cationic solid lipid nanoparticles (cSLNs) can be useful to introduce large nucleic acids to target cells. A careful selection of components and their amounts is critical to obtain a successful delivery system. In this study, solid Witepsol nanoparticles were formulated, characterized, and evaluated in vitro for gene delivery purposes. Materials and Methods: Solid Witepsol nanoparticles were formulated through the microemulsion dilution technique using two grades of Witepsol and three surfactants, namely Cremephor RH40, Kolliphor HS15, and Peceol. Dimethyldioctadecylammonium bromide was incorporated into the system as a cationic lipid. Twelve combinations of these ingredients were formulated. The obtained nanoparticles were then evaluated for particle size, zeta potential, DNA binding and protection ability, cytotoxicity, and transfection ability. Results: Particle sizes of the prepared cationic cSLNs were between 13.43±0.06 and 68.80±0.78 nm. Their zeta potential, which is important for DNA binding efficiency, was determined at >+40 mV. Gel retardation assays revealed that the obtained cSLNs can form a compact complex with plasmid DNA (pDNA) encoding green fluorescent protein and that this complex can protect pDNA from DNase I-mediated degradation. Cytotoxicity evaluation of nanoparticles was performed on the L929 cell line. In vitro transfection data revealed that solid Witepsol nanoparticles could effectively transfect fibroblasts. Conclusion: Our findings indicate that solid Witepsol nanoparticles prepared using the microemulsion dilution technique are promising non-viral delivery systems for gene therapy.


INTRODUCTION
Genetic treatment approaches show promise for diseases ranging from cancer to inherited illnesses. The use of naked nucleic acids is not therapeutically effective due to their hydrophilic character, anionic charge, and high molecular weight, which hinder them from crossing cell membranes. 1,2 To overcome these challenges, viral or non-viral gene delivery systems are used. Production of non-viral delivery systems that can carry large amounts of genetic material, have low toxicity and a low risk for immune responses, and be easily produced is one of the research areas of pharmaceutical sciences. 3,4 In recent years, lipid-based carrier systems, such as liposomes, emulsions, nanostructured lipid carriers, and solid lipid nanoparticles (SLNs), have gained interest as delivery systems for gene therapy. Of these, SLNs have emerged to the forefront as promising systems in the field of non-viral gene delivery owing to their various advantages. 5 Specifically, SLNs are suitable for large scale production and exhibit longterm stability. They are usually prepared by using non-toxic, biodegradable lipids. 6,7 SLNs can be produced in nanosize and with cationic properties, which enable binding to nucleic acids and increase cellular influx and/or intracellular transition due to electrostatic interactions. 8 The size and charge of SLNs can be modified by changing production strategies based on the purpose of production. 9 Essential components for SLN formulations include lipids, emulsifiers, and an aqueous phase. The melting point of lipids is usually higher than room and body temperatures. Witepsol, which is mainly used in pharmaceutical research and development as an excipient, is a lipid that can be used for SLN formulation. A range of grades of Witepsol that comprise different proportions of triglycerides, diglycerides, and monoglycerides are commercially available to meet the requirements of pharmaceutical formulation and production and biopharmacy. Witepsol grades are classified in categories, namely H, W, S, and E. For example, Witepsol H series lipids consist mostly of triglycerides with a proportion of, at most, 15% diglycerides and not more than 1% monoglycerides. They are characterized by hydroxyl values up to 15 and have a small gap between the melting and solidification temperatures. By contrast, Witepsol W series lipids consist of a mixture of triglycerides (65%-80%), diglycerides (10%-35%), and monoglycerides (1%-5%). Their hydroxyl values are between 20-50 and exhibit a bigger difference between melting and solidification points. 10,11 Because of the diversity in their composition, Witepsol grades exhibit different characteristics; therefore, the selection of the most suitable grade during formulation development must be conducted according to these properties. 11,12 Surfactants used in this study include Kolliphor HS15 (macrogol-15-hydroxystearate) and Cremephor RH40 (macrogolglycerol hydroxystearate). They are non-ionic oil-in-water (o/w) solubilizing and emulsifying agents with hydrophilic-lipophilic balance (HLB) values of 14-16, making them suitable for o/w emulsion formulations. 11,13 However, they exhibit different emulsifying capacity depending on the formulation components.
The aim of this study was to develop stable, solid Witepsol nanoparticles suitable for gene delivery. Therefore, the microemulsion dilution technique was employed and various formulation parameters were evaluated to produce nontoxic, biocompatible, cationic SLNs, with an optimal size and surface properties, which can be used in gene delivery. The obtained SLNs were first characterized and then complexed with plasmid DNA (pDNA). Then, protection potential against DNase I, cytotoxicity, and transfection ability were investigated. According to our findings, this study is one of the first on the development of solid Witepsol nanoparticles using the microemulsion dilution technique for gene delivery.

Plasmid DNA
The model plasmid pEGFP-C1 (Invitrogen, USA), which encodes the green fluorescent protein (GFP) under the CMV promoter (pDNA), was amplified in Escherichia coli DH5α. The Maxiprep DNA purification kit (Invitrogen, USA) was used for plasmid purification. Plasmid integrity was evaluated by restriction enzyme digestion and visualized using a horizontal electrophoresis system. Furthermore, the purity and concentration of the plasmid were evaluated by measuring its absorbance at 260/280 nm. Finally, purified plasmid DNA was diluted to 100 µg/mL, aliquoted, and stored at -20°C until use.

Formulation of solid Witepsol nanoparticles
The microemulsion dilution technique was used to develop SLNs. 2 First, Witepsol was used as an internal oil phase to obtain an o/w microemulsion system. CRH40 or KHS15 and Pec (2:1, w:w) were used as surfactants (S) and ethanol as the co-surfactant (CoS). The cationic lipid DDAB was added into the solid lipid phase to achieve cationic microemulsion. 14 SLNs prepared with DDAB were abbreviated as cSLN. Compositions of the designed SLNs are listed in Table 1. All components were weighted as that the amount of solid lipids were 50 mg per formulation and heated to 50°C (10°C above the melting temperature of Witepsol). Previously warmed UPH 2 O at an equivalent temperature was then added onto the lipid and S:CoS (1:1, w:w) mixture with nearly 2% of total weight increase per addition. Transparent regions were indicated on the phase diagram to determine the o/w microemulsion formation area. A microemulsion consisting of 4% oil, 36% S:CoS, and 60% water was selected for further studies for all developed SLNs. The acquired hot o/w microemulsion was then rapidly dispersed in 8 mL UPH 2 O (0°C-4°C), stirring at 1000 rpm at a ratio of 1:5 (v/v). Once hot microemulsion droplets were applied to cold water, SLNs were formed. The concentration for the final SLNs was 7.5 mg/mL for all formulations, with respect to the amounts of solid lipids.

Characterization of solid Witepsol nanoparticles
Particle size, polydispersity index (PDI), and zeta potential values of SLNs, cSLNs, and cSLN:pDNA complexes, with various formulation parameters, were measured using dynamic light scattering [(DLS), Zetasizer Nano ZS, Malvern Instruments Ltd., UK)]. 15 Disposable polystyrene micro cuvettes were used to measure particle size and PDI for each sample. The zeta potential was measured in standard zeta cuvettes and calculated using the software by employing the Smoluchowski equation. Measurements were repeated at least three times for each sample.

Stability of cationic, solid Witepsol nanoparticles
Particle size and zeta potential measurements of cSLNs were followed in terms of stability for 3 months. The measurements were performed at various time intervals using DLS (Zeta sizer Nano ZS, Malvern Instruments Ltd., UK). Samples were stored at 4°C during this period.

Gel retardation assay
The cSLN:pDNA complexes were generated through electrostatic interactions between cationic nanoparticles and anionic pDNA. 16 The complex formation ability of pDNA with cSLN was determined through 1% (w/v) agarose gel electrophoresis. The purity and concentration of the pDNA were spectrophotometrically assayed at 260 and 280 nm, respectively, using Nanovette (Beckman Coulter, USA). The ratio of absorbance at 260/280 nm was 1.752 (~1.8), indicating pDNA purity. 17 A stock solution of pDNA (100 µg/mL) was prepared in nuclease-free UPH 2 O. The cSLN:pDNA complexes were prepared by incubating pDNA (100 µg/mL) with cSLNs for 30 min at 25°C on a benchtop shaker for increasing cSLN:pDNA ratios [0.5:1, 1:1, 2:1, and 3:1 (v/v)] to enable binding of pDNA to cSLNs electrostatically. Glycerol (2%) was added to each sample and electrophoresis was performed at 100 V for 60 min. Then, ethidium bromide solution (500 ng/mL) was used to stain the gel. The stained pDNA bands were then visualized under the ultraviolet transilluminator (Vilber Lourmat, France) to determine the optimal pDNA:cSLN ratio. Naked pDNA was used as a control.

In vitro DNase I protection assay
The protection of pDNA by cSLNs was evaluated using the in vitro DNase I protection assay. For this purpose, DNase I was added to freshly prepared cSLN:pDNA systems to get 1 IU DNase I/2.5 µg pDNA as the final concentration and incubated at 37°C for 30 min. To terminate the enzymatic reaction and to obtain the release of pDNA, SDS (1%, v/v) was added. The integrity of pDNA was visualized by horizontal gel electrophoresis. 18

Cytotoxicity analysis
Cytotoxicity evaluation of the formulations was performed on L929 cell lines. Dulbecco's modified Eagle's medium

Transfection studies
The transfection ability of the developed formulations is important for the transport of therapeutic genes into the cells.
For this study, CoS-7 cells were cultured in 6-well plates at a density of 5x10 4 cells/mL and incubated until 70% confluency. The medium was then removed and the cells were washed with PBS (pH: 7.4). Fresh growth medium (500 µL) was instantly added to prevent cells from desiccation. Predetermined doses of the cSLN:pDNA complex (2:1, v/v), which contains 2.5 µg of pDNA, were applied to the cells based on cytotoxicity test results and incubated for 4 h at 37°C in a humidified atmosphere of 5% CO 2 . Then, the medium containing the SLN suspension was replaced with fresh growth medium and cells were incubated for 48 h to increase GFP production. Transfection efficiency was determined c under a fluorescence microscope (Olympus, Japan).

Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, Inc., USA). Results are expressed as means ± standard error of mean. Cytotoxicity results were analyzed using Student's t-test. The significance level was set at p<0.05.

RESULTS AND DISCUSSION
Due to the rapid progress in molecular biology and genetics, interest in using nanoparticles as vehicles in drug and gene delivery systems has increased. In this regard, the use of SLNs is important. During the last decade, SLNs have mostly been used as drug delivery systems. [20][21][22] Even cationic SLNs, developed using cationic lipids, can be used as gene carrying systems; however, instances of their application in gene therapy are few. [23][24][25] Solid lipids with relatively higher melting points, such as glyceryl behenate, behenic acid, and stearic acid (melting point >60°C), were used as the solid lipid matrix in the preparation of SLNs. 9,14 However, in this study, Witepsol, which has a melting point of approximately 37°C, has been used. The low melting point of the solid lipid matrix brings several advantages. First, the energy requirement for large scale production is decreases, thereby reducing the production cost of nanoparticle carriers and providing an advantage to enter the pharmaceutical market. 23 Furthermore, following endocytosis, the genetic material-loaded SLNs presumably encounter consecutive pH drop in the endosome and lysosome due to the acidic character of endosomal media. 26 At this step, the low melting point of Witepsol, which is close to body temperature, might facilitate the release of the genetic material into the cytoplasm compared with lipids with high melting points. 2 Here, we aimed to produce SLNs using Witepsol in the solid matrix. For this purpose, plotting pseudo-ternary phase diagrams, which is the initial process of SLN production using the microemulsion dilution method, was performed at the lipid melting temperature. 27

Preparation of pseudo-ternary phase diagrams
For the formation of the o/w microemulsion system, the HLB of the system was adjusted between 10 and 12 with non-toxic surfactants. One of the surfactants used in these formulations is Pec. It is a readily dispersible, solubilizing agent, consisting of a mixture of mono-and diglycerides of oleic acid. Because the HLB value of Pec is quite low (approximately 3), the HLB value of surfactant mixtures was increased by using CRH40 and KHS15 to allow for the formation of an o/w microemulsion. The pseudoternary phase diagrams constituted by titration with UPH 2 O into the oil (Witepsol), S (CRH40:Pec or KHS15:Pec, 2:1, w:w), and CoS (ethanol) mixtures are presented in Figure 1. According to the construction of the phase diagram, the final formulations were selected providing microemulsion requirements, such as being o/w type and transparent, as well as having a high proportion of the solid lipid and a low proportion of S:CoS. 28,29 Because the o/w microemulsion formation areas are similar for all formulations, the same ratios were selected to compare the effect of solid lipids (WH35/WW35) and S:CoS (CRH40:Pec/ KHS15:Pec). Thus, the o/w microemulsions consisting 4 wt % solid lipid, 36 wt % S:CoS, and 60 wt % water were prepared and used for SLN preparation for all formulations.

Physicochemical characterization
To examine the particle size and zeta potential of nanoparticles, DLS measurements were performed. As shown in Table 2, the obtained solid Witepsol nanoparticles are in the nanometer size range (13.43-80.49 nm). Complex formation with pDNA increased the particle size of the system. SLNs without cationic lipids had a zeta potential between -2.4 and 13.7 mV. When CRH40 was used as the surfactant, the zeta potential values of nanoparticles were +13.7 and +12.7 mV for SLN 1 and SLN 3 , respectively. By contrast, when KHS15 was used as the surfactant, the zeta potential decreased to -0.5 and -2.4 for SLN 2 and SLN 4 , respectively, which is attributable to the glycerol group in CRH40. Furthermore, following DDAB incorporation, the zeta potential of cSLNs was >40 mV for all formulations. These results were as expected due to the cationic amphiphilic character of DDAB, which is commonly used in non-viral gene delivery studies. 30 Then, the final zeta potential of the formulations was decreased by complexing with pDNA; however, it remained >30 mV for all four formulations. The obtained positive zeta potential of the formulations is important for achieving an interaction between the negatively charged nucleic acid and cell membrane for transfection. 31,32 The PDI values of the produced SLNs increased after DDAB was incorporated into the system as a cationic lipid. However, polydispersity of the formulation decreased after complex formation with pDNA occurred. Particle sizes of the final cSLN:pDNA vector systems were <100 nm, which is necessary for i.v. injection. Moreover, they are small enough to provide stability to the dispersal system owing to Brownian motion. 33 The physicochemical stability of the developed cSLNs was evaluated. For this experiment, cSLNs were stored at 4°C and particle size and zeta potential were measured for up to 90 d ( Table 3). The results revealed that particle size and zeta potential of cSLNs remained stable for 21 d. Particle size of cSLNs increased dramatically within 90 d. The particle size of only cSLN 3 remained at less than 100 nm. Thus, the developed formulations can be used for complexation with pDNA for at least 21 d because their particle size is <100 nm and zeta potential is >40 mV. 34

Gel retardation assay
The gel retardation assay is used to evaluate the nucleic acidbinding ability of cationic nanoparticles. 16 cSLNs were evaluated for their complex formation ability with pDNA using the agarose  gel retardation assay to identify the optimal cSLN:pDNA ratio. The migration of naked pDNA and cSLN:pDNA complexes for different Witepsol nanoparticles in agarose gel is shown in Figure 2. The obtained cSLNs showed pDNA binding ability and the migration of pDNA in agarose gel stopped when the ratio of cSLN:pDNA reached 2:1 (v/v) for all cSLNs.

Protection ability of nanoparticles against DNase I degradation
The agarose gel photograph of cSLN:pDNA complexes incubated with DNase I is shown in Figure 3. Lane 1 contains the untreated plasmid (negative control). As seen in lane 2, naked pDNA was completely digested by DNase I. Lanes 3-5 indicate the protection of the cSLN formulations against DNase I at three cSLN:pDNA ratios, namely 1:1, 2:1, and 3:1 (v:v). This evidence confirmed that the obtained cSLNs efficiently protected the pDNA from DNase I-mediated degradation. 35

Cytotoxicity
The cytotoxicity assay was performed to identify non-toxic doses of the obtained nanoparticles before transfection. The results   obtained from the alamarBlue cytotoxicity assay on the L929 cell line supported that all excipients used in this study have low cytotoxicity. Significant cytotoxicity was not determined on L929 cells in at concentrations ranging from 225 to 1125 µg/ mL. The applied doses showed >70% cell viability, which is considered the minimum reference cell viability dose for further transfection studies. 33 Furthermore, concentration dependent cytotoxicity was observed for all formulations (Figure 4).

Cellular uptake studies
The uptake of Witepsol nanoparticles in the CoS-7 cell line was determined using the pEGFP-C1 plasmid and visually investigated under a fluorescence microscope ( Figure 5). Green shows the fluorescence signal from GFP. According to transfection results, all obtained formulations have transfection ability. 36,37 Since the developed nanoparticles showed efficient transfection in cell culture, they can be used for different genetic diseases by loading disease specific therapeutic genes.

CONCLUSION
We prepared and characterized solid Witepsol nanoparticles. The obtained cationic nanoparticles exhibited pDNA binding and protection ability. The characterization of formulations in terms of particle size, zeta potential, and PDI revealed that the obtained nanoparticles are in the nanometer size range. Cytotoxicity results confirmed the suitability of the nanoparticles for gene delivery. Transfection studies showed that the developed nanoparticles can carry genetic material into cells efficiently. Thus, solid Witepsol nanoparticles can be considered promising delivery vehicles for non-viral gene therapy.

ACKNOWLEDGMENTS
This study has been financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under grant code 218S840 and İzmir Katip Çelebi University Research Fund under grant code 2020-ÖDL-ECZF-0001. This paper is dedicated to the memory of our co-author dear Selen Isar, who sadly passed away on Wednesday, December 9 th , 2020.

Conflicts of interest:
No conflict of interest was declared by the authors. The authors alone are responsible for the content and writing of the paper.