Characterization of an amino acid based biodegradable surfactant facilitating the incorporation of DNA into lipophilic delivery systems

Journal of Colloid and Interface Science 566 (2020) 234–241

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Characterization of an amino acid based biodegradable surfactant facilitating the incorporation of DNA into lipophilic delivery systems Julian Dominik Wolf a,b, Markus Kurpiers a,b, Randi Angela Baus b, Roman Xaver Götz b, Janine Griesser a,b, Barbara Matuszczak c, Andreas Bernkop-Schnürch a,b,⇑ a b c

Thiomatrix Forschungs– und Beratungs GmbH, Research Center Innsbruck, Trientlgasse 65, A-6020 Innsbruck, Austria Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 13 September 2019 Revised 13 January 2020 Accepted 23 January 2020 Available online 24 January 2020 Keywords: Biodegradable Cationic surfactant Gene therapy Hydrophobic ion pairing SEDDS Transfection

a b s t r a c t Hypothesis: Lysine based cationic surfactants are well-tolerated tools for hydrophobic ion pairing (HIP) with DNA and its incorporation into lipophilic delivery systems. Experiments: Di-Boc-lysine was esterified with 1-hexadecanol and the Boc-residues were cleaved off resulting in hexadecyl lysinate (HL). Subsequently, its Log POctanol/water and the critical micelle concentration (CMC) were determined. Degradability was evaluated utilizing trypsin and pancreas lipase as well as Caco-2 cells. Afterwards, the viability of Caco-2 cells upon incubation with HL was investigated. Finally, HL was ion-paired with plasmid DNA (pDNA, 6159 bp) and the obtained complex was incorporated into self-emulsifying drug delivery systems (SEDDS) for transfection studies on HEK-293 cells. Findings: HL was synthesized with a yield of 53% and subsequent characterization revealed a Log PWater/Octanol of 0.05 and a CMC of 2.7 mM. Enzymatic degradation studies showed rapid degradation

Abbreviations: ACN, Acetonitrile; CMC, Critical micelle concentration; CPC, Cetylpyridinium chloride; CTAB, Cetyltrimethylammonium bromide; DCM, Dichloromethane; DMAP, 4–Dimethylaminopyridine; DTAB, Dodecyltrimethylammonium bromide; FBS, Fetal bovine serum; HIP, Hydrophobic ion pairing; HL, Hexadecyl lysinate; MEM, Eagles minimum essential medium; OPA, Phthaldialdehyde; PBS, Phosphate buffered saline; pDNA, Plasmid DNA; rH, relative humidity; SEDDS, Self-emulsifying drug delivery system; TFA, Trifluoroacetic acid. ⇑ Corresponding author at: Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria. E-mail address: [email protected] (A. Bernkop-Schnürch). https://doi.org/10.1016/j.jcis.2020.01.088 0021-9797/Ó 2020 Elsevier Inc. All rights reserved.

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of HL by isolated enzymes and Caco-2 cells and cell viability experiments revealed no toxic effect of HL even in a concentration of 250 mg
ml1 within 24 h. HIP with pDNA was the most efficient in a molar ratio of 6159:1 (HL:pDNA) equalling a charge ratio of 1:1. Formed complexes could be incorporated into SEDDS facilitating successful transfection of HEK-293 cells. Ó 2020 Elsevier Inc. All rights reserved.

1. Introduction Surfactants are defined as substances bearing the property of adsorbing onto the surfaces or interfaces of a system, whereby they alter the surface or interfacial free energies of these boundaries in a considerable way [1]. Due to this property, surfactants became an indispensable part of our everyday life. Besides their application in products as detergents or motor oils, they play a crucial role in biotechnology and microelectronics but also in the pharmaceutical field. In case of the latter, surfactants are frequently utilized to enhance the solubility or to improve the stability of drugs and dosage forms. A more recently developed application represents a process called hydrophobic ion pairing (HIP) allowing for both solubility enhancement in nonpolar solvents and stability improvement of hydrophilic drugs such as peptides or DNA [2,3]. According to the old adage ‘opposites attract’, ionic surfactants interact with oppositely charged moieties of hydrophilic drugs resulting in a lipophilic complex as the respective charges are shielded by the hydrophobic tail of the surfactant. Applied on DNA or peptides, this modification facilitates the incorporation of considerable amounts of drugs into lipophilic carriers, such as self-emulsifying drug delivery systems (SEDDS) or liposomes, while maintaining their biological activity [2,4–8]. However, ionic surfactants utilized for HIP usually exhibit a major safety concern. It is well known, that cationic surfactants are the most toxic followed by anionic and amphoteric ones, whereas non-ionic structures are tolerated best [9,10]. In case of DNA possessing a great density of negatively charged phosphate moieties, it is necessary to apply cationic surfactants in comparatively high concentrations for efficient complexation [3]. Considering repeated administrations and high DNA-surfactant complex payloads, this could favour accumulation of cationic surfactants at the site of action resulting in unintended damage to the patient. A promising approach to achieve a more favourable safety profile represents the use of biodegradable surfactants based on amino acids [11–13]. Claffey et al., for instance, described arginine esters with octanol as well as dodecanol suitable for HIP with DNA and subsequent transfection [14]. At the same time, these esters were less toxic than tetradecyltrimethylammonium bromide as intended. Another study with arginine esters of medium and long chain aliphatic alcohols further supported these findings and described successful HIP with peptide drugs [15]. Furthermore, Tang et al. demonstrated a high potential of ornithine esters for liposomal DNA delivery [16]. So far, however, the plain hydrophobic complexes between hydrophilic macromolecular drugs and the amino acid esters were characterized but not incorporated into the advanced SEDDS carrier, which has previously been shown to provide improved transfection with incorporated DNA complexes [3,6]. Based on this background, it was hypothesized that an appropriate amino acid ester utilized for HIP could facilitate the incorporation of hydrophilic DNA into hydrophobic SEDDS while providing improved cell compatibility. Therefore, the aim of the current study was to synthesize and characterize a novel biodegradable cationic surfactant based on lysine being suitable for HIP with DNA due to its two amino groups.

The amino acid was esterified with 1-hexadecanol as the respective ester had previously shown promising absorption enhancing characteristics [17]. Afterwards, the obtained hexadecyl lysinate (HL) was characterized including degradation studies with isolated enzymes as well as Caco-2 cells and cell viability studies. Finally, HIP complexes with pDNA were prepared and incorporated into SEDDS for successful transfection of HEK-293 cells. 2. Materials and methods 2.1. Materials Na,Ne–Di–Boc–L–lysine (98%) was purchased from Alfa Aesar (Germany). Capmul PG-8 (NF), Captex 300 (EP/NF), Captex 355 (EP/NF) and Captex 8000 (EP/NF) were obtained from Abitec Corporation (USA). Brij O10 was provided by Croda International (UK) and phosphate buffered saline tablets (BioReagent grade), 5 M hydrochloric acid (volumetric solution, factor  0.995 and  1.005, Opti-MEMÒ as well as trifluoroacetic acid (99%) were bought from Fisher Scientific (Austria). The plasmid extraction kit (QIAGEN Plasmid Midi Kit) was supplied by Qiagen (Germany) and Lipofectin was obtained from Life Technologies (USA). Eagles minimum essential medium (MEM) was obtained from Carl Roth (Germany) and pcDNA3-EGFP was a gift from Doug Golenbock (Addgene plasmid # 13031). Cell culture microplates were supplied by Greiner Bio-One (Austria). Further cell culture supplies were obtained from Biochrom (Germany). Phthaldialdehyde Reagent - Solution Complete (OPA reagent), 1–hexadecanol (99%), trypsin from bovine pancreas (47 IU per mg protein) as well as lipase from porcine pancreas (type II, 47 triacetin units per mg protein) were purchased from Sigma-Aldrich (Austria). All other chemicals, reagents, and solvents were of analytical grade and obtained from commercial sources. 2.2. Synthesis of hexadecyl lysinate In order to obtain HL, a previously described synthesis procedure was adapted as displayed in Fig. 1 [17]. In brief, 2.5 g (7.22 mmol) of Na,Ne–Di–Boc–L–lysine as free carboxylic acid and 1.75 g (7.22 mmol) of 1–hexadecanol were dissolved in 37.5 ml of dichloromethane (DCM). Afterwards, 1.49 g (7.22 mmol) of N,N’–dicyclohexylcarbodiimide (DCC) for activation of the carboxylic acid moieties and 97.69 mg (0.80 mmol) of 4–dimethylaminopyridine (DMAP) as catalyst were added to the mixture. After stirring for 24 h at room temperature, the precipitated dicyclohexylurea was removed by filtration and both DCM as well as DMAP were evaporated utilizing a rotary evaporator (Heidolph VAP Value G3B, Heidolph Instruments, Germany). The protecting groups of the obtained Na,Ne–di–Boc–L–lysine hexadecyl ester were cleaved by dissolution of the intermediate product in 31 ml of dioxane, addition of 3.75 ml of 5 M HCl and stirring for 24 h at room temperature. Subsequently, dioxane and HCl were removed under vacuum. Thereafter, the obtained product was purified via C18 reversed-phase silica gel flash chromatography using a mobile phase of 55% (v/v) acetonitrile (ACN), 35% water and 10% 5 M HCl. The chemical structure of HL and cleavage of the protecting groups

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were centrifuged for 5 min at 9800 g. Subsequently, 100 ml aliquots were withdrawn from both phases and each was mixed with 100 ml of OPA reagent as well as 200 ml of methanol. After incubation for 3 min, 100 ml of n-octanol or water were added to the aqueous and the lipophilic phase, respectively. Samples were further diluted 1 to 12 with ACN and the HL derivate was immediately quantified via HPLC as described above. The partition coefficient was calculated using the following equation:

LogP ¼ log

Fluorescence HLin Fluorescence HLin

octanol phase aqueous phase

2.5. Critical micelle concentration

Fig. 1. Synthetic pathway for hexadecyl lysinate (HL). DCC: N,N’–dicyclohexylcarbodiimide; DCM: dichloromethane; DMAP: 4–dimethylaminopyridine.

The critical micelle concentration (CMC) represents an important characteristic of surfactants. Hence, the CMC was determined by combination of two previously described methods [18,19]. In brief, a methanolic solution of pyrene was prepared in a concentration of 1.2 mg
ml1 and 100 ml of it were transferred into each well of a black 96-well microplate (Greiner Bio-One, Germany). After methanol evaporation, 200 ml of aqueous HL solutions in various concentrations were added to the remaining pyrene. The plate was shaken for 2 min and the fluorescence intensities at 500 nm as well as 362 nm after excitation at 319 nm (bandwidths: 20 nm) were measured utilizing a multimode microplate reader (Tecan SparkÒ, Tecan Trading AG, Switzerland). Sodium dodecyl sulfate as well as cetyltrimethyl ammonium bromide (CTAB) served as control to confirm a sufficient accuracy of the method. 2.6. Degradation by isolated enzymes

were confirmed by FT-IR (Bruker Alpha equipped with platinum ATR module) and 1H NMR (Varian Gemini 200 spectrometer, 1H: 200 MHz). As internal standard the center of the solvent multiplet (DMSO d6, d 2.49 ppm) was used and related to tetramethylsilane. The molecular weight was determined by a Hitachi Chromaster 5610 mass spectrometer equipped with a positive electrospray ionization ion source. 2.3. HPLC analysis Analyses were performed using a Hitachi Elite LaChrom HPLCSystem equipped with L–2130 pump, L–2200 autosampler and L– 2480 fluorescence detector. Due to insufficient UV absorbance of HL, pre-column derivatization of the ester was necessary to facilitate an appropriate sensitivity for following experiments. Therefore, a fluorophore was introduced by reaction of samples with an equal volume of OPA reagent. After an incubation period of 3 min, samples were further diluted as described in the respective chapters and 10 ml of the mixture were immediately injected onto a Kinetex F5 column (150  4.6 mm, 5 mm, 100 Å; Phenomenex, Germany) at 25 °C. Separation was achieved by elution with a mobile phase composed of ACN/water (71% to 29%, v/v) containing 0.1% (v/v) trifluoracetic acid (TFA) at a flow rate of 1 ml
min1 and a runtime of 5 min. The ester derivate was detected by fluorescence utilizing excitation and emission wavelengths of 340 and 455 nm, respectively. A calibration curve was established in a concentration range from 0.42 to 41.67 lg
ml1 (R2 = 0.997). 2.4. Log POctanol/Water determination The Log POctanol/Water was determined by dissolving of 2.5 mg of HL in 1 ml of a 1:1 mixture of n-octanol and water. After incubation for 24 h at room temperature while shaking at 300 rpm, samples

Enzymatic degradation studies were adapted from a method previously described by our research group [20]. In brief, HL was dissolved in 25 mM BIS-TRIS buffer pH 6.8 in a concentration of 5 mg
ml1. Lipase and trypsin solutions were prepared to obtain activities of 70 U
ml1 (referring to triacetin) and 8.8 IU
ml1, respectively. Solutions were preheated to 37 °C and enzymatic degradation was initiated by addition of 500 ml of each enzyme solution to 500 ml of HL solution resulting in a final HL concentration of 2.5 mg
ml1 and enzyme activities of 35 U
ml1 (lipase, triacetin) or 4.4 IU
ml1 (trypsin). The mixtures were incubated in a thermomixer at 300 rpm and 37 °C and aliquots of 50 ml were withdrawn at predetermined time points. Samples were immediately treated with 100 ml of 0.5% (v/v) TFA in ACN to stop the enzymatic reaction. After incubation with an equal volume of OPA reagent, samples were diluted 1 to 10 with ACN and analysed via HPLC as described above. 2.7. Degradation by Caco-2 cells Degradation studies with Caco-2 cells were performed to prove the biodegradability of HL not only by isolated enzymes but also by cells. Briefly, Caco-2 cells were cultured in 24-well plates in a density of 2  105 cells per well at 37 °C, 5% CO2 and 90% relative humidity (rH) in 500 ml of MEM containing phenol red, 10% fetal bovine serum (FBS) as well as 1% penicillin–streptomycin. MEM was changed every 48 h for 14 days, whereof the last 5 days MEM was only supplemented with antibiotics anymore. When a monolayer was observed, cells were washed once with 500 ml of 25 mM BIS-TRIS buffer pH 6.8 containing also 5.55 mM glucose, 136.7 mM sodium chloride, 1 mM calcium chloride and 5 mM potassium chloride. Afterwards, 500 ml of HL dissolved in BISTRIS buffer in a concentration of 250 mg∙ml1 were added and aliquots of 50 ml were withdrawn at predetermined times. Samples were immediately mixed with 100 ml of stop solution containing

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0.5% TFA (v/v) in ACN and stored at –80 °C until analysis. Finally, HL was quantified by HPLC analysis after derivatization with an equal volume of OPA reagent and 1 to 10 dilution with ACN. For quantification of the residual amount of HL, the initial concentration was utilized as the 100% value.

precipitation efficiency was calculated utilizing the following equation:

Precipitation efficiencyð%Þ ¼ 100   ConcentrationpDNA after HIP  1 ConcentrationpDNA before HIP

2.8. Cell viability studies In order to confirm a reduced cytotoxic potential of HL, resazurin assays on Caco-2 cells were performed [21]. Within this assay, metabolically active cells reduce the nonfluorescent dye resazurin to the highly fluorescent resofurin enabling assessment of cell viability [22]. Briefly, Caco-2 cells were cultured in 24well plates as described above utilizing only MEM supplemented with phenol red, FBS and antibiotics. After washing the cells twice with phosphate buffered saline (PBS) pH 7.4, 500 ml of HL dissolved in MEM in concentrations of 10, 50, 100 and 250 mg
ml1 were added. As references, common cationic surfactants, namely DTAB and cetyl pyridinium chloride (CPC), were applied in equal concentrations, whereas MEM and 2% (v/v) TritonÒ X–100 in MEM served as negative and positive controls, respectively. Thereafter, cells were incubated for 4 or 24 h before discarding the supernatant and washing twice with PBS. Subsequently, 300 ml of 44 mM resazurin in MEM were added to each well and incubated for 3 h at 37 °C. Finally, fluorescence of the supernatants was measured at 590 nm after excitation at 540 nm utilizing the microplate reader. Cell viability was calculated utilizing the following equation, where F is referring to the fluorescence intensity:

Cell

v iabilityð%Þ ¼

F Sample  F TritonÒX100  100 F MEM  F TritonÒX100

2.9. Hydrophobic ion pairing with plasmid DNA For HIP and subsequent transfection studies the enhanced green fluorescence plasmid pcDNA3-EGFP was utilized. It was propagated and handled as described before [3]. In order to evaluate the potential of the novel cationic surfactant for HIP, precipitation studies with pDNA according to Hauptstein et al. were performed; CTAB and dodecyltrimethylammonium bromide (DTAB) served as references [6]. In brief, aqueous solutions of pDNA in a concentration of 1.0 mg
ml1 were mixed with equal volumes of surfactant solutions. Considering the 12,318 anionic charges within the backbone of the plasmid, various surfactant concentrations representing differing molar ratios were applied. The complex formed immediately during vortex mixing apparent from a white precipitate and was separated by centrifugation for 10 min at 12,100 g. The remaining amount of plasmid in the supernatant was quantified with the NanoQuant Plate of the microplate reader and the complex pellet was lyophilized (FreeZone 6, Labconco, USA) and stored at –20 °C. The percentile

2.10. Transfection studies The novel complexes were incorporated into SEDDS recently described by Griesser et al. and transfection was performed by a modified method [3]. In brief, HEK–293 cells were seeded into 24-well plates in a density of 2  105 cells per well and incubated at 37 °C, 5% CO2 and 90% rH until a confluency of 60–80% was observed. Afterwards, pDNA complexes prepared with HL and CTAB in molar ratios of 6159:1 and 12318:1, respectively, were incorporated into SEDDS referring to a pDNA concentration of 0.60 mg
ml1 (Table 1). Obtained SEDD formulations were diluted to 0.1% (v/v) with Opti-MEMÒ and 250 ml of resulting emulsions were added to each well. After incubation at 37 °C, 5% CO2 and 90% rH for 6 h, the samples were diluted with 250 ml of MEM. Plates were further incubated for 18 h before a green fluorescent proteinlive-cell-assay was performed. The fluorescence intensity was measured at an excitation wavelength of 483 nm and emission at 535 nm using the multimode microplate reader. Naked pDNA in a concentration of 0.60 mg
ml1 and pDNA plus the transfection reagent LipofectinÒ were utilized as controls to adjust the method. 2.11. Statistical data analysis All studies and tests were carried out with n  3 for each experimental setup. Statistical data analyses were performed using the Student t-test with p < 0.05 as the minimal level of significance. Level of p  0.05 was set for significant, p  0.01 for very significant and p  0.001 for highly significant. 3. Results and discussion 3.1. Synthesis A novel surfactant was prepared by esterification of Bocprotected lysine with 1-hexadecanol and subsequent cleavage of the protecting groups. Thereby, HL dihydrochloride was obtained as white powder with a yield of 53%. Successful deprotection was confirmed via FT–IR (Fig. 2) as the intermediate product displayed a characteristic band at 1677 cm1 referring to the carbonyl group of Boc, which was not apparent within the spectrum of the final synthesis product. Furthermore, the unprotected HL exhibited an increased absorption at wavenumbers > 3000 cm1 due to the NAH stretching of free amino moieties. The band at 3363 cm1 within the spectrum of the intermediate product resulted from

Table 1 Composition of SEDDS as described by Griesser et al. [3]. Values are indicated in percent (v/v). Excipient

Propylene glycol Capmul PG-8 Captex 8000 Captex 300 Captex 355 Cremophor EL Cremophor RH 40 Brij O10

Description

Propane-1,2-diol Propylene Glycol Monocaprylate Glyceryl Tricaprylate Glyceryl Tricaprylate/Tricaprate Glyceryl Tricaprylate/Tricaprate PEG-35 castor oil PEG-40 hydrogenated castor oil PEG-10 oleyl ether

Formulation A

B

C

15 25 15 15 – 30 – –

20 25 20 – – – 20 15

20 25 – – 20 – 20 15

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Fig. 2. FT-IR spectra of HL before (blue, upper graph) and after (red, lower graph) cleavage of the Boc protecting groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

water in the sample. Additionally, 1H NMR, 13C NMR and LC-MS analysis confirmed the successful synthesis of HL. All applied analysis techniques including TLC and HPLC indicated the high purity of the target product also demonstrating that the acidic mobile phase does not cause a detectable hydrolytic cleavage of the ester substructure during chromatographic purification. Hexadecyl lysinate: MW = 370.4 (calculated 370.6 for C22H46N2O2). 1H NMR (DMSO d6, 200 MHz): 0.84 [t, CH3], 1.10– 1.80 [m, 17  CH2], 2.74 [t, CH2N], 3.96 [t, CHN], 4.14 [t, OCH2), 8.13 [s, NH2]. 13C NMR (DMSO d6, 100 MHz): 14.0 [CH3], 21.2– 31.3 [17  CH2], 38.2 [CH2N], 51.6 [CHN], 65.7 [OCH2], 169.4 [COO]. 3.2. Log POctanol/Water An important property of a surfactant is its partition coefficient between n-octanol and water, giving a measure of lipophilicity and hydrophilicity. The Log POctanol/Water of HL was 0.05 ± 0.01 indicating amphiphilic properties being characteristic for surfactants. Therefore, dissolution of an appropriate quantity of HL in aqueous media necessary for application as ion pairing compound seems feasible. Simultaneously, it enhances the lipophilicity of formed API-surfactant complexes enabling their incorporation in hydrophobic carrier systems.

Fig. 3. Fluorescence intensity ratios I(500 nm)/I(362 nm) of pyrene (3 mM) in aqueous solutions of HL in concentrations from 0.1 to 10 mM. Indicated values are means (±SD, n = 3) and the dashed vertical line designates the CMC.

3.3. Critical micelle concentration For the CMC determination of HL the fluorescence characteristics of pyrene, a four ring polycyclic aromatic hydrocarbon, were exploited. As the fluorescence intensity of its monomer vibrational band as well as the formation of an excimer depend on the solvent environment, changes in the fluorescence properties at two characteristic wavelengths can be observed. After calculating the intensity ratio of both wavelengths measured at different surfactant concentrations, the CMC is displayed by the peak of the resulting graph [18]. As shown in Fig. 3, the CMC of HL was found to be 2.7 mM equal to 1.20 mg
ml1 being in the range of common cationic surfactants [23,24].

3.4. Degradation by isolated enzymes The biodegradability of HL was analysed utilizing two enzymes, namely trypsin and lipase, known to cleave ester bonds. As illustrated in Fig. 4, after 5 min only one third and two thirds of the initial HL amounts were still present within the mixtures upon incubation with trypsin and lipase, respectively. After rapid

Fig. 4. Enzymatic degradation of HL (2.5 mg
ml1) by lipase (j, 35 U
ml1 referring to triacetin) and trypsin (▲, 4.4 IU
ml1) in 25 mM BIS–TRIS buffer pH 6.8 over 180 min. Indicated values are means (±SD, n = 3).

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hydrolysis within the first 15 min, the reaction slowed down noticeably explainable by the Michaelis-Menten kinetics [25]. However, trypsin mediated hydrolysis exhibited a more pronounced deceleration in comparison to lipase probably due to self-digestion of the enzyme [26]. Sufficient biodegradability, as shown for HL, is crucial to prevent an enrichment of the surfactant and therefore harmful effects at the site of application. Nevertheless, these experiments took place in an aqueous medium, whereas a complex prepared via HIP between drug and HL is supposed to be administered within a lipophilic dosage form. In case of SEDDS, for example, a protective effect against enzymatic degradation of an incorporated peptide-surfactant complex has been shown previously [27–29]. Hence, an increased stability of HL being part of a complex within the oily droplets can be assumed. Upon release from the delivery system and disintegration of the complex, the subsequent rapid enzymatic degradation of HL avoids any undesirable side effects.

3.5. Degradation by Caco-2 cells After proving a good degradability of HL by isolated enzymes, degradation studies on Caco-2 cells were performed. A sufficient biodegradation was expected due to the presence of specific enzymes, such as trypsin, being secreted by these cells [30]. As shown in Fig. 5, a good HL degradability by Caco-2 cells could be confirmed as <5% of the initial HL amount could be detected after 4 h of incubation. Furthermore, a comparable curve shape as for incubation with isolated enzymes could be observed. This finding indicated a high compatibility of HL with tissues expressing appropriate enzymes.

Fig. 6. Cell viability assay after incubation of Caco-2 cells for 4 h (A) and 24 h (B) with HL (j dark grey bars), DTAB ( grey bars) and CPC (h white bars) dissolved in MEM in given concentrations. Indicated values are means (±SD, n = 4). The difference between HL and DTAB at a concentration of 10 mg
ml1 after 24 h is statistically very significant (p-value = 0.007).

3.6. Cell viability studies The aim of the current study was to generate a surfactant with an improved safety profile. According properties of lysine based surfactants have been previously described in connection with microorganisms and were also proven by toxicity studies on Caco-2 cells as HL was significantly better tolerated by the cells compared to DTAB and CPC [31]. After incubation for 4 h, concentrations of 50 mg
ml1 for CPC and 100 mg
ml1 in case of DTAB resulted in viabilities below 50%, whereas regarding HL even the highest tested concentration of 250 mg
ml1 showed no toxic effects (Fig. 6A). As illustrated in Fig. 6B, an incubation period of 24 h revealed very significant differences between HL and DTAB as well as CPC already at the lowest investigated concentration of 10 mg
ml1. Furthermore, the utmost HL level still revealed no changes in the cell viability. These findings are best explained by the rapid degradation of HL by Caco-2 cells into lysine and hexadecanol both being well tolerated and resulting in a high compatibility and no observable time-dependent toxicity.

Fig. 5. Degradation of HL (250 mg
ml1) by Caco-2 cells in 25 mM BIS–TRIS buffer pH 6.8 over 4 h. Indicated values are means (±SD, n = 4).

3.7. Hydrophobic ion pairing with plasmid DNA The hydrophobicity of macromolecular drugs can be altered by different mechanisms such as hydrogen bonding and ion pairing. In case of hydrogen bonding, interaction between H-bond acceptors and donors of the macromolecule as well as an applied surfactant results in a shielding of hydrophilic structures by the surfactant’s hydrophobic residue [32]. This process has been shown to facilitate an increased lipophilicity of a peptide drug [33]. In contrast, HIP is based on strong attractive forces due to electrostatic interactions between cationic and anionic structures [34]. The hydrophobic tail of an added surfactant shields the charged structures from interaction with water, thus drastically increasing their hydrophobicity and enabling their incorporation into lipophilic carrier systems, such as SEDDS. By utilizing CTAB as counterion for phosphate moieties present in the backbone of the pcDNA3-EGFP plasmid, for instance, a 5250-fold raised lipophilicity with regard to the Log POctanol/Water has been observed previously [3]. HL showed a high precipitation efficiency of almost 90% (Fig. 7). This value was achieved at a molar ratio of 6159:1 (surfactant to pDNA) as the plasmid exhibits 6159 base pairs and HL possesses two cationic charges. However, by further increasing the molar ratio, the precipitation efficiency decreased again explainable by the formation of solubilizing micelles as described previously for peptide drugs [4,34,35]. Application of DTAB or CTAB as counter ion resulted in a comparable HIP formation, although higher molar ratios were required because of their monovalent character. Due to stronger hydrophobic interactions, CTAB effectively precipitated the plasmid at a lower ratio in comparison to DTAB being in good accordance with results from Dias and co-workers [36].

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4. Conclusion

Fig. 7. Precipitation efficiency observed by hydrophobic ion pairing of pDNA (6159 bp, 1 mg
ml1) with HL (j), CTAB (▲) and DTAB (d). After addition of indicated molar ratios of surfactants in aqueous solution, mixtures were centrifuged and the remaining pDNA in the supernatant was analyzed utilizing the NanoQuant Plate. Indicated values are means (±SD, n = 3).

3.8. Transfection studies Since the novel ester revealed an improved safety profile and a sufficient precipitation efficiency during hydrophobic ion pairing studies, transfection studies were performed. Complexes with GFP encoding pDNA were incorporated into three different SEDDS and applied to HEK-293 cells. Upon successful transfection, viable cells translated the genetic information and the resulting fluorescence could be quantified. As a pDNA-CTAB complex within SEDDS has previously shown high transfection rates, it was chosen as a reference [3,6]. Results illustrated in Fig. 8 showed a similar improvement by SEDDS in comparison to naked pDNA as described within both studies utilizing the according reference complex incorporated into SEDDS. Concerning complexes prepared with HL successful transfection with the GFP encoding plasmid was observed. Compared to the CTAB complex, formulations A and C showed no statistic significant differences in the transfection capacity, whereas CTAB revealed a significant higher fluorescence for SEDDS B after the incubation period compared to HL. Nevertheless, the novel amino acid based cationic surfactant facilitated successful transfection of a cell culture while exhibiting distinctly enhanced cell compatibility. Following the principle ‘safety first’, this property might be favoured over a less pronounced improvement in the transfection efficacy.

A major drawback of cationic surfactants represents their deficient safety profile. This shortcoming was in the past more and more often addressed by development of ‘‘green” surfactants based on amino acids, which are easily enzymatically cleaved into nontoxic products. Within this study, an according amino acid based surfactant, namely hexadecyl lysinate, was successfully synthesized and revealed high biodegradability being reflected in enhanced cell compatibility. Furthermore, the lysine ester showed sufficient HIP properties for pDNA resulting in the formation of pDNA-HL complexes as it was achieved previously with comparable surfactants [14,16]. Prepared pDNA complexes could then be incorporated into SEDDS, a lipophilic drug delivery system, allowing for effective transfection of HEK–293 cells. Therefore, HL represents a promising and well-tolerated tool to facilitate the application of hydrophilic drugs within lipophilic carrier systems. Having shown its first potential, further drug delivery associated applications of HL might be explored including HIP with a broader range of drugs and modulation of the zeta potential of nanoparticulate as well as self-emulsifying systems. Furthermore, different hydrophobic residues as well as bonding pathways bear the capability to further improve the performance of lysine based surfactants [37]. CRediT authorship contribution statement Julian Dominik Wolf: Conceptualization, Methodology, Formal analysis, Investigation, Visualization. Markus Kurpiers: Validation, Methodology, Investigation, Data curation. Randi Angela Baus: Methodology, Investigation. Roman Xaver Götz: Investigation, Formal analysis. Janine Griesser: Methodology. Barbara Matuszczak: Investigation. Andreas Bernkop-Schnürch: Conceptualization, Supervision. Acknowledgements/Declaration of interests The authors sincerely thank Christina Tenhaken and Michael Moser for their assistance and inspiring discussions. The research leading to these results has received funding from the Österreichischen Forschungsgemeinschaft mbH (FFG), Austria under the grant number 7677912/856696. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. References

Fig. 8. Transfection efficiency of naked pDNA as well as SEDDS (A, B, C) containing complexes of pDNA with HL ( grey bars) and CTAB (j dark grey bars). Indicated values are means (±SD, n = 4). The difference between preparations with HL and CTAB within SEDDS B is statistically very significant (p-value = 0.0012).

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