Article pubs.acs.org/Langmuir
In Vitro Interaction of Polyelectrolyte Nanocapsules with Model Cells Sylwia Łukasiewicz† and Krzysztof Szczepanowicz*,‡,§ †
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-348 Krakow, Poland ‡ Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, 30-239 Krakow, Poland § Institute of Pharmacology, PAS, 31-343 Krakow, Poland S Supporting Information *
ABSTRACT: The nanocapsules based on a liquid core with polyelectrolyte shells prepared by the technique of sequential adsorption of polyelectrolytes (LbL) were investigated to verify capsules bioacceptance. Using AOT (docusate sodium salt) as emulsifier, we obtained liquid cores, stabilized by the interfacial complex AOT/PLL (poly-L-lysine hydrobromide). These liquid cores were encapsulated by sequential adsorption of polyelectrolytes using biocompatible polyanion PGA (poly-L-glutamic acid sodium salt) and biocompatible polycation PLL. The average size of the formed capsules was 60−80 nm. The influence of a number of polyelectrolytes layer in the shell (thickness of polyelectrolytes shell), surface charge, and capsule doses on cell viability was studied in a cellular coculture assay. In order to improve nanocapsules biocompatibility, the PEG-ylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). For the most toxic nanocapsules (with only one polycation layer) about 90% of cells could survive when the concentration of nanocapsules was below 0.2 × 106 per one cell. That suggests that they use as a delivery vehicles is quite safe for living cells. Analysis of internalization of AOT(PLL/PGA)4-g-PEG in HEK 293 cells indicates that tested nanocapsules can easily penetrate cells membrane.
1. INTRODUCTION Nanoencapsulation has high application potential in medicine since it can be used to improve the compatibility of lipophilic, poorly water-soluble or even water-insoluble active compounds with physiological fluids and can protect therapeutic molecules from the destructive influence of an external environment.1 The ability to penetrate cells by nanocapsules makes them a promising candidate for drug delivery system. Moreover, they can be functionalized to achieve “intelligent targeting”, i.e., the delivery to the specific cells or organs. Therefore, it is envisaged that the nanoparticulate system with functionalized surfaces for targeted delivery will play a central role in modern therapies (personalized medicine). Different types of nanoparticles such as for example liposomes,2 solid lipid nanoparticles,3 micellar nanoparticles,4 gold,5 carbon nanotubes,6 and quantum dots7 are widely described as potential drug carriers. One of the most promising drug delivery system is based on using polymeric nanoparticles. In particular, the technique of nanocapsules shell formation by the sequential adsorption of polyelectrolytes, called layer-by-layer technique (LbL),8−10 is considered as the most versatile one. Such methodology of capsules formation was originally proposed by Sukhorukov in 1998.11 He described formation of polyelectrolytes multilayer shell on latex microparticles. Further on, the method has been used to create polyelectrolytes multilayer shell on liquid nanocores.12−16 Polyelectrolytes are polymers, which in aqueous solution dissociate into polyions. Lots of biomacromolecules such as nucleic acids (DNA, RNA), proteins, polysaccharides, and © 2014 American Chemical Society
other biopolymers are polyelectrolytes. Their applications in the preparation of nanocarriers may cause increase of nanoparticulate systems bioacceptance. Similar properties possess synthetic polyelectrolytes like poly(amino acid)s (e.g., PLL, polyarginine, or PGA). Therefore, such polyelectrolytes used to form shells of nanocapsules being easy to synthesize and purify may ideally fulfill the requirements, such as biodegradability, low toxicity, and ability to alter the biodistribution of drugs, for the materials used for controlled drug delivery systems. Further advantages of polymeric nanoparticles system are their small size, which can make them compatible with various administration routes, including intravenous injection, and the relatively high drug encapsulation capacity.17 Recent studies have shown that the uptake of nanoparticles into cells may cause a cytotoxic effect.18−20 Therefore, planning a new nanoparticulate system, especially for drug delivery, the main task is controlling the balance between the effective internalization into the cell and inducing toxic effect on the cells. Interaction between nanoparticles and cell membrane is the main factor that affects this process. The interaction depends on charge, shape, size, surface area, flexibility, surface chemical properties, and amphipathic character of nanoparticles.21−24 When nanoparticles are charged, the electrostatic interaction mainly determine their Received: September 19, 2013 Revised: January 10, 2014 Published: January 12, 2014 1100
dx.doi.org/10.1021/la403610y | Langmuir 2014, 30, 1100−1107
Langmuir
Article
interplay with lipid membrane.21,22 Moreover, it is important to know that nanoparticles size and surface properties can significantly change when they are transferred into biological systems.25−28 They can aggregate either due to the ionic strength of physiological media or due to chemical reactions with components of the cell media and protein adsorption.28−31 To minimize this effect, coating of nanoparticles surface with a hydrophilic, flexible, and nonionic polymer is used. The most popular coating material is the Food and Drug Administrationapproved water-soluble polymer poly(ethylene glycol) (PEG). PEG chains have the potential to reduce both nonspecific binding of proteins and cytotoxicity. Such corona masks the presence of capsules for the immune system (stealth effect) and prevents unspecific binding to cell membrane. One of the proposed methods of PEG corona formation on polyelectrolytes nanocapsules through adsorption of copolymers containing PEG chains was described by Szczepanowicz et al.14 They used PEG-ylated polyelectrolytes (PGA-g-PEG, PEG chains grafted on poly(glutamic acid) backbone) as an outermost layer of capsule’s shell. In the present study we have focused on the influence of thickness of polyelectrolyte shell, surface charge, and presence of PEG corona on cell viability and ability to deliver the “cargo” inside the specific cells (HEK 293 cell line). We used liquid cores and biocompatible and biodegradable polymers PLL and PGA to form nanocapsules shells, which were further modified by PEG-ylation. Basic in vitro investigations of the synthesized nanocarriers presented in this paper are a necessary prerequisite for indication that the nanocapsules are good candidates for further biomedical experiments as an effective drug delivery system.
solutions of ionic strength 0.015 M to obtain PE solution of concentration 2 g/L, without any pH adjustment. Nanocapsules were formed by addition of oil phase (AOT/chloroform) to polycation (PLL) solution during mixing with the magnetic stirrer at 300 rpm. The optimal ratio of surfactant (AOT) to polycation concentrations was determined by measuring the zeta potential of emulsion drops and examining their stability.12 It was found optimal when the zeta potential reached a value close to the zeta potential of the same polyelectrolyte in solution. Then the capsules cores (emulsion drops stabilized by AOT/PLL) were encapsulated in polyelectrolyte multilayer shells, and the consecutive layers of polyelectrolytes were formed the by layer-by-layer technique, using the saturation method;11,33 i.e., the multilayer shells were constructed by subsequent adsorption of polyelectrolytes from their solutions without the intermediate rinsing step. Because of the nature and the size of the core, the standard LbL methods involving rinsing steps together with suspension centrifugation or filtration are not effective because of the destruction, destabilization, or aggregation of emulsion drops. They have been successfully used for the formation of polyelectrolyte multilayer shell on solid cores in the micrometer range. Since nanoobjects and emulsions have started to be promising candidates for the capsules’ cores, the saturation method of sequential adsorption of polyelectrolytes has gained importance.12 Capsules suspension was added to the polyelectrolyte solution while mixing with a magnetic stirrer. Volumes of polyelectrolyte solution used to form each layer were chosen empirically by analyzing the results of the simultaneous zeta potential measurements. They were found optimal when the zeta potential reached the value close to the zeta potential of the same polyelectrolyte in solution. For the preparation of fluorescently labeled nanocapsules FITC-PAH or RODPLL was used instead of one PLL layer. For the model drug encapsulation, at the beginning of the procedure Coumarine 6 was dissolved in chloroform (0.8 g/L) prior to emulsification with AOT. Particle Size Analysis, Zeta Potential, and Nanocapsules Concentration Measurements. The size distribution (the hydrodynamic diameter) of capsules was determined using a Zetasizer Nano Series from Malvern Instruments by DLS (dynamic light scattering) with the detection angle of 173°. Each value was obtained as an average from three runs with at least 10 measurements. The zeta potential of polyelectrolytes, emulsion drops, and capsules was determined by the microelectrophoretic method, using a Malvern Zetasizer Nano ZS apparatus. Each value was obtained as an average from three subsequent runs of the instrument with at least 20 measurements. The zeta potential of capsules as well as of polyelectrolytes in solution was measured in 0.015 M NaCl solution. Nanocapsules concentration was determined by NTA (Nanoparticle Tracking Analysis) using a NS500 instrument from NanoSight. The concentration of nanocapsules was measured in 0.015 M NaCl. All measurements were performed at 25 °C. SEM (Scanning Electron Microscopy). Microscopic SEM images of nanocapsules were performed with a JEOL JSM-7500F field emission scanning electron microscope at an operation voltage of 15 keV. Capsules were deposited on the copper cylinder by immersing it into nanocapsules suspension for 10 s and dried overnight. UV−vis. UV/vis absorption spectra of the capsules were acquired using a UV-1800 spectrophotometer (Shimadzu). UV−vis spectrophotometry was applied to confirm encapsulation of model drugs. Stability Studies (Colloidal and Biogolical). To evaluate the colloidal and biological stability of nanocapsules, their freshly prepared suspensions were stored in 0.015 M NaCl and FBS solutions at room temperature. Size distribution (hydrodynamic diameter) and zeta potential of nanocapsules were measured after preparation and after appropriate storage time as described above. Cell Cullture. HEK 293 cells were grown in Dulbecco’s modified essential medium (DMEM) supplemented with 1% L-glutamine and 10% heat-inactivated fetal bovine serum (FBS). The cells were cultured at 37 °C in the atmosphere of 5% CO2. Two days before cell viability and cytotoxicity measurements, the cells were seeded into proper 96-well plates at a density of 3 × 104 cells per well. In case of confocal microscopy imaging cells were plated on 35 mm plates with
2. EXPERIMENTAL SECTION Materials. The biocompatible polyelectrolytes used in our studies were the polycation poly-L-lysine hydrobromide PLL (MW ∼ 15 000− 30 000) and the polyanion poly-L-glutamic acid sodium salt PGA (MW ∼ 15 000−50 000) for fluorescence measurements, fluorescently labeled polycations poly(fluorescein isothiocyanate allylamine hydrochloride) FITC-PAH (MW ∼ 70 000), and fluorescently labeled polyL-lysine hydrobromide (ROD-PLL) were used. ROD-PLL was synthesized via coupling of Lissamine rhodamine B sulfonyl chloride according to the protocol described in ref 32. All polyelectrolytes chloroform, Coumarine 6, and sodium chloridewere obtained from Sigma-Aldrich. All materials were used as received without further purification. The distilled water used in all experiments was obtained with the three-stage Millipore Direct-Q 3UV purification system. PGAg-PEG was synthesized according to the procedure described by Szczepanowicz et al.14 The obtained coupling rate of approximately 31% corresponded to a grafting ratio of g = 3.2. HEK 293 cells were obtained from the American Type Culture Collection (Manassas, VA). All cell culture materials were purchased from GIBCO and Sigma. The ATP-lite one-step test was purchased from PerkinElmer, the MTT reagent from Sigma-Aldrich, and LDH cytotoxicity detection kit from Clontech. Nanocapsules Preparation. Nanocapsules were prepared using the method described by Szczepanowicz et al.,12 i.e., the direct encapsulation of emulsion drops in polyelectrolyte multilayer shells. The Food and Drug Administration-approved, negatively charged oilsoluble surfactant AOT (docusate sodium salt) was used as emulsifier to obtain oil emulsion droplets (capsules cores), which were stabilized by AOT/polycation interfacial complexes. The oil phase for capsules preparation was prepared by dissolution of 340 g/L AOT in chloroform. Because of the toxicity issue, chloroform was evaporated from suspensions of nanocapsules after preparation of emulsion. The amount of the chloroform after evaporation determined by GC-ECD analysis was 0.0397 mg/L. Polyelectrolytes were dissolved in NaCl 1101
dx.doi.org/10.1021/la403610y | Langmuir 2014, 30, 1100−1107
Langmuir
Article
medium supplemented with 2% FBS at 37 °C. Before imagining FDA (fluorescein diacetatewhich serves as a viable probe and enables for the visualization of the interior of the cell) was added to the cells. The cells were incubated with AOT(PLL/PGA)4-g-PEG nanocapsules at dose 0.15 × 106/cell. FDA was excited at 488 nm (Ar) and ROD-PLL at 561 nm (HeNe). Emission detection: 500−550 and 580−650 nm, respectively. Data were registered in sequential mode.
15 mm diameter glass coverslips at the density 105, 1 day before the experiment. Cell Viability and Cytotoxicity Assays. MTT Reduction Test. Evaluation of cell viability was performed using the MTT reduction test. Various types of nanoparticles (with various number of polyelectrolyte layers) resuspended in 0.015 M NaCl were added in various doses to the fresh medium into each well. Doses of 1 × 106, 0.8 × 106, 0.5 × 106, and 0.2 × 106 nanocapsules/cell were used. After 24 h treatment the cell culture media with nanoparticles was removed and cells were incubated with 2 g/L MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide), resuspended in the serum-free media for 4 h at 37 °C, 5% CO2 in air. After incubation the medium was removed, and then 100 μL of DMSO was added to each well. A yellow tetrazolium salt is converted by viable cells to purple formazan, which was dissolved in dimethyl sulfoxide. The absorbance was detected at 570 nm using microplate reader (TECAN Infinitive200Pro). Untreated cells served as a control. The results obtained described the average cell viability from different experiments repeated 10 times. Statistical analysis was performed using the Student’s t test. Luminescence ATP Detection Assay (ATPlite Firstep). Luminescent cell viability assay is a homogeneous method to determine the number of viable cells in culture. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. The amount of ATP in cells correlates with their viability. The experiments were performed as described above (MTT assay). After 24 h of treatment, the medium with nanoparticles was removed and F12 medium with 2% FBS was added to the cells. The microplate was equilibrated at room temperature before the incubation of the cells with freshly prepared ATPlite compound. The test was performed according to the instruction including in the ATPlite firstep kit (PerkinElmer). The luminescence was measured using microplate reader (TECAN Infinitive200). Untreated cells served as a control. The results obtained described the average cell viability from different experiments repeated 10 times. Statistical analysis was performed using the Student’s t test. LDH Cytotoxicity Detection Kit. Cytotoxicity assay was performed in HEK 293 cells after 4 h incubation with various types and doses of nanocapsules (the same as described above). The LDH release into the cell culture medium was determined using the Cytotoxicity Detection Kit (LDH) (Clontech). The plates were centrifuged at 250g for 10 min, and 100 μL of supernatant was taken to quantify the LDH. The test was performed according to the protocol including in the kit. The absorbance was measured at 490 nm (TECAN Infinitive200). The results represented the cytotoxic effect from different experiments repeated four times. Statistical analysis was performed using the Student’s t test. Intracellular Nanocapsules Accumulation. Accumulation of nanocapsules inside the HEK 293 cells was measured in a 96-well plate using TECAN infinitive 200pro plate reader similarly to the procedure described for vibrant phagocytosis assay kit (Molecular Probes). Three hours before experiments, cells were seeded at a density of 105 cells/ well. Then the culture medium was replaced by 100 μL of HBSS buffer (with 10% FBS or without serum) containing 0.15 × 106 fluorescently labeled nanocapsules per cell. After 1 h incubation the buffer was removed, and 100 μL of trypan blue (0.25 g/L as a fine suspension in citrate-balanced salt solution, pH 4.4) was added to the well for 1 min. This technique enables detection of the intracellular fluorescence emitted by nanocapsules as well as the effective fluorescence quenching of the extracellular probe by trypan blue. Fluorescence was measured at excitation wavelength 485 nm and emission wavelength 520 nm. Experiment was done on multiple replicates. Signal from wells containing only nanocapsules suspension was used as a negative control. By subtracting the average fluorescence intensity of a group of negative control, the ability of nanocapsules to accumulate in the cells was obtained. Confocal Microscopy Imaging. Confocal microscopy was used to analyze the localization of the fluorescently tagged nanocapsules in HEK 293 cells. Images were acquired using Leica LSC SP5 laser scanning confocal microscope (Leica) and 63× HCX PL APO NA 1.4 oil immersion lens (Leica). The measurements were performed in F12
3. RESULTS AND DISCUSSION In our previous work we found that it was possible to control the size of the emulsion droplets by changing only the concentration of polycation during preparation while maintaining the same ratio of AOT to polycation and the size of droplets can varied between 70 and 200 nm. The second parameter important during emulsion preparation is the ratio of surfactant (AOT) to polycation since it affects the zeta potential and stability of formed droplets. Therefore, that optimal ratio was determined by measuring zeta potential of emulsion droplets and examining their stability. Figure 1a presents variations in zeta potential of formed emulsion droplets stabilized by the AOT/PLL complex with increasing concentration of PLL and information about their stability. The optimal dosage of PLL when the zeta potential of emulsion drops reached a value close to the zeta potential of the PLL in solution just after overcharging is marked by arrow. The
Figure 1. Changing of the zeta potential of (a) formed emulsion droplets with increasing amount of polycation (PLL) used to form droplets (condition marked by arrow represents first stable sample after overcharging), (b) formed AOT(PLL/PGA) capsules for various amounts of PGA used to form layer (condition marked by arrow represents first stable sample after overcharging), and (c) formed PEG-ylated nanocapsules for various amount of PGA-g-PEG used to form PEG-ylated layer (condition marked by arrow represents first stable sample after overcharging). 1102
dx.doi.org/10.1021/la403610y | Langmuir 2014, 30, 1100−1107
Langmuir
Article
average drop size was around 60 nm (green line in Figure S1 in Supporting Information) with polydispersity index (PDI)
