Investigation on Physicochemical Characteristics of a Nanoliposome-Based System for Dual Drug Delivery
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Synergistic effects of multiple drugs with different modes of action are utilized for combinatorial chemotherapy of intractable cancers. Translation of in vitro synergistic effects into the clinic can be realized using an efficient delivery system of the drugs. Despite a few studies on nano-sized liposomes containing erlotinib (ERL) and doxorubicin (DOX) in a single liposome vesicle, reliable and reproducible preparation methods as well as physicochemical characteristics of a non-PEGylated nanoliposome co-encapsulated with ERL and DOX have not been yet elucidated. In this study, ERL-encapsulated nanoliposomes were prepared using the lipid film-hydration method. By ultrasonication using a probe sonicator, the liposome diameter was reduced to less than 200 nm. DOX was loaded into the ERL-encapsulated nanoliposomes using ammonium sulfate (AS)-gradient or pH-gradient method. Effects of DOX-loading conditions on encapsulation efficiency (EE) of the DOX were investigated to determine an efficient drug-loading method. In the EE of DOX, AS-gradient method was more effective than pH gradient. The dual drug-encapsulated nanoliposomes had more than 90% EE of DOX and 30% EE of ERL, respectively. Transmission electron microscopy and selected area electron diffraction analyses of the dual drug-encapsulated nanoliposomes verified the highly oriented DOX-sulfate crystals inside the liposome as well as the less oriented small crystals of ERL in the outermost region of the nanoliposome. The nanoliposomes were stable at different temperatures without an increase of the nanoliposome diameter. The dual drug-encapsulated nanoliposomes showed a time-differential release of ERL and DOX, implying proper sequential releases for their synergism. The preparation methods and the physicochemical characteristics of the dual drug delivery system contribute to the development of the optimal process and more advanced systems for translational researches.
KeywordsNanoliposome Dual drug delivery system Ultrasonication Remote loading technology Time-differential release
Enhanced permeability and retention
1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)
Selected area electron diffraction
Standard error of mean
Transmission electron microscopy
Intractable cancers such as triple-negative breast cancers have limits to treatment with standard therapies because of DNA damage response highly interconnected with various signaling networks [1, 2, 3]. Therapeutic strategies increasing initial chemo-sensitivity of such recalcitrant tumors have been studied to challenge the limits [4, 5]. A recent study suppressing oncogenic signaling pathways while using a DNA damage agent is one of the combinatorial therapeutic strategies [6, 7, 8]. The study suggested that pretreatment of a growth factor inhibitor to resistant cancer cells synergize their apoptotic response to a genotoxic drug [9, 10]. There is a highly important clinical need for therapeutic strategies to target multiple cancer cell-specific survival pathways for enhancement of the extent of tumor cell killing and potential reduction of total drug exposure during treatment [11, 12]. Meanwhile, in order to translate in vitro synergistic effects of two kinds of drugs into the clinic, a delivery system that can deliver the both drugs and release them in time-differential manner is essential due to the difference between pharmacokinetic (PK) properties of the drugs and the difficulty in targeting the same cancer cells in proper temporal sequence [13, 14, 15, 16].
A nanoliposomal delivery system is one of the dual drug delivery systems which can be applied to combination therapy. The major components of liposomes are in general phospholipids similar to those of cell membranes. The phospholipids form a bilayered concentric sphere with an inner compartment and bilayer membranes . The nanoliposomes can allow the drugs having different physicochemical properties to be loaded into the inner compartment and the bilayer membrane of the liposomes and then delivered to the lesion. Thus, in vivo synergistic effect of the two drugs delivered using the nanoliposomal system can be achieved through enhanced co-localization of the drugs to cancer cells by not only reconciliation of the PK properties of each drug but also the so called enhanced permeability and retention (EPR) effect of tumor tissue. Besides the encapsulation of drugs in the inner compartment, the nanoliposome can solubilize hydrophobic drugs, enhance their stability, modulate their blood circulation properties, and hence induce their accumulation to tumor tissues .
Despite the recent studies on nanoliposome-based combination chemotherapy delivery systems, the preparation processes of the systems need to be optimized for reliable and reproducible fabrication, and physicochemical characteristics of them should be elucidated for the development of more advanced systems [13, 19]. In our study, a nanoliposomal delivery system co-encapsulated with elrotinib (ERL; ERL free base, logP = 3.3) and doxorubicin (DOX; DOX HCl, logP = 1.27) in a single liposome vesicle was prepared as a model nanoliposome system according to a previous report except for a so-called non-PEGylated liposome formulation , and physicochemical characteristics of the system were studied. In vitro drug release studies were carried out to investigate a time-differential release of the drugs. Among several methods such as extrusion, sonication, and high-pressure homogenization [20, 21, 22], ultrasonication using a probe sonicator was utilized to reduce liposome diameter due to its more efficient processability. A comparative study about effects of drug-loading technologies such as pH- or ammonium sulfate-gradient method on encapsulation efficiency (EE) of the drug was performed to optimize the drug encapsulation. In addition, the most effective process for drug encapsulation was determined through investigating the effects of process conditions on EE of the drug. The preparation conditions for nanoliposomal dual drug delivery system encapsulated with both ERL and DOX and releasing the drugs in a proper sequence were optimized. The optimized preparation methods and the characteristics of the dual drug delivery system suggest a platform of a reliable and reproducible preparation processes and a more advanced delivery system for translational studies.
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (POPG) were supplied from Avanti Polar Lipids, Inc. (Alabaster, Al, USA). Cholesterol was from Sigma-Aldrich, Corp. (St. Louis, MO, USA). Doxorubicin (DOX) hydrochloride and erlotinib (ERL) free base were purchased from Boryung Co., Ltd. (Seoul, Korea) and Shanghai Send Pharmaceutical Technology Co., Ltd. (Shanghai, China), respectively. Ammonium sulfate (AS) and citric acid (CA) anhydrous were supplied from Daejung Chemicals & Metals Co., Ltd. (Siheung-si, Korea). All the other reagents were of reagent grade and supplied from Sigma-Aldrich, Corp. (St. Louis, MO, USA).
Preparation of Dual Drug-Encapsulated Nanoliposomes
A lipid mixture composed of DSPC, cholesterol, and POPG at a ratio of 27:20:3 (w/w/w) was mixed with ERL free base at a weight ratio of the drug to the total lipid, 3:50. Those mixtures were dissolved in 9 mL of a mixed solvent of chloroform:methanol = 2:1 (v/v). The lipid solution was placed in a round-bottomed flask, and to form a lipid membrane, the solvent was removed using a rotary evaporator (Rotavapor R-210; BÜCHI Labortechnik AG, Flawil, Switzerland) under vacuum at 40 °C. The membrane was dried in a vacuum overnight to remove all the residual solvent. In these processes, ERL molecules are inserted into the lipid membrane via hydrophobic attractions and then spontaneously encapsulated into the lipid bilayer membrane of the liposomes, which are formed by the following hydration process.
In order to hydrate the ERL-containing lipid membrane, 8 mL of 250–400 mM AS or 300 mM citric acid buffer (pH 3.9) were added to the membrane formed at the inner surface of round-bottomed flask. After incubation of the membrane at 65 °C for 30 min by rotation of the flask, the liposome suspension was sonicated using a bath sonicator (Branson 3510E-DTH; Branson, Danbury, CT, USA). To reduce the liposome diameter, the liposome suspension was sonicated using the probe sonicator at the conditions such as a pulse of 5 s on and 2 s off, an amplitude of 20% and an energy of 36 J per pulse in an ice-water bath, or a water bath under magnetic stirring. To remove AS which was unloaded into the nanoliposomes, dialysis of the nanoliposomes was performed overnight in phosphate buffered saline (PBS, pH 7.4) with a dialysis tubing cellulose membrane (14,000 molecular weight cutoff (MWCO); Sigma-Aldrich Corp., St. Louis, MO, USA). In the case of nanoliposome suspension prepared using the citric acid buffer, the pH of the solution was adjusted to about 6.5 using 300 mM sodium bicarbonate buffer to make a gradient between the inside and outside of the nanoliposomes.
DOX-Loading into ERL-Encapsulated Nanoliposomes
To encapsulate DOX into ERL-encapsulated nanoliposomes, DOX hydrochloride was first dissolved in 0.9% NaCl aqueous solution at 65 °C. In general, 2 mL of 1.5 mg/mL DOX solution were added to 8 mL of the AS- and ERL-encapsulated or citric acid- and ERL-encapsulated nanoliposome suspension. Drug-loading processes of the DOX-added nanoliposome suspension were composed of incubation, sonication using the bath sonicator, equilibration at room temperature (RT), and dialysis against PBS (pH 7.4) using the dialysis tubing cellulose membrane to remove unencapsulated DOX from the nanoliposome suspension. Effects of DOX-loading conditions on DOX’s EE were investigated using four experimental groups. Group 1 was the mixture of DOX solution and the liposomes, which was treated in the sequence of an incubation at 65 °C for 30 min, a sonication at 65 °C for 5 min, and an overnight dialysis. Group 2 was incubated at 65 °C for 30 min, sonicated at 65 °C for 5 min, equilibrated for 30 min at RT, and then dialyzed overnight. Group 3 was incubated at 65 °C for 30 min, sonicated at 65 °C for 15 min, and then dialyzed overnight. Group 4 was incubated at 65 °C for 30 min, sonicated at 65 °C for 15 min, equilibrated for 30 min at RT, and then dialyzed overnight.
Diameter, Morphology, and Physical Stability of Dual Drug-Encapsulated Nanoliposomes
Diameter of the drug-encapsulated nanoliposomes was measured at 25 °C using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd., Osaka, Japan). Morphology and mean diameter of the single or dual drug-encapsulated nanoliposomes were observed using a field emission transmission electron microscope (FE-TEM) (JEM 2100F; JEOL Ltd., Tokyo, Japan, installed at Korea Basic Science Institute) at 200 kV. In order to prepare the sample, the nanoliposome suspension was drop-casted onto a carbon-coated copper grid, and the grid was air-dried at RT before viewing under the microscope. Physical stability of the dual drug-encapsulated nanoliposomes incubated in PBS (pH 7.4) at different temperatures of 4, 25, and 37 °C was investigated by monitoring a change in the liposome diameter as a function of time. The liposome diameter was measured using a particle size analyzer (Zetasizer Nano ZS, Malvern Instruments Limited, Worcestershire, UK).
EE of Drugs
Results are presented as mean ± SEM, unless otherwise noted.
Results and Discussion
Effect of Preparation Processes on Nanoliposome Characteristics
Effect of Drug-Loading Method on EE
When the changes in the EEs of DOX according to the AS concentrations were compared, the EE was the highest and SEM of the EE was the lowest at 350 mM AS. AS-gradient or pH-gradient method utilizes a drug translocation mechanism, which induces the drugs outside the nanoliposomes to migrate into inner compartment of the nanoliposomes because of a driving force generated from a transmembrane ion concentration gradient of the nanoliposomes [29, 30]. The EE of DOX by AS-gradient method was, however, quite higher than that of pH-gradient method. These results can be ascribed to the fact that AS-gradient method is more effective in developing crystalline complexes than pH-gradient method due to higher crystallizability between the ionized DOX and the counter ions inside the nanoliposomes [31, 32].
Effect of Drug-Loading Conditions on EE
Morphology and Physical Stability of Dual Drug-Encapsulated Nanoliposomes
Time-Differential Drug Release
As shown in Fig. 8, the ERL release was 36 ± 0.01%, while the DOX release was less than 10% until 8 h of the release test period. In particular, during this period, the release rate of ERL was much faster than that of DOX. By 48 h, 65 ± 0.07% of ERL were released from the nanoliposomes in contrast to 30 ± 0.01% release of the DOX. The release rates of ERL and DOX are shown in Fig. 8b. Until 8 h, the release rate of ERL was more than 4% per hour in contrast to less than 1% per hour of the DOX release rate. After 8 h, the release rate of ERL was slowed down. Compared with the release of ERL, DOX showed a slow release and had an almost zero-order release rate. These results suggest that during the initial period of release test, much more amounts of ERL were released from the liposomes than those of DOX and there was a time-differential release between ERL and DOX. The sequential release of the drugs was thought to be originated from the difference between the physicochemical states of each drug in the liposomes [28, 32, 47]. These results on the dual drug-encapsulated nanoliposome system can contribute to translation of in vitro synergistic effects of two kinds of drugs into the clinic through overcoming both the difference between PK properties of each drug and the difficulty in targeting the same cancer cells in proper temporal sequence.
As a dual drug delivery system, a nanoliposomal delivery system encapsulating both ERL and DOX was prepared and characterized. The liposome diameter was controllable by ultrasonication, and the sonication for diameter reduction was effective when carried out after the film-hydration and before DOX encapsulation. The nanoliposome diameter decreased remarkably during an initial period of ultrasonication. DOX was loaded into ERL-encapsulated nanoliposomes through pH- or AS-gradient method. AS-gradient method showed higher EE of DOX than pH-gradient, and the AS concentration for higher EE of DOX was determined. Equilibration of a mixture of DOX solution and ERL-encapsulated nanoliposomes in DOX-loading process was advantageous for EE increase of DOX. By HR-TEM and SAED analyses of the dual drug-encapsulated nanoliposomes, not only the highly oriented crystals formed between protonated DOX cations and divalent sulfate anions inside the liposome but also the less oriented small crystals of ERL in the outermost layer of the nanoliposome were identified. ERL and DOX co-encapsulated in the nanoliposomes showed a time-differential release, indicating much faster release of ERL than that of DOX from the liposomes. The elucidated preparation methods of the nanoliposomal dual drug delivery system can contribute to development of advanced dual drug delivery systems and translational researches of the combination therapies exhibiting synergistic effects via sequential actions of the drugs.
This research has been performed as a project no. SKO1707C04, a project no. KK1703-F00, and a project no. KK1803-F00 supported by the Korea Research Institute of Chemical Technology (KRICT).
Availability of Data and Materials
The authors declare that the materials, data, and associated protocols are promptly available to readers without undue qualifications in material transfer agreements. All data generated and analyzed during this study are included in this article.
JHN performed the experiments, drafted the manuscript, and revised it. SYK carried out additional experiments, analyzed the data obtained, and revised the manuscript. HS proposed the initial work, finalized the manuscript, and supervised the work at the same time. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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