This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.
Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.
The number of studies investigating efficient drug delivery systems has risen in recent years. However, limitations such as rapid clearance, poor biodistribution, and solubility at physiological pH and insufficient cellular uptake still need to be surpassed. The use of nanosystems has emerged as recent progress in cancer therapeutics, applied to increase the intracellular concentration of drugs inside cancerous cells while minimizing toxicity in healthy cells. Moreover, nanoparticles obtained from a different range of materials (i.e., polymers, dendrimers, liposomes, viruses, carbon nanotubes, and metals such as iron oxide and gold) are currently being applied to enhance anticancer effects and reduce systemic toxicity1. Liposome nanocapsules in particular have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. In recent years, various nutraceutical products such as vitamins, enzymes, and herbal extracts have been formulated using liposome technology2.
Liposomes are spherical vesicles consisting of one or more concentric lipid bilayers spontaneously formed by the dispersion of phospholipids in aqueous media3,4. The polar heads of the phospholipids are located on the outer and inner surfaces of the membranes, in contact with the aqueous environment. In contrast, fatty acid chains form the hydrophobic core of the membranes and are protected from water5. Some attributes of liposomes that make them attractive drug delivery systems include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds6.
Liposomes can be prepared using various processes steps such as agitation, sonication, extrusion, lyophilization, freezing, and thawing. Classical methods include reverse phase evaporation, solvent injection, and detergent dialysis. The most applied method is thin lipid film hydration, also known as Bangham's method, which is used to obtain vesicular-lipid forms7,8,9,10,11. Lamellarity (the number of phospholipid bilayers) and particle size are classical parameters used to characterize liposomes as either 1) unilamellar vesicles (ULVs), formed by a unique phospholipid bilayer and varying in size as follows: i) small unilamellar vesicles (SUVs, ~0.02-0.20 µm), ii) large unilamellar vesicles (LUVs, ~0.2-1.0 µm), and iii) giant unilamellar vesicles (GUVs, >1 µm); or 2) multilamellar vesicles (MLVs, >0.1 µm)3,12. Vesicle size is an important parameter when considering for therapeutic use, such as in cancer treatment, in which sizes of <200 nm are ideal to allow nanovesicles to cross the endothelial barrier and reach tumoral tissues4.
Herein, the encapsulation procedure following the classical hydration of a thin lipid film technique7 was described using tarin, a plant lectin characterized as a hydrophilic globular protein13,14,15. Nanosized vesicles are produced by including sonication and extrusion steps in the main technique, resulting in stable liposomal nanovesicles with high entrapment efficiency16.
1. Preparation of tarin liposomal nanocapsules16
NOTE: All preparations should be prepared in triplicate in order to obtain a larger volume (7 mL) and enable the sample to be centrifuged in an ultracentrifuge (see details below).
2. Encapsulation efficiency
NOTE: Determine the encapsulation efficiency using Peterson's protocol17in order to avoid lipid interference in protein quantification. All samples (BSA standards and liposome supernatant) should be analyzed in triplicate. Also prepare a blank tube.
3. Size and stability determination
NOTE: Size distribution and polidispersity index (PdI) of the liposomal preparations are evaluated by dynamic light scattering (DLS). A PdI close to 0.1 indicates a homogeneous preparation. For stability determination, store liposomes at 4 °C and check size distribution and size average regularly.
4. Morphological characterization
NOTE: Liposome characterization is performed according to Murtey and Ramasamy19. Samples containing nanoliposomes obtained in step 1.10 are prepared in triplicate.
Figure 1 describes the nanoliposome preparation16,20,21. Phospholipids, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS), the main liposome constituents, were first dissolved in chloroform to obtain the lipid film. The lipid film was then rehydrated in ammonium sulfate solution containing the hydrophilic protein (tarin) to be entrapped, and the incubation was performed overnight. Then, sonication and extrusion techniques were applied to generate small unilamellar vesicles. The ultracentrifugation step separated the liposomal preparation from free lipids and unencapsulated protein, while the supernatant was used for the determination of entrapment efficiency.
Nanoliposomes produced using the aforementioned methodology exhibited a size distribution ranging from 51−396 nm and an average size of 155 nm (Table 2). The preparation was homogeneous, since the polydispersity index was 0.168. A high entrapment efficiency of 83% can be reached if the liposomes are extruded through a 0.2 µm pore size membrane (Table 2).
Morphological nanoliposome characteristics were evaluated by SEM. Figure 2A,B displays round-shaped liposomal vesicles in the range of 121 nm and analyzed at 20 kV, whereas Figure 2C,D displays inadequately prepared samples. Nanoliposomes were simply air dried without previous fixation or any other treatment described in this study. As a result, larger and damaged vesicles in the range of 332 µm and analyzed at 5 kV were observed.
Figure 1: Schematic representation of nanoliposome preparation. DOPE, PEG, and CHEMS, the main liposome constituents, were first dissolved in chloroform to obtain the lipid film (1, 2, 3). The lipid film was then rehydrated in a saline buffer containing the hydrophilic protein (tarin) to be entrapped, and the incubation was performed overnight (4). Then, sonication and extrusion techniques were applied to generate SUVs (5, 6). The ultracentrifugation step separated the liposomal preparation from free lipids and unencapsulated protein, while the supernatant was used for the determination of entrapment efficiency (7). This figure has been modified from Correa et al.16. Please click here to view a larger version of this figure.
Figure 2: Nanoliposome photomicroscopy by SEM. (A,B) Images of round-shaped liposomal vesicles in the range of 121 nm and analyzed at 20 kV. (C,D) Images of inadequately prepared samples. Mistreated samples allowed for the observation of larger and/or damaged vesicles, which cannot resist vacuum and/or voltage conditions at 5 kV. This figure has been modified from Correa et al.16. Please click here to view a larger version of this figure.
Liposome Components | Weight (g) | Concentration (mM) | Final volume |
DOPE | 0.0420 | 5.7 | 10 mL |
MPEG 2000-DSPE | 0.1059 | 3.8 | |
CHEMS | 0.0024 | 0.5 |
DOPE - 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine); MPEG 2000-DSPE - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol)-2000] (ammonium salt); CHEMS – cholesterylhemisuccinate.
Table 1: Preparation of tarin liposomal nanocapsules.
Membrane pore size (µm) | Size distribution (nm) | Average size (nm) | Polydispersity index (PdI) | Peak (nm) | Entrapment Efficiency |
0.2 | 51 - 396 | 155 | 0. 168 | 94 ± 39 | 0.83 |
Size and polydispersity index were evaluated by dynamic light scattering, while encapsulation efficiency was determined according to Peterson17.
Table 2: Size, polidispersity index, and entrapment efficiency of the nanoliposome preparation.
The protocol described herein was tested by Correa et al.16 to encapsulate tarin, an immunomodulatory and antitumoral lectin purified from Colocasia esculenta22. The methodology yielded successful results, allowing for the production of stable nanoliposomes of appropriate size for therapeutic applications. The formulation presents controlled release at different pH levels under physiological conditions. It also potentiates tarin pharmacological properties, such as inhibition of human glioblastoma U-87 MG and breast cancer MDA-MB-231 cell lines and stimulation of mice bone marrow cells. The liposomal preparation exhibited no toxic effects in healthy mice cells16.
The classical method, first described by Bangham et al.7, allows for the production of large multilamellar liposome vesicles, heterogeneous in size and shape. Adaptations of this method, as reported in the present study, are successfully applied by including additional steps such as sonication and extrusion through a 0.2 µm polycarbonate membrane. This allows production of a more homogeneous dispersion regarding size in the nanometer range16,23,24. Therefore, to ensure successful results, the encapsulation protocol and liposomal formulation described here should be strictly followed.
The nanoliposome composition was carefully selected in order to ensure the formation of a bilayer membrane with DOPE, MPEG 2000-DSPE, and CHEMS as the main constituents. These are natural animal membrane bilayer constituents and the latter can confer fluidity to nanoliposome architecture, ensuring broad application for bioactive compound delivery in human beings.
Nanoliposome pegylation is essential to guarantee liposome structure stability. The absence of PEG leads to size enlargement, a high polydispersity index, and low entrapment efficiency. Optimal results can be obtained with DOPE as the main liposome component. However, this is a high-cost phospholipid. The financial costs of nanoliposome production can be achieved by replacing DOPE with other similar lipids such as DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). CHEMS is a cholesterol molecule naturally found in animal cell membranes, which should not be excluded from the formulation, since it is important to ensure lipid bilayer fluidity and malleability16.
Other aspects of the encapsulation protocol can also be adapted. The chloroform used to dissolve the liposomal components can easily be replaced by methanol with no effects on size average, homogeneity, and entrapment efficiency. However, some protein leakage can occur at storage under 4 °C16. The overnight incubation step with ammonium sulfate solution containing tarin is not mandatory; however, for convenience it can be performed with no damage to nanoliposomal biophysical characteristics, encapsulation, or stability efficiency losses, as demonstrated by Correa et al.16. The extrusion step is performed at room temperature, which can decrease flow rate between the syringes if a 0.1 µm pore size membrane is used.
To overcome this issue, use of a 0.2 µm pore size membrane or heating of the extruder holder above the lipid transition temperature should be considered. The analyst must be careful not to damage the lipids or protein that can be inactivated and lose biological activity. Alternatively, liposomal preparation can be dialyzed against HBS instead of ultracentrifugation, using a cut-off membrane according to protein molecular weight. The choice of chemical nature of the buffer in which nanoliposomes are suspended after ultracentrifugation is directly related to its subsequent application. Since perspectives of this study include in vivo and in vitro assays, suspension in HEPES buffered saline was adequate to ensure no cytotoxic effects and a pH range close to physiological conditions.
Liposomes should be finely treated, similar to living cells, to obtain higher quality SEM images. Fixation and drying procedures are important to ensure the visualization of smaller intact vesicles that support values higher than 20 kV under vacuum conditions. Figure 2A,B displays nanosized vesicles compatible with the extrusion procedure. Visualization of vesicles ranging from 51-396 nm is possible if adequate sample preparation following this procedure is performed. The steps include fixation, drying by increasing ethanol concentrations, and chemical dehydration to avoid the formation of aggregates and ruptured vesicles caused by the vacuum and electron beam. On the other hand, Figure 2C,D shows liposome vesicles dried under room temperature and not subjected to any treatments described here, which means that they were prepared inadequately. As a result of the inadequate procedure, giant vesicles are formed, even after extrusion through a 0.2 µm pore size membrane. Ruptured vesicles are also observed in both panels as a result of vacuum and electron beam damage.
Nanoliposome vesicles have been explored as an encapsulation and delivery system for hydrophobic molecules, including resveratrol (3,5,4'-trihydroxystilbene), a bioactive compound against colorectal cancer cells. The encapsulation procedure can overcome the poor solubility of lipophilic compounds in addition to providing biocompatibility, biodegradability, non-immunogenicity, and non-toxicity characteristics inherent to liposome nanocapsules25. Protocol adaptations must be taken into consideration depending on the administration route and purpose, such as the development of new liposome formulations for oral administration.
The authors are thankful to the COPPE/UFRJ, Electronic Microscopy Laboratory, and Multiuser Materials Characterization Laboratory facilities; to Dr. Adalberto Vieyra, Dr. Jennifer Lowe, and Rafael Lindoso, professors at Universidade Federal do Rio de Janeiro, UFRJ, Brazil, for use of the ultracentrifuge; to Dr. Alexandre Guedes Torres and Daniel Perrone, professors at the Universidade Federal do Rio de Janeiro, UFRJ, Brazil, for use of the rotary evaporator; to Professor Roland Bodmeier and Dr. Andriy Dashevskiy from Freie Universität in Berlin, who helped with resources, provided new methodologies, and supervised ACNTF during a 6 month Erasmus+ fellowship in Germany; to Dr. Rossana Thiré and Aline Fernandes, professor and technician at the Universidade Federal do Rio de Janeiro, UFRJ, Brazil, for use of Zetasizer Malvern; to Bluma Guenther and Taissa Rodrigues, professor and technician at the Universidade Federal do Rio de Janeiro, UFRJ, Brazil, for use of the SEM; to Dr. Rachel Ann Hauser Davis, researcher at Fundação Oswaldo Cruz, for narration. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES) - Finance Code 001 (grant No. 1627392; 1811605); by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (grant No. E-26/202.815/2018; E-26/202.815/2018; E-26/203.039/2015 and E-26/202.860/2016); by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant No. 406601/2018-6), and Financiadora de Estudos e Projetos (FINEP).
Name | Company | Catalog Number | Comments |
Ammonium Sulfate | Sigma-Aldrich Co | A4418 | |
Analitycal Ballance Mettler H10Tw | Mettler Inc. | 417870 | |
Beckman DU-640 Spectrophotometer | Beckman Coulter | 8043-30-1090 | |
Bovine serum albumin (BSA) | Sigma-Aldrich Co | 5470 | |
BUCHI Rotavapor R-300 Rotary Evaporator with Controller and V-300 Pump | Thermo Fischer Scientific | 05-001-022PM | |
CHEMS (cholesterylhemisuccinate) | Sigma-Aldrich Co | C6512 | |
Chloroform | Sigma-Aldrich Co | 48520-U | CAUTION |
Copper (II) Sulfate (Pentahydrate) | Sigma-Aldrich Co | 209198 | |
Coverslips (13mm diameter) | Thermo Scientific Nunc | EW-01839-00 | |
DOPE(1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine) | Lipoid GMBH | 565600.1 | |
Ethanol Absolute | Sigma-Aldrich Co | 32205 | |
Folin -Ciocalteu phenol reagent | Sigma-Aldrich Co | F9252 | |
Glutaraldehyde | Sigma-Aldrich Co | G5882 | |
HEPES | Sigma-Aldrich Co | H3375 | |
Hexamethyldisilazane (HMDS) | Sigma-Aldrich Co | 440191 | CAUTION |
JEOL JSM-6460 LV Sacnning Electron Microscope | JEOL LTD | ||
Mini Extruder 7 | Avanti Polar Lipids | 610000 | |
MPEG 2000-DSPE 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) | Lipoid GMBH | 588200.1 | |
Optima L-90k Ultracentrifuge | Beckman Coulter | PN LL-IM-12AB | |
Phosphate Buffer | Sigma-Aldrich Co | P3619 | |
Poli-L-lysine | Sigma-Aldrich Co | P8920 | |
Potassium L-tartrate monobasic | Sigma-Aldrich Co | 243531 | |
Sodium Carbonate | Sigma-Aldrich Co | S7795 | |
Sodium chloride | Sigma-Aldrich Co | S7653 | |
Sodium Deoxycholate (DOC) | Sigma-Aldrich Co | D6750 | |
Sodium Dodecyl Sulfate | Sigma-Aldrich Co | L3771 | |
Sodium Hydroxide | Sigma-Aldrich Co | S8045 | |
Sodium phosphate dibasic anhydrous | Sigma-Aldrich Co | RES20908-A7 | |
TESCAN VEGA 3 Scanning Electron Microscope | Tescan | #657874 | |
Trichloroacetic Acid (TCA) | Sigma-Aldrich Co | 91230 | |
Zetasizer Nano ZSP | Malvern Panalytical LTD | ||
Ultrasonic cleaning bath model 2510 | Branson |
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