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 Universita&#776;t in Berlin, who helped with resources, provided new methodologies, and supervised ACNTF during a 6 month Erasmus+ fellowship in Germany; to Dr. Rossana Thir&#233; 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 Funda&#231;&#227;o Oswaldo Cruz, for narration. This study was financed in part by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior, Brasil (CAPES) - Finance Code 001 (grant No. 1627392; 1811605); by Funda&#231;&#227;o Carlos Chagas Filho de Amparo &#224; 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 Cient&#237;fico e Tecnol&#243;gico (CNPq) (grant No. 406601/2018-6), and Financiadora de Estudos e Projetos (FINEP).

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).
<ol>
	<li>Weigh the liposome components using an analytical balance, as shown in Table 1.</li>
	<li>Dissolve the lipid components in chloroform using a 250 mL volumetric flask that fits in a rotary evaporator to avoid loss of material.</li>
	<li>Stir the mixture at 150 rpm for 15 min.</li>
	<li>Remove the chloroform using a rotary evaporator under the following conditions:
	<ol>
		<li>Adjust the volumetric flask mouth to the standard position (25&#176;) for optimal efficiency, while in contact with the water from the heating bath. 
		NOTE: The equipment arm should be inclined at 25&#176; to maintain contact between the volumetric flask and water bath, while not affecting evaporation efficiency or damaging the sample. The standard position can vary according to the equipment brand.</li>
		<li>Set the condenser temperature to a minimum of 3 &#176;C.</li>
		<li>Set the heating bath temperature to 40 &#177; 1 &#176;C.</li>
		<li>Set the rotation to 120 rpm.</li>
		<li>Adjust the vacuum to 207 mbar and boiling point to 20 &#176;C.</li>
		<li>After ~25 min, remove the flask and discard the evaporated solvent, remaining in the condenser, appropriately. 
		NOTE: A thin and opaque film, consisting of the liposome components, is formed in this step and can be easily visualized. The evaporated solvent remaining in the condenser must be stored into disposal recipients (chlorinated) to be handled by a specialized company for appropriate discarding.</li>
	</ol>
	</li>
	<li>Hydrate the lipid film to reach a 0.01 M lipid concentration in 0.3 M ammonium sulfate solution (pH = 7.4) containing tarin at 1 mg/mL to a final volume of 10 mL.
	<ol>
		<li>Stir the mixture for 40 min and incubate overnight at 4 &#176;C. 
		NOTE: This step can be considered a stop point. Overnight incubation is not obligatory.</li>
	</ol>
	</li>
	<li>After incubation, sonicate the suspension for 1 min at 25 &#176;C (room temperature; RT) to reduce vesicle size and avoid aggregation. 
	NOTE: Size reduction was performed in an ultrasonic sonicator under the following conditions: 130 W and 40 kHz.</li>
	<li>Perform a 12-cycle extrusion through a 0.2 &#181;m polycarbonate pore membrane. 
	NOTE: Before the extrusion process, test the mini-extruder assembly using water to avoid sample leakage. A 0.1 &#181;m pore membrane is also suitable. In this case, pre-heat the mini-extruder holder above the lipid transition temperature to facilitate extrusion, while maintaining the physicochemical characteristics of both lipids and proteins.
	<ol>
		<li>Fit the parts of the mini extruder, as described in the manufacturer&#39;s manual.</li>
		<li>Place the polycarbonate membrane between two pre-wet filter supports and place it into the holder.</li>
		<li>Insert a 1 mL empty syringe into the device, fill the other syringe to its total volume with the liposomal suspension, and insert it on the opposite side.</li>
		<li>Perform a 12-cycle extrusion through a 0.2 &#181;m polycarbonate pore membrane. Push the sample from one syringe to another, slowly. Collect the extruded suspension in a pre-cooled tube. 
		NOTE: The polycarbonate membrane should be replaced only when sample transference from one syringe to another becomes difficult. The liposomal suspension should become clear during the extrusion process as a result of size reduction due to the formation of SUVs. About 0.2 mL of the sample can be lost during this step.</li>
	</ol>
	</li>
	<li>Separate liposomes by ultracentrifugation. 
	NOTE: Maintain the samples in an ice bath until the ultracentrifuge is ready to be used. Separate SUVs from the remaining components and ammonium sulfate by ultracentrifugation using an ultracentrifuge with a swing-bucket rotor (see details below).
	<ol>
		<li>Weigh the sample in the titanium tube that fits the rotor and balance the tubes. Check the minimum volume required according to the rotor used to avoid damaging the tubes, and adjust the liposomal suspension volume with ammonium sulphate, if necessary. 
		NOTE: The swing bucket should always be supported on the stand when outside of the centrifuge to avoid scratching the &#34;zebra stripes&#34; (i.e., the black and white stripes on the bottom), which are used by the centrifuge to determine rotation speed.</li>
		<li>Turn on the vacuum before using the centrifuge to allow it to refrigerate.</li>
		<li>Fit the titanium tubes into the swing bucket. 
		NOTE: Lift the tubes to the position they assume when running to ensure that they are perfectly fitted.</li>
		<li>Release the vacuum, open the centrifuge door, and place the rotor inside. 
		NOTE: Pay attention to the circle mark on the bottom of the rotor, which must fit in the opposite direction of the same circle mark into the centrifuge itself.</li>
		<li>Close the centrifuge door, press Vacuum and wait until the vacuum reaches from 200 to &#60;20 microns or from 26 Pa to &#60;3 Pa.</li>
		<li>Adjust the parameters in the ultracentrifuge display to 150,000 x g (the equivalent to 29,600 rpm for the aforementioned swing bucket) for 90 min at 4 &#176;C (acceleration: max, deceleration: max). 
		NOTE: Always convert the speed to x g if the specific centrifuge is set in rpm. Use the centrifuge website to convert the unit according to the rotor used.</li>
		<li>Press Recall, check the conditions, and press Start to run. 
		NOTE: Wait until the centrifuge reaches the desired speed.</li>
		<li>After 90 min, release the vacuum by pressing the Vacuum button and open the centrifuge door when the vacuum reaches 200-700 microns (equivalent to 26-93 Pa).</li>
		<li>Switch off the centrifuge, remove the rotor from the inside, and leave it on the bench with the buckets to dry.</li>
		<li>Maintain the ultracentrifuged samples on ice.</li>
	</ol>
	</li>
	<li>Carefully, separate supernatant from the pellet by turning the tube upside down into a disposable 15 mL centrifuge tube to separate the supernatant and pellet. 
	NOTE: Store the supernatant containing the unencapsulated protein at 4 &#176;C. It will be used to determine the encapsulation efficiency. The pellet appears as a translucent jelly.</li>
	<li>Suspend the pellet containing the encapsulated protein in HEPES buffered saline (3 mL of 1x HBS). 
	NOTE: The HBS 2x (stock solution) is prepared by diluting the following amounts of reagents in distilled water: 140 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, then adjusting the pH to 7.4 and final volume to 100 mL. Na2HPO4 can be replaced by NaHCO3or omitted to avoid interference if liposome concentration is to be determined. The HBS 2x stock solution should be diluted in distilled water to obtain HBS 1x before use.</li>
</ol>
2. Encapsulation efficiency
NOTE: Determine the encapsulation efficiency using Peterson&#39;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.
<ol>
	<li>Preparation of stock reagents and working solutions17
	<ol>
		<li>For stock reagents, prepare copper-tartrate-carbonate (CTC) by mixing 10 mL of 20% sodium carbonate, 200 &#181;L of 0.1% copper sulfate, 200 &#181;L of 0.2% potassium tartrate with 9.6 mL of distilled water. Prepare 100 mL of 10% sodium dodecyl sulfate (SDS) and 0.8 N sodium hydroxide (NaOH).</li>
		<li>For working solutions, prepare 10 mL of 0.15% sodium deoxycholate (DOC) and 72% trichloroacetic acid (TCA). Dissolve 10 mg of bovine serum albumin (BSA) in 10 mL of distilled water to obtain a 1 mg/mL standard solution. Prepare reagent A by adding equal parts of CTC, NaOH, SDS, and H2O. Prepare reagent B by diluting Folin-Ciocalteu phenol reagent 1:5 in distilled water. 
		NOTE: Reagent A requires 1 mL for each reaction tube, while reagent B requires 0.5 mL. To determine the final volumes of reagents A and B, first define the number of reaction tubes to be used, considering three distinct concentrations of BSA, blank, and samples in triplicate. Reagent A must be well-homogenized before use and can be stored at 25 &#176;C (RT) for 2 weeks. Reagent B is also stable at 25 &#176;C (RT) if stored in an amber bottle.</li>
	</ol>
	</li>
	<li>Precipitation 
	NOTE: This step is performed in microcentrifuge tubes.
	<ol>
		<li>Dilute the liposome supernatant with water to a final volume of 1 mL containing 5-100 &#181;g of protein. 
		NOTE: The blank tube should be filled with 1 mL of distilled water.</li>
		<li>Add 0.1 mL of 0.15% DOC, homogenize by vortexing, and incubate for 10 min at RT.</li>
		<li>Add 0.1 mL of 72% TCA, mix well, and centrifuge at 3,000 x g and RT for 15 min. 
		NOTE: DOC-TCA promotes protein precipitation, forming two distinct phases. The target protein can be recovered by centrifugation, avoiding lipid interference.</li>
		<li>Carefully discard the supernatant by verting the tube downwards and laying it on an absorbent paper. Save the pellet for the subsequent step. 
		NOTE: The pellet can be very difficult to see, but the tube should be turned upside down even if it is not visible.</li>
	</ol>
	</li>
	<li>Spectrophotometry
	<ol>
		<li>Suspend the pellet obtained from step 2.2.4 in 1 mL of distilled water. Mix thoroughly to make sure the pellet is dissolved and transfer the sample to a new test tube.</li>
		<li>Prepare dilutions of albumin (BSA) standards to a final volume of 1 mL. 
		NOTE: Protein standards must be prepared between 5-100 &#181;g/mL.</li>
		<li>Add 1 mL of reagent A to the tubes from step 2.3.1 and 2.3.2 without exception, mix well, and incubate for 10 min at RT. 
		NOTE: SDS can relieve possible lipid interferences while aiding in protein solubilization.</li>
		<li>Add 0.5 mL of reagent B to the tubes from step 2.3.1 and 2.3.2, mix well, and incubate for 30 min at RT while protected from the light. 
		NOTE: The Follin-Ciocalteu phenol reagent is a mixture of phosphomolybdate and phosphotungstate used for colorimetric assays of some nitrogen-containing compounds, such as proteins. Copper complexation increases the reactivity of phenols towards this reagent, producing a blue/purple complex according to protein concentration.</li>
		<li>Determine absorbances at 750 nm using a spectrophotometer.</li>
		<li>Calculate the protein concentration in the supernatant based on the standard curve as follows.
		<ol>
			<li>Plot absorbance value (Abs) vs. BSA concentration (mg/mL) to obtain the angular coefficient (k) considering a linear tendency line.</li>
			<li>Determine the supernatant protein concentration (C) by the ratio between the absorbance value and angular coefficient (k), then multiply by the total volume as follows: 
			<img alt="Equation 1" src="/files/ftp_upload/59900/59900eq1.jpg" /></li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Determine encapsulation efficiency according to the following formula: 
	<img alt="Equation 2" src="/files/ftp_upload/59900/59900eq2.jpg" /></li>
	<li>where loaded protein = 10 mg, nonencapsulated protein = value of C obtained in step 2.3.6.2. 
	NOTE: In this case, a total of 10 mg tarin dissolved in ammonium sulfate solution (1 mg/mL) is used to perform the encapsulation procedure, since this concentration is sufficient to obtain satisfactory in vitro effects13,16,18.</li>
</ol>
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 &#176;C and check size distribution and size average regularly.
<ol>
	<li>Turn on the DLS equipment 30 min before use to warm up the laser lamp.</li>
	<li>Transfer the liposomal preparation obtained in step 1.10 to a disposable sizing cuvette.</li>
	<li>Set the equipment parameters as follows: dispersant type = water (RI = 1.33); material = lipids (RI = 1.45); and RT.</li>
	<li>Press Start and wait while the equipment finishes its reading.</li>
	<li>Remove the cuvette and turn off the equipment. 
	NOTE: Either transfer the sample from the cuvette back to the disposable 15 mL centrifuge tube for subsequent analyses, or discard it if there is a sufficient amount for new reads.</li>
</ol>
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.
<ol>
	<li>Fix the glass coverslips (13 mm diameter) in the bottom of a Petri dish with double-sided tape. Cut the tape into small pieces (appropriate size to fix the coverslips), remove the protective paper underneath, and fix it on the bottom of the Petri dish. With the aid of tweezers, remove the protective paper on top of the tape and fix the coverslips on it. 
	NOTE: Be careful in the following steps not to release the coverslips, and use a strong tape.</li>
	<li>Coat the coverslips with poly-L-lysine. Place wet filter papers inside the Petri dish to maintain moisture and incubate for 1 h at RT (25 &#176;C).</li>
	<li>After coating, rinse the coverslips with distilled water.</li>
	<li>Fill the coverslips with a drop of the sample from step 1.10 and allow them to dry for 1 h at RT.</li>
	<li>To fix the samples, cover them with 4% glutaraldehyde prepared in 0.1 M phosphate buffer, pH = 7.2. Place wet filter papers inside the Petri dish and seal the dish to maintain moisture levels. Incubate at 4 &#176;C for 48 h.</li>
	<li>Rinse the coverslips 3x for 5 min with the same phosphate buffer.</li>
	<li>Dehydrate samples as follows: 35% ethanol 1x for 15 min, 50% ethanol 1x for 15 min, 75% ethanol 1x for 15 min, 95% ethanol 2x for 15 min, and absolute ethanol 3x for 20 min.</li>
	<li>Chemically dry the samples by immersion 2x in 1&#8722;2 mL of hexamethyldisilazane (HMDS) for 10 min. 
	NOTE: The HMDS should be manipulated carefully inside a fume hood. Samples should be allowed to dry overnight inside a desiccator or inside the fume hood at RT.</li>
	<li>Mount the dried samples on a stub with a carbon conductive adhesive tape.</li>
	<li>Sputter the surface of the coverslip in a vacuum with an electrically conductive layer (20 nm thickness) of gold-palladium.</li>
	<li>Record images with a scanning electron microscope (SEM) at low vacuum mode and low voltage (20 kV).</li>
</ol>

<ol>
	<li>Wang, X., Wang, Y., Chen, Z. G., Shin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+of+cancer+therapy+by+nanotechnology.">Advances of cancer therapy by nanotechnology.</a> Cancer Research and Treatment: Official Journal of Korean Cancer Association. 41 (1), 1 (2009).</li><li>Keller, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+in+nutrition.">Liposomes in nutrition.</a> Trends in Food Science &amp; Technology. 12 (1), 25-31 (2001).</li><li>Fr&#233;zard, F., Schettini, D. A., Rocha, O. G., Demicheli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipossomas:+propriedades+f&#237;sico-qu&#237;micas+e+farmacol&#243;gicas,+aplica&#231;&#245;es+na+quimioterapia+&#224;+base+de+antim&#244;nio.">Lipossomas: propriedades f&#237;sico-qu&#237;micas e farmacol&#243;gicas, aplica&#231;&#245;es na quimioterapia &#224; base de antim&#244;nio.</a> Quimica Nova. 28 (3), 511-518 (2005).</li><li>Ferreira, D. d. S., Lopes, S. C. d. A., Franco, M. S., Oliveira, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-sensitive+liposomes+for+drug+delivery+in+cancer+treatment.">pH-sensitive liposomes for drug delivery in cancer treatment.</a> Therapeutic Delivery. 4 (9), 1099-1123 (2013).</li><li>Papachristos, A., Pippa, N., Ioannidis, K., Sivolapenko, G., Demetzos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+forms+of+anticancer+agents+beyond+anthracyclines:+present+and+future+perspectives.">Liposomal forms of anticancer agents beyond anthracyclines: present and future perspectives.</a> Journal of Liposome Research. 25 (2), 166-173 (2015).</li><li>Akbarzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome:+classification,+preparation,+and+applications.">Liposome: classification, preparation, and applications.</a> Nanoscale Research Letters. 8 (1), 102 (2013).</li><li>Bangham, A., Standish, M. M., Watkins, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+univalent+ions+across+the+lamellae+of+swollen+phospholipids.">Diffusion of univalent ions across the lamellae of swollen phospholipids.</a> Journal of Molecular Biology. 13 (1), 238-252 (1965).</li><li>Szoka, F., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+preparation+of+liposomes+with+large+internal+aqueous+space+and+high+capture+by+reverse-phase+evaporation.">Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation.</a> Proceedings of the National Academy of Sciences of the United States of America. 75 (9), 4194-4198 (1978).</li><li>Batzri, S., Korn, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+bilayer+liposomes+prepared+without+sonication.">Single bilayer liposomes prepared without sonication.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 298 (4), 1015-1019 (1973).</li><li>Zumbuehl, O., Weder, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+of+controllable+size+in+the+range+of+40+to+180+nm+by+defined+dialysis+of+lipid/detergent+mixed+micelles.">Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid/detergent mixed micelles.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 640 (1), 252-262 (1981).</li><li>Szebeni, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+and+denaturation+of+hemoglobin+encapsulated+in+liposomes.">Oxidation and denaturation of hemoglobin encapsulated in liposomes.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 798 (1), 60-67 (1984).</li><li>Coelho, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+systems:+Advanced+technologies+potentially+applicable+in+personalized+treatments.">Drug delivery systems: Advanced technologies potentially applicable in personalized treatments.</a> EPMA Journal. 1 (1), 164-209 (2010).</li><li>Pereira, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+the+lectin+from+taro+(Colocasia+esculenta)+and+its+effect+on+mouse+splenocyte+proliferation+in+vitro+and+in+vivo.">Purification and characterization of the lectin from taro (Colocasia esculenta) and its effect on mouse splenocyte proliferation in vitro and in vivo.</a> The Protein Journal. 33 (1), 92-99 (2014).</li><li>Pereira, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+and+binding+properties+of+isoforms+of+tarin,+the+GNA-related+lectin+from+Colocasia+esculenta.">Structural analysis and binding properties of isoforms of tarin, the GNA-related lectin from Colocasia esculenta.</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1854 (1), 20-30 (2015).</li><li>Pereira, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structures+of+Colocasia+esculenta+tarin+lectin.">High-resolution crystal structures of Colocasia esculenta tarin lectin.</a> Glycobiology. 27 (1), 50-56 (2016).</li><li>Correa, A., Vericimo, M. A., Dashevskiy, A., Pereira, P. R., Paschoalin, V. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+Taro+Lectin+Nanocapsules+Control+Human+Glioblastoma+and+Mammary+Adenocarcinoma+Cell+Proliferation.">Liposomal Taro Lectin Nanocapsules Control Human Glioblastoma and Mammary Adenocarcinoma Cell Proliferation.</a> Molecules. 24 (3), 471 (2019).</li><li>Peterson, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplification+of+the+protein+assay+method+of+Lowry+et+al.+which+is+more+generally+applicable.">A simplification of the protein assay method of Lowry et al. which is more generally applicable.</a> Analytical Biochemistry. 83 (2), 346-356 (1977).</li><li>Merida, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarin+stimulates+granulocyte+growth+in+bone+marrow+cell+cultures+and+minimizes+immunosuppression+by+cyclo-phosphamide+in+mice.">Tarin stimulates granulocyte growth in bone marrow cell cultures and minimizes immunosuppression by cyclo-phosphamide in mice.</a> PLoS ONE. 13 (11), e0206240 (2018).</li><li>Murtey, M. D., Ramasamy, P., Janecek, M., Kral, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+Preparations+for+Scanning+Electron+Microscopy+-+Life+Sciences.">Sample Preparations for Scanning Electron Microscopy - Life Sciences.</a> Modern Electron Microscopy in Physical and Life Sciences. , (2016).</li><li>Andrade, C. A., Correia, M. T., Coelho, L. C., Nascimento, S. C., Santos-Magalh&#227;es, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+of+Cratylia+mollis+lectin+encapsulated+into+liposomes.">Antitumor activity of Cratylia mollis lectin encapsulated into liposomes.</a> International Journal of Pharmaceutics. 278 (2), 435-445 (2004).</li><li>dos Santos Ferreira, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+bone-targeted+pH-sensitive+liposomal+formulation+containing+doxorubicin:+physicochemical+characterization,+cytotoxicity,+and+biodistribution+evaluation+in+a+mouse+model+of+bone+metastasis.">Development of a bone-targeted pH-sensitive liposomal formulation containing doxorubicin: physicochemical characterization, cytotoxicity, and biodistribution evaluation in a mouse model of bone metastasis.</a> International Journal of Nanomedicine. 11, 3737 (2016).</li><li>Pereira, P. R., Corr&#234;a, A. C. N. T. F., Vericimo, M. A., Paschoalin, V. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarin,+a+Potential+Immunomodulator+and+COX-Inhibitor+Lectin+Found+in+Taro+(Colocasia+esculenta).">Tarin, a Potential Immunomodulator and COX-Inhibitor Lectin Found in Taro (Colocasia esculenta).</a> Comprehensive Reviews in Food Science and Food Safety. 17 (4), 878-891 (2018).</li><li>Olson, F., Hunt, C., Szoka, F., Vail, W., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 557 (1), 9-23 (1979).</li><li>Mui, B., Chow, L., Hope, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+technique+to+generate+liposomes+of+defined+size.">Extrusion technique to generate liposomes of defined size.</a> Methods in Enzymology. 367, 3 (2003).</li><li>Soo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+delivery+and+cytotoxicity+of+resveratrol+through+a+dual+nanoencapsulation+approach.">Enhancing delivery and cytotoxicity of resveratrol through a dual nanoencapsulation approach.</a> Journal of Colloid and Interface Science. 462, 368-374 (2016).</li></ol>

The authors have nothing to disclose.

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 &#181;m 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 &#176;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 &#181;m pore size membrane is used.
To overcome this issue, use of a 0.2 &#181;m 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 &#181;m 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&#39;-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 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&#39;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 &#181;m), ii) large unilamellar vesicles (LUVs, ~0.2-1.0 &#181;m), and iii) giant unilamellar vesicles (GUVs, &#62;1 &#181;m); or 2) multilamellar vesicles (MLVs, &#62;0.1 &#181;m)3,12. Vesicle size is an important parameter when considering for therapeutic use, such as in cancer treatment, in which sizes of &#60;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&#160;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.

<table><tbody><tr><td>Ammonium Sulfate</td><td>Sigma-Aldrich Co</td><td>A4418</td><td></td></tr><tr><td>Analitycal Ballance Mettler H10Tw</td><td>Mettler Inc.</td><td>417870</td><td></td></tr><tr><td>Beckman DU-640 Spectrophotometer</td><td>Beckman Coulter</td><td>8043-30-1090</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich Co</td><td>5470</td><td></td></tr><tr><td>BUCHI Rotavapor R-300 Rotary Evaporator with Controller and V-300 Pump</td><td>Thermo Fischer Scientific</td><td>05-001-022PM</td><td></td></tr><tr><td>CHEMS (cholesterylhemisuccinate)</td><td>Sigma-Aldrich Co</td><td>C6512</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich Co</td><td>48520-U</td><td>CAUTION</td></tr><tr><td>Copper (II) Sulfate (Pentahydrate)</td><td>Sigma-Aldrich Co</td><td>209198</td><td></td></tr><tr><td>Coverslips (13mm diameter)</td><td>Thermo Scientific Nunc</td><td>EW-01839-00</td><td></td></tr><tr><td>DOPE(1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine)</td><td>Lipoid GMBH</td><td>565600.1</td><td></td></tr><tr><td>Ethanol Absolute</td><td>Sigma-Aldrich Co</td><td>32205</td><td></td></tr><tr><td>Folin -Ciocalteu phenol reagent</td><td>Sigma-Aldrich Co</td><td>F9252</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich Co</td><td>G5882</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich Co</td><td>H3375</td><td></td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Sigma-Aldrich Co</td><td>440191</td><td>CAUTION</td></tr><tr><td>JEOL JSM-6460 LV Sacnning Electron Microscope</td><td>JEOL LTD</td><td></td><td></td></tr><tr><td>Mini Extruder 7</td><td>Avanti Polar Lipids</td><td>610000</td><td></td></tr><tr><td>MPEG 2000-DSPE 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt)</td><td>Lipoid GMBH</td><td>588200.1</td><td></td></tr><tr><td>Optima L-90k Ultracentrifuge</td><td>Beckman Coulter</td><td>PN LL-IM-12AB</td><td></td></tr><tr><td>Phosphate Buffer</td><td>Sigma-Aldrich Co</td><td>P3619</td><td></td></tr><tr><td>Poli-L-lysine</td><td>Sigma-Aldrich Co</td><td>P8920</td><td></td></tr><tr><td>Potassium L-tartrate monobasic</td><td>Sigma-Aldrich Co</td><td>243531</td><td></td></tr><tr><td>Sodium Carbonate</td><td>Sigma-Aldrich Co</td><td>S7795</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich Co</td><td>S7653</td><td></td></tr><tr><td>Sodium Deoxycholate (DOC)</td><td>Sigma-Aldrich Co</td><td>D6750</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich Co</td><td>L3771</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich Co</td><td>S8045</td><td></td></tr><tr><td>Sodium phosphate dibasic anhydrous</td><td>Sigma-Aldrich Co</td><td>RES20908-A7</td><td></td></tr><tr><td>TESCAN VEGA 3 Scanning Electron Microscope</td><td>Tescan</td><td>#657874</td><td></td></tr><tr><td>Trichloroacetic Acid (TCA)</td><td>Sigma-Aldrich Co</td><td>91230</td><td></td></tr><tr><td>Zetasizer Nano ZSP</td><td>Malvern Panalytical LTD</td><td></td><td></td></tr><tr><td>Ultrasonic cleaning bath model 2510</td><td>Branson</td><td></td><td></td></tr></tbody></table>

preparation and characterization of nanoliposomes for the entrapment of bioactive hydrophilic globular proteins

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.

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&#8722;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 &#181;m 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 &#181;m and analyzed at 5 kV were observed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59900/59900fig1.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59900/59900fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59900/59900fig2.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59900/59900fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Liposome Components</td>
			<td>Weight (g)</td>
			<td>Concentration (mM)</td>
			<td>Final volume</td>
		</tr>
		<tr>
			<td>DOPE</td>
			<td>0.0420</td>
			<td>5.7</td>
			<td rowspan="3">10 mL</td>
		</tr>
		<tr>
			<td>MPEG 2000-DSPE</td>
			<td>0.1059</td>
			<td>3.8</td>
		</tr>
		<tr>
			<td>CHEMS</td>
			<td>0.0024</td>
			<td>0.5</td>
		</tr>
	</tbody>
</table>
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 &#8211; cholesterylhemisuccinate.
Table 1: Preparation of tarin liposomal nanocapsules.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Membrane pore size (&#181;m)</td>
			<td>Size distribution (nm)</td>
			<td>Average size (nm)</td>
			<td>Polydispersity index (PdI)</td>
			<td>Peak (nm)</td>
			<td>Entrapment Efficiency</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>51 - 396</td>
			<td>155</td>
			<td>0. 168</td>
			<td>94 &#177; 39</td>
			<td>0.83</td>
		</tr>
	</tbody>
</table>
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.

Watch this Scientific Journal Video about Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins at JoVE.com

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

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 ...

preparation-characterization-nanoliposomes-for-entrapment-bioactive

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

23.5K Views.  Federal University of Rio de Janeiro. 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.

Video: Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Chemistry

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Biochemistry

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Summary

Explore More Videos

Chapters in this video

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform