Name: preparation and characterization of lipophilic doxorubicin pro-drug micelles
Uploaded: 2016-08-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.

1. Synthesis of DOX-PA
<ol>
	<li>Weigh 390 mg of doxorubicin and 243 mg of palmitic acid hydrazide, and transfer to a round bottom flask.</li>
	<li>Add 150 ml of anhydrous methanol to the flask with a glass syringe. Add 39 &#181;l of trifluoroacetic acid (TFA) with a pipette. Using a magnetic stirrer, stir the reaction mixture for 18 hr at RT in the dark. 
	NOTE: The quantities of reaction materials can be scaled up or down to obtain different amounts of DOX-PA. The ratio of reactants should be maintained in the same proportions. Reactions using DOX quantities in the range of 78 mg to 1,170 mg can be performed in a regular chemistry laboratory.</li>
	<li>Purification of DOX-PA using a silica gel column.17
	<ol>
		<li>Remove the solvent in the reaction mixture with a rotary evaporator. Add 3 g of silica gel after the volume of the mixture is reduced to around 20 ml. Continue rotary evaporation to yield dry powders and to allow the adsorption of products onto the silica gel. Keep the sample under a vacuum for an additional 30 min after the dry powders are formed.</li>
		<li>Pack 50 g of silica gel into a column using dichloromethane as a solvent. Carefully add the silica gel sample containing adsorbed product to the column.</li>
		<li>Elute the column with a mixture of dichloromethane and methanol, while gradually increasing the percentage of methanol, thereby increasing solvent polarity (Table 1).</li>
		<li>Collect fractions of eluent in test tubes (25 ml/tube) and monitor the progress by thin layer chromatography (TLC).</li>
		<li>Combine all fractions containing pure DOX-PA and remove the solvent using a rotary evaporator until dry powder is formed. Further dry the product under a vacuum O/N.</li>
	</ol>
	</li>
	<li>Analysis of DOX-PA by TLC.
	<ol>
		<li>Cut a 4 cm x 8 cm section of TLC plate. Spot sample solutions 0.5 cm from the bottom of the plate with TLC spotting capillaries using methanol as the solvent.</li>
		<li>Place the TLC plate into a developing chamber containing a mixture of dichloromethane and methanol (3/1, v/v). The depth of solvent should be just less than 0.5 cm.</li>
		<li>Remove the plate from the developing chamber when the solvent front reaches the top of the plate. Mark the location of the solvent front with a pencil and allow the plate to dry. Place the TLC plate into a staining chamber containing saturated iodine vapor in order to visualize samples.</li>
	</ol>
	</li>
	<li>Analysis of DOX-PA with 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR).18
	<ol>
		<li>Dissolve 15 mg of DOX-PA in 1 ml of methyl sulfoxide-d6 (DMSO) and transfer the sample into an NMR tube.</li>
		<li>Insert the NMR tube into the magnet of the NMR instrument. Measure the proton spectrum, selecting DMSO as a solvent. Remove the NMR tube from the magnet. Analyze the NMR result18.</li>
	</ol>
	</li>
</ol>
2. Preparation of DOX-PA Micelles by Film-dispersion Method
<ol>
	<li>Dissolve DSPE-PEG (40 mg) and DOX-PA (4 mg) with 2 ml of methanol in a 10 ml glass vial.</li>
	<li>Remove the organic solvent under a vacuum using a rotary evaporator until a thin film forms in the vial. 
	NOTE: Alternatively, evaporate the organic solvents under inert gas (e.g., argon or nitrogen gas) to form a film and keep the vial in a vacuum desiccator to further remove residual solvent.</li>
	<li>Transfer 2 ml of Dulbecco&#39;s phosphate buffered saline (pH 7.4, DPBS) to the glass vial.</li>
	<li>Place the vial in an ultrasonic bath for 3 min at RT to generate micelles. 
	NOTE: Ultrasonic power varies among different models of ultrasonic baths. Select a unit which can generate enough ultrasonic power to disperse the thin polymer/drug film. The output power of ultrasonic bath used in this protocol is 110 W.</li>
	<li>Keep micelles at 4 &#176;C for short-term storage and -20 &#176;C for long-term storage. 
	NOTE: Alternatively, micelles can also be freeze-dried and reconstituted with water before use. Usually, no cryoprotectant or lyoprotectant is needed for this formulation.</li>
</ol>
3. Characterization of DOX-PA Micelles
<ol>
	<li>Determination of DOX-PA concentration in micelles and drug encapsulation efficiency
	<ol>
		<li>Dissolve DOX-PA synthesized in previous steps in DMSO to prepare DOX-PA solutions of five different concentrations: 1 &#181;g/ml, 5 &#181;g/ml, 20 &#181;g/ml, 50 &#181;g/ml, and 100 &#181;g/ml. Measure the absorption of DOX-PA solutions with a UV-VIS spectrometer at 490 nm. Generate a standard curve based on the DOX-PA drug concentrations and their corresponding absorption at 490 nm.</li>
		<li>Dilute 25 &#181;l of drug-loaded micelle with 500 &#181;l of DMSO. Measure the absorption at 490 nm with a UV-VIS spectrometer. Calculate drug concentrations with the standard curve generated in 3.1.1.</li>
		<li>Calculate encapsulation efficiency using the following equation: 
		Drug Encapsulation Efficiency (%) = (amount of drugs in micelles)/(amount of added drug) &#215; 100%</li>
	</ol>
	</li>
	<li>Characterization of particle size with dynamic light scattering (DLS)
	<ol>
		<li>Dilute micelles with DPBS (pH 7.4) to a final DSPE-PEG concentration of 1 mg/ml. Analyze a 2 ml sample with a particle size analyzer to obtain the Z-average size and polydispersity index (PDI).</li>
	</ol>
	</li>
	<li>Evaluation of in vitro anticancer activity 
	NOTE: Use appropriate sterile technique and operate inside a biosafety cabinet.
	<ol>
		<li>Remove the cell culture medium from a cell culture flask (e.g., T25) containing DU-145 human prostate cancer cells and rinse the cells with 2 ml of DPBS (pH 7.4).</li>
		<li>Aspirate DPBS, add 1 ml of trypsin solution (0.25%), and incubate for 2 min at 37 &#176;C to detach the cells.</li>
		<li>Add 10 ml of cell culture medium (RPMI 1640 + 10% Fetal Bovine Serum + 1% Antibiotic-Antimycotic), when most of cells are detached from the flask. Transfer cells to a 15-ml centrifuge tube and centrifuge the cells at 1,000 x g for 5 min.</li>
		<li>Re-suspend the cell pellet with 5 ml of cell culture medium and remove a sample for counting cell numbers with a hemocytometer. Dilute the cells with cell culture medium to a density of 50,000 cells/ml. Add the diluted-cell suspensions into a 96-well cell culture plate (100 &#181;l/well). Incubate the cells in a cell culture incubator (37 &#176;C, 5% CO2) for 18 hr to allow cell attachment.</li>
		<li>Dilute DOX dimethyl sulfoxide (DMSO) solution and DOX-PA DMSO solution with cell culture medium to obtain final drug concentrations of 0.1 &#181;M, 0.5 &#181;M, 2 &#181;M, 5 &#181;M, and 10 &#181;M, respectively. Keep final DMSO concentration in all above samples to 0.5%. Dilute DOX-PA micelle with cell culture medium to obtain final drug concentrations of 0.1 &#181;M, 0.5 &#181;M, 2 &#181;M, 5 &#181;M, and 10 &#181;M, respectively. Use the blank cell culture medium a control.</li>
		<li>Remove the 96-well cell culture plate from the incubator and replace the cell culture medium with 100 &#181;l of medium containing different treatment agents prepared in step 3.3.5 (n = 4 for each group). Incubate the cells in the cell culture incubator (37 &#176;C, 5% CO2) for an additional 72 hr.</li>
		<li>Aspirate the medium and add 100 &#181;l of medium containing 0.5 mg/ml of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).</li>
		<li>Incubate the cells in the cell culture incubator for additional 2 hr. Carefully remove the medium and add 100 &#181;l of DMSO to dissolve formazan crystals.</li>
		<li>Measure absorbance with the microplate spectrophotometer at a wavelength of 570 nm and a reference wavelength of 670 nm.</li>
		<li>Calculate the cell viability using the following equation: 
		Cell Viability (%) = (ATest&#160; &#8260;&#160; Acontrol) &#215; 100% 
		NOTE: Compare the cell viability between different groups using a one-way analysis of variance (ANOVA) statistical test. Calculate the IC50 based on cell viability vs. drug concentration data.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hennenfent, K. L., Govindan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+formulations+of+taxanes:+a+review.+Old+wine+in+a+new+bottle?.">Novel formulations of taxanes: a review. Old wine in a new bottle?.</a> ESMO. 17 (5), 735-749 (2006).</li><li>Paliwal, S. R., Paliwal, R., Agrawal, G. P., Vyas, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanomedicine+for+breast+cancer+therapy.">Liposomal nanomedicine for breast cancer therapy.</a> Nanomedicine. 6 (6), 1085-1100 (2011).</li><li>Mahapatro, A., Singh, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanoparticles+are+excellent+vehicle+for+site+directed+in+vivo+delivery+of+drugs+and+vaccines.">Biodegradable nanoparticles are excellent vehicle for site directed in vivo delivery of drugs and vaccines.</a> J Nanobiotechnology. 9 (55), (2011).</li><li>Danquah, M., Li, F., Duke, C. B., Miller 3rd, ., D, D., Mahato, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micellar+delivery+of+bicalutamide+and+embelin+for+treating+prostate+cancer.">Micellar delivery of bicalutamide and embelin for treating prostate cancer.</a> Pharm Res. 26 (9), 2081-2092 (2009).</li><li>Li, F., Danquah, M., Mahato, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+amphiphilic+lipopolymers+for+micellar+drug+delivery.">Synthesis and characterization of amphiphilic lipopolymers for micellar drug delivery.</a> Biomacromolecules. 11 (10), 2610-2620 (2010).</li><li>Li, F., Danquah, M., Singh, S., Hao, W., Mahato, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paclitaxel-+and+lapatinib-loaded+lipopolymer+micelles+overcome+multidrug+resistance+in+prostate+cancer.">Paclitaxel- and lapatinib-loaded lipopolymer micelles overcome multidrug resistance in prostate cancer.</a> Drug Deliv. and Transl. Res. 1 (6), 9 (2011).</li><li>Li, F., Lu, Y., Li, W., Miller, D. D., Mahato, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+formulation+and+in+vitro+evaluation+of+a+novel+microtubule+destabilizer,+SMART-100.">Synthesis, formulation and in vitro evaluation of a novel microtubule destabilizer, SMART-100.</a> J Control Release. 143 (1), 151-158 (2010).</li><li>Minko, T., Kopeckova, P., Pozharov, V., Kopecek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPMA+copolymer+bound+adriamycin+overcomes+MDR1+gene+encoded+resistance+in+a+human+ovarian+carcinoma+cell+line.">HPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line.</a> J Control Release. 54 (2), 223-233 (1998).</li><li>Rosenholm, J. M., Mamaeva, V., Sahlgren, C., Linden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+in+targeted+cancer+therapy:+mesoporous+silica+nanoparticles+entering+preclinical+development+stage.">Nanoparticles in targeted cancer therapy: mesoporous silica nanoparticles entering preclinical development stage.</a> Nanomedicine. 7 (1), 111-120 (2012).</li><li>Kaur, I. P., 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=Issues+and+concerns+in+nanotech+product+development+and+its+commercialization.">Issues and concerns in nanotech product development and its commercialization.</a> J Control Release. 193, 51-62 (2014).</li><li>Jones, M., Leroux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+micelles+-+a+new+generation+of+colloidal+drug+carriers.">Polymeric micelles - a new generation of colloidal drug carriers.</a> Eur J Pharm Biopharm. 48 (2), 101-111 (1999).</li><li>Wang, H., Li, F., Du, C., Mahato, R. I., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+and+lapatinib+combination+nanomedicine+for+treating+resistant+breast+cancer.">Doxorubicin and lapatinib combination nanomedicine for treating resistant breast cancer.</a> Mol Pharm. 11 (8), 2600-2611 (2014).</li><li>Ma, P., Rahima Benhabbour, S., Feng, L., Mumper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2'-Behenoyl-paclitaxel+conjugate+containing+lipid+nanoparticles+for+the+treatment+of+metastatic+breast+cancer.">2'-Behenoyl-paclitaxel conjugate containing lipid nanoparticles for the treatment of metastatic breast cancer.</a> Cancer Lett. 334 (2), 253-262 (2013).</li><li>Lundberg, B. B., Risovic, V., Ramaswamy, M., Wasan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lipophilic+paclitaxel+derivative+incorporated+in+a+lipid+emulsion+for+parenteral+administration.">A lipophilic paclitaxel derivative incorporated in a lipid emulsion for parenteral administration.</a> J Control Release. 86 (1), 93-100 (2003).</li><li>Perche, F., Patel, N. R., Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+and+toxicity+of+antibody-targeted+doxorubicin-loaded+PEG-PE+micelles+in+ovarian+cancer+cell+spheroid+model.">Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell spheroid model.</a> J Control Release. 164 (1), 95-102 (2012).</li><li>Gill, K. K., Kaddoumi, A., Nazzal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+micelles+of+PEG(2000)-DSPE+and+vitamin-E+TPGS+for+concurrent+delivery+of+paclitaxel+and+parthenolide:+enhanced+chemosenstization+and+antitumor+efficacy+against+non-small+cell+lung+cancer+(NSCLC)+cell+lines.">Mixed micelles of PEG(2000)-DSPE and vitamin-E TPGS for concurrent delivery of paclitaxel and parthenolide: enhanced chemosenstization and antitumor efficacy against non-small cell lung cancer (NSCLC) cell lines.</a> Eur J Pharm Sci. 46 (1-2), 67-71 (2012).</li><li>Still, W. C., Kahn, M., Mitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Chromatographic+Technique+for+Preparative+Separations+with+Moderate+Resolution.">Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution.</a> J. Org. Chem. 43 (14), 2923-2925 (1978).</li><li>Morton, L. A., Saludes, J. P., Yin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constant+pressure-controlled+extrusion+method+for+the+preparation+of+Nano-sized+lipid+vesicles.">Constant pressure-controlled extrusion method for the preparation of Nano-sized lipid vesicles.</a> J Vis Exp. (64), (2012).</li><li>Ulbrich, K., Etrych, T., Chytil, P., Jelinkova, M., Rihova, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPMA+copolymers+with+pH-controlled+release+of+doxorubicin:+in+vitro+cytotoxicity+and+in+vivo+antitumor+activity.">HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity.</a> J Controlled Release. 87 (1-3), 33-47 (2003).</li><li>Patil, 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=Cellular+Delivery+of+Doxorubicin+via+pH-Controlled+Hydrazone+Linkage+Using+Multifunctional+Nano+Vehicle+Based+on+Poly(beta-L-Malic+Acid).">Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(beta-L-Malic Acid).</a> Int J Mol Sci. 13 (9), 11681-11693 (2012).</li><li>Hu, X., Liu, S., Huang, Y., Chen, X., Jing, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+block+copolymer-doxorubicin+conjugates+via+different+linkages:+preparation,+characterization,+and+in+vitro+evaluation.">Biodegradable block copolymer-doxorubicin conjugates via different linkages: preparation, characterization, and in vitro evaluation.</a> Biomacromolecules. 11 (8), 2094-2102 (2010).</li><li>Huynh, L., Neale, C., Pomes, R., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+approaches+to+the+rational+design+of+nanoemulsions,+polymeric+micelles,+and+dendrimers+for+drug+delivery.">Computational approaches to the rational design of nanoemulsions, polymeric micelles, and dendrimers for drug delivery.</a> Nanomedicine. 8 (1), 20-36 (2012).</li><li>Shi, C., 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+drug-specific+nanocarrier+design+for+efficient+anticancer+therapy.">A drug-specific nanocarrier design for efficient anticancer therapy.</a> Nat Commun. 6, 7449 (2015).</li></ol>

The authors have nothing to disclose.

In this work, we describe an uncomplicated, rapid film-dispersion method for the preparation of micelles. This method utilizes the self-assembly properties of an amphiphilic polymer (e.g., DSPE-PEG) to form core-shell structured micelles in an aqueous environment. This micelle preparation method has several advantages. 1. It involves a simple formulation process, which avoids the use of complicated size-reduction steps (such as extrusion or homogenization) commonly used in the preparation of liposomes, nanoparticles, and nanoemulsions.19 2. It has a good reproducibility. Excellent batch-to-batch consistency can be achieved once the formulation has been optimized and established. The protocol is tolerant to variations in preparation process variables, such as sonication strength and time. Although other methods (such as the dialysis approach or the emulsion-solvent evaporation approach) are available for preparing micelles, the film dispersion method is more convenient and efficient. Therefore, it has a great potential to be adapted for use in large-scale manufacturing of micelles in the pharmaceutical industry. The limitation of this method is that it depends on the self-assembly properties of carrier materials and thus is restricted to materials with such properties. The inappropriate selection of polymer materials may lead to failure in generating satisfactory micelle formulations. In addition, the preparation method relies on ultrasonication to disperse the polymer/drug film and to facilitate the formation of micelles. Most commercially available ultrasonic baths are suitable because only a weak ultrasonic power is needed to form micelles. However, it might be a problem if an ultrasonic bath with a very weak power is used.
Poor drug loading and premature drug release are major concerns for micelle drug delivery. In this protocol, we developed a lipophilic pro-drug strategy to enhance the loading of doxorubicin into DSPE-PEG micelles. Conjugation of doxorubicin with lipids significantly improved compatibility and interaction of the drug with the lipid core of DSPE-PEG micelles. The pro-drug, DOX-PA, was synthesized by conjugating doxorubicin with lipid through an acidic pH-responsive hydrazone linker. This linker is stable at neutral pH and cleavable at acidic pH.20,21 Thus, DOX is stably loaded in DSPE-PEG micelles as a pro-drug at neutral pH and has minimal drug leakage during storage and during circulation in the blood. Once DOX-PA micelles enter tumor cells through endocytosis, DOX-PA pro-drug will be cleaved and converted into free DOX in response to the acidic endosome environment (pH 5-6). Since DOX is more hydrophilic than DOX-PA, this conversion will disrupt the interactions between DOX and the hydrophobic core of micelles, and thus facilitate the quick release of DOX from micelles. This innovative design feature will avoid incomplete drug release, which is a major problem associated with drug conjugates using non-cleavable or slow-cleaving linkers. 22 This approach may also be applied to the delivery of other drugs. The selection of an appropriate linker is critical for the success of this approach. The linkers should be stable in a physiological environment in order to maximize the stability of the formulation. The linkers should also be efficiently cleaved in response to triggers in the tumor microenvironment (e.g., pH, enzymes, etc.) to release the parent drug.
In this protocol, DOX-PA was efficiently loaded into micelles with high encapsulation efficiency (~ 100%). The micelle formulation also demonstrated superior stability and remained intact for at least three weeks without drug precipitation or reduction in drug concentration. This is due to the enhanced interaction between lipid moieties in the pro-drug and the lipid core of DSPE-PEG micelles. Engineering the compatibility of carrier molecules with drugs to enhance their interaction is a promising strategy for improving the performance of micelles and other nanocarriers. This approach can enhance drug loading and minimize pre-mature drug release for these nanocarriers.23 The selection of an appropriate drug/polymer pair is a critical step in designing the micelle formulation. The structure of drug molecules can be modified (pro-drug approach) or the design of carrier materials can be optimized in order to improve compatibility and to enhance drug/carrier interactions.2324 In addition, computational modeling can be a useful tool to assist in the optimization of nanocarriers and make it possible to prepare a tailor-designed nanocarrier for specific a specific drug.
In summary, we describe here a method to synthesize a lipophilic pro-drug of doxorubicin. Protocols for the preparation and characterization of drug loaded micelles are also described. The film-dispersion method is a simple and promising method for preparing a variety of nanoscale self-assembly delivery systems. The characterization methods described here can be used as standard in vitro assays to determine the properties of nanomedicines, thus can facilitate the optimization of formulation and product development processes.

Chemotherapy is commonly used to treat various forms of cancers. Most, if not all, chemotherapy drugs have toxic side effects which may vary from manageable minor conditions, such as nausea and diarrhea, to more life threatening conditions. Because most anticancer drugs are toxic, non-selective exposure of these drugs to normal tissue inevitably causes toxicity. Therefore, there is a great need for a therapeutic approach that can selectively deliver drugs into cancer cells. Another challenge with the administration of anticancer drugs is their poor water solubility. Usually, solubilizing agents are needed to formulate these poorly soluble drugs. However, most solubilizing agents, such as dimethyl sulfoxide (DMSO), Cremophor EL, and Polysorbate 80 (Tween 80) may cause liver and kidney toxicity, hemolysis, acute hypersensitivity reactions and peripheral neuropathies.1 Therefore, safe and biocompatible formulations are needed for the clinical use of poorly soluble anticancer drugs. Nanocarriers are promising drug delivery systems for addressing the above challenges. These nanocarriers include liposomes,2 nanoparticles,3 micelles,4-7 polymer-drug conjugates,8 and inorganic materials.9 Several nanomedicine products (e.g., Doxil, Abraxane, and Genexol) have been approved by the regulatory agencies to treat cancer patients.10
Polymeric micelles are promising nano-scale drug delivery carriers, which have been successfully used for the delivery of anticancer drugs.4-7,11,12 Typical polymeric micelles are prepared from amphiphilic polymers through a self-assembly process. The core-shell structured polymeric micelles include a hydrophilic shell and a hydrophobic core. The hydrophilic shell can sterically stabilize micelles and prolong their circulation in blood stream. The hydrophobic core can effectively encapsulate hydrophobic drugs. Because of the small size of micelles (typically less than 200 nm) and long-circulation properties, polymeric micelles are believed to achieve tumor targeting through enhanced permeability and retention (EPR) effects (passive tumor targeting).
Drug loading stability is critical for the tumor targeting ability of micelles. To achieve optimal tumor targeting, micelles should have minimal drug leakage before reaching the tumor site, yet quickly release the drug after entering cancer cells. In addition, formulation stability is also an essential requirement for product development, because formulation stability determines the feasibility of product development, as well as the shelf-life of developed products. Recently, much effort has been made to improve the loading of drugs into delivery carriers. The lipophilic pro-drug approach is a strategy which has been explored to improve drug loading into lipid nanoparticles and emulsions.13,14 The conjugation of lipids with drugs can significantly improve their lipophilicity and enhance loading and retention in the lipophilic components of nanocarriers.
Here, we describe a protocol for preparing lipophilic doxorubicin pro-drug loaded micelles. First, the synthesis procedure for doxorubicin lipophilic pro-drug is described. Then, a protocol for generating micelles with a film-dispersion method is introduced. This method has been successfully used in our previous studies.5 DSPE-PEG was selected as the carrier material for preparing micelles because it has been successfully used for micelle drug delivery.15,16 Finally, we describe several in vitro assays used to characterize micelle formulations and to evaluate anticancer activity.

<table><tbody><tr><td>DSPE-PEG2K</td><td>Cordenpharm</td><td>LP-R4-039</td><td>&gt;95%</td></tr><tr><td>Doxorubicin</td><td>LC Laboratories</td><td>D-4000</td><td>&gt;99%</td></tr><tr><td>Palmitic Acid Hydrazide</td><td>TCI AMERICA&amp;nbsp;&amp;nbsp;</td><td>P000425G</td><td>&gt;98.0%</td></tr><tr><td>Methanol</td><td>ACROS Organics</td><td>610981000</td><td>Anhydrous</td></tr><tr><td>Methylene chloride&amp;nbsp;</td><td>FISHER&amp;nbsp;</td><td>D151-4</td><td>99.90%</td></tr><tr><td>Methyl sulfoxide-d&lt;sub&gt;6&lt;/sub&gt;</td><td>ACROS Organics</td><td>AC320760075</td><td>NMR solvent</td></tr><tr><td>Trifluoroacetic Acid&amp;nbsp;</td><td>ACROS Organics</td><td>AC293811000</td><td>99.50%</td></tr><tr><td>Silica Gel</td><td>FISHER&amp;nbsp;</td><td>L-7446</td><td>230-400 mesh</td></tr><tr><td>Baker Flex TLC Plates</td><td>FISHER&amp;nbsp;</td><td>NC9990129</td><td></td></tr><tr><td>DPBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>DU 145&amp;nbsp; Prostate Cancer Cells</td><td>ATCC</td><td>HTB-81</td><td></td></tr><tr><td>MTT</td><td>ACROS Organics</td><td>158990050</td><td>98%</td></tr><tr><td>RPMI 1640 Medium</td><td>MEDIATECH INC&amp;nbsp;</td><td>10041CV</td><td></td></tr><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>LIFE TECHNOLOGIES&amp;nbsp;</td><td>15240062</td><td>100x stock solution</td></tr><tr><td>Fetal Bovine Serum</td><td>LIFE TECHNOLOGIES&amp;nbsp;</td><td>10437077</td><td></td></tr><tr><td>Nuclear Magnetic Resonance Spectroscopy</td><td>Varian, Inc</td><td></td><td>300 NMR&amp;nbsp;</td></tr><tr><td>B&amp;uuml;chi R-3 Rotavapor</td><td>Buchi</td><td>1103022V1&amp;nbsp;</td><td>Rotary evaporator</td></tr><tr><td>Ultrasonic Bath</td><td>BRANSON ULTRASONICS CORPORATION&amp;nbsp;</td><td>CPX952318R</td><td></td></tr><tr><td>UV-VIS spectrometer Biomate 3</td><td>Thermo Spectronic</td><td></td><td></td></tr><tr><td>Zetasizer Nano ZS90&amp;nbsp;</td><td>Malvern Instruments</td><td></td><td>Particle Size Analyer</td></tr><tr><td>Microplate Spectrophotometer&amp;nbsp;</td><td>Rio-Rad</td><td>Benchmark Plus&amp;nbsp;</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Napco</td><td>CO2 6000</td><td></td></tr><tr><td>Biological Safety Cabinet</td><td>Nuaire</td><td></td><td></td></tr><tr><td>SigmaPlot&amp;nbsp;</td><td>Systat Software, Inc.</td><td></td><td>Analytical Software</td></tr><tr><td>96-Well Cell Culture Plate</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Trypsin&amp;nbsp; 0.25%</td><td>Corning Cellgro</td><td>25-053-CI</td><td></td></tr></tbody></table>

preparation and characterization of lipophilic doxorubicin pro-drug micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

Figure 1 shows the synthesis scheme of DOX-PA. DOX-PA was synthesized by conjugation of palmitic acid with doxorubicin through a pH-sensitive hydrazone bond. A slight excess of palmitic acid hydrazide was used to facilitate the completion of the reaction. This reaction method has a very high efficiency and only a small amount of doxorubicin remained after an 18 hr reaction (Figure 2). The yield was approximately 88%. At the end of the reaction, DOX-PA was purified using a silica gel column and dried to obtain a pure red solid product. DOX-PA was analyzed with TLC, which showed a single spot for purified DOX-PA (Figure 2). Figure 3 shows the 1H-NMR spectrum of DOX-PA. Characteristic NMR peaks from both doxorubicin and palmitic acid were observed, which further confirmed success of the conjugation reaction.
The appearance of micelles prepared in this protocol is shown in Figure 4. Blank DSPE-PEG micelles without drug appear as a transparent liquid. DOX-PA DSPE-PEG micelles appear as a red liquid; the red color is due to the DOX-PA loaded in the micelles. Representative results of particle size analysis are shown in Figure 5. The Z-average mean particle size for blank DSPE-PEG micelles was 17.0 &#177; 0.5 nm (PDI = 0.034 &#177; 0.019). The loading of DOX-PA increased micelle particle size slightly; the Z-average mean particle size for DOX-PA DSPE-PEG micelles was 25.7 &#177; 1.6 nm (PDI = 0.407 &#177; 0.035).
The DOX-PA concentration in the micelle formulation was determined based on the absorption at 490 nm. DSPE-PEG has negligible absorption at this wavelength, thus does not interfere in the analysis of DOX-PA concentration. The DOX-PA concentration in the micelle formulation was 1.99 &#177; 0.11 mg/ml and the drug loading efficiency was 99.3 &#177; 5.7%. The high encapsulation efficiency is due to the lipophilic feature of DOX-PA, which enhances its retention in the hydrophobic core of micelles. The formulation showed excellent stability. There was no visible precipitation when stored at - 4 &#176;C for 3 weeks. No significant change in drug concentration was observed during storage.
DU-145 human prostate cancer cells were treated with different concentrations of free doxorubicin, free DOX-PA, and DOX-PA DSPE-PEG micelles. Cell viability was determined at the end of a 72 hr treatment using a MTT assay (Figure 6). A concentration-dependent reduction in cell viability was achieved by treating DU145 cells with free doxorubicin (IC50 = 0.10 &#181;M), free DOX-PA (IC50 = 0.33 &#181;M), or DOX-PA micelles (IC50 = 0.25 &#181;M). Although free doxorubicin is more effective than free DOX-PA or DOX-PA micelles at lower concentrations (0.1 &#181;M and 0.5 &#181;M), DOX-PA groups showed greater reduction in cell viability than free doxorubicin at higher concentrations (2-10 &#181;M). Also, there appeared to be no significant difference between DOX-PA and DOX-PA micelles at both higher and lower concentrations. Blank DSPE-PEG micelles showed no toxicity (data not shown), indicating good biocompatibility and safety of DSPE-PEG as a drug delivery carrier material.
<img alt="Figure 1" src="/files/ftp_upload/54338/54338fig1.jpg" /> 
Figure 1. Synthesis of lipophilic pro-drug of doxorubicin (DOX-PA). Reagents and conditions: doxorubicin (DOX), palmitic acid hydrazide (PA) and trifluoroacetic acid (TFA) dissolved in methanol were stirred for 18 hr at RT in the dark. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54338/54338fig2.jpg" /> 
Figure 2. Thin layer chromatography (TLC). TLC of (1) doxorubicin, (2) raw reaction mixture, (3) palmitic acid hydrazide, and (4) DOX-PA conjugate. Samples were developed with a mixture of dichloromethane and methanol (3/1, v/v) and stained with iodine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54338/54338fig3.jpg" /> 
Figure 3. 1H-NMR of DOX-PA in deuterated DMSO. The presence of characteristic peaks from both DOX and PA demonstrates the success of the conjugation reaction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54338/54338fig4.jpg" /> 
Figure 4. Appearance of micelles. (A) DSPE-PEG micelles and (B) DOX-PA DSPE-PEG micelles. Representative figures are presented to demonstrate the appearance of blank DSPE-PEG micelles and DOX-PA DSPE-PEG micelles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54338/54338fig5.jpg" /> 
Figure 5. Particle size and size distribution of micelles. (A) DSPE-PEG micelles and (B) DOX-PA DSPE-PEG micelles. The micelle size is determined by dynamic light scattering. Representative figures are presented to demonstrate particle size and size distribution. Data presented are Mean &#177; SD (n = 3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54338/54338fig6.jpg" /> 
Figure 6. Anticancer activity of micelle formulations. DU145 cells were treated for 72 hr and cell viability was measured using the MTT assay. Data presented in the figure are Mean &#177; SD (n =4). *, P &#60; 0.01, compared to DOX treated groups. There is no statistical difference between DOX-PA and DOX-PA micelles treated groups. IC50 was calculated based on cell viability vs. drug concentration data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54338/54338table1.jpg" />
Table 1. CH2Cl2/CH3OH eluting solvents used in the purification of DOX-PA.

Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

preparation-characterization-lipophilic-doxorubicin-pro-drug

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.8K Views.  Hampton University. A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

Video: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Cancer Research

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 ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

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.

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 ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...