Name: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles (Video) | JoVE Journal
Uploaded: 2016-08-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Learn to prepare and characterize lipophilic doxorubicin prodrug micelles using film dispersion methods. This protocol enhances drug loading, stability, and tumor targeting for improved cancer nanomedicine applications.

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5 minutes

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>

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</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, nanoparti...

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

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...

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 ...

The overall goal of this protocol is to describe procedures for preparing and characterizing lipophilic doxorubicin prodrug loaded micell formulations. This method provides a simple and reliable approach for micell formulation. The synthesis of lipophilic prodrug of doxorubicin can enhance drug loading and stability, thus addressing a significant challenge in developing micell formulation.The micell formulation can enhance drug targeting into tumors and therefore can reduce toxicities of cancer treatment. In addition to the application described in this project, this technical can also be used to prepare the nanomedicine for the agents. To synthesize lipophilic doxorubicin prodrug DOX-PA, weigh 390 milligrams of doxorubicin and 243 milligrams of palmitic acid hydrozide, and transfer them to a round bottom flask.Add 150 milliliters of anhydrous methanol to the flask with a glass syringe. Then add 39 microliters of trifluoroacetic acid, or TFA, with a pipette. Using a magnetic stirrer, stir the reaction mixture for 18 hours at room temperature in the dark.To purify DOX-PA using a silica gel column, first remove the solvent in the reaction mixture with a rotary evaporator. Add three grams of silica gel after the volume of the mixture is reduced to around 20 milliliters. Continue rotary evaporation to yield dry powders and to allow the absorption of products onto the silica gel.Keep the sample under a vacuum for an additional 30 minutes after the dry powders are formed. Pack 50 grams of silica gel into a column using dichloromethane as a solvent. Carefully add the silica gel sample containing absorbed product to the column.Elute the column with a mixture of dichloromethane and methanol while gradually increasing the percentage of methanol, thereby increasing solvent polarity. Collect fractions of elluent in test tubes at 25 milliliters per tube, and monitor the progress by thin layer chromatography, or TLC. 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 overnight. To prepare micelles, dissolve 40 milligrams of DSPE-PEG and four milligrams of DOX-PA with two milliliters of methanol in a 10 milliliter glass vial. Remove the organic solvent under a vacuum using a rotary evaporator, until a thin film forms in the vial.Next, transfer two milliliters of Dulbecco's phosphate buffered saline, pH 7.4 to the glass vial. Select an ultrasonic bath that can generate enough ultrasonic power to disperse the thin polymer drug film. The output power of the ultrasonic bath used in this protocol is 110 watts.Place the vial in an ultrasonic bath for three minutes at room temperate to generate micelles. Keep micelles at four degrees Celsius for short term storage, and minus 20 degrees celsius for long term storage. Once micelles are prepared, perform various characterizations to determine drug concentration in micelles, particle sizes, and in vitro anticancer activities.To determine the DOX-PA concentration and drug encapsulation efficiency, dilute 25 microliters of drug loaded micelles with 500 microliters of DMSO. Measure the absorption at 490 nanometers with a UV-VIS spectrometer, and calculate drug concentrations and encapsulation efficiency as described in the text protocol. To evaluate in vitro anticancer activity, add the diluted cell suspensions into a 96 well cell culture plate at 100 microliters per well.Incubate the cells in a cell culture incubator for 18 hours to allow cell attachment. Next, dilute a DOX DMSO solution and a DOX-PA DMSO solution with cell culture medium to obtain final drug concentrations. Keep the final DMSO concentration in all samples at 0.5%Then dilute DOX-PA micelles with cell culture medium to obtain final drug concentrations.Use the blank cell culture medium as a control. Remove the 96 well cell culture plate from the incubator and replace the cell culture medium with 100 microliters of medium containing different treatment agents. Incubate the cells in the cell culture incubator for an additional 72 hours.Next, aspirate the medium and add 100 microliters of medium containing 0.5 milligrams per milliliter of MTT. Incubate the cells in the cell culture incubator for an additional two hours. Carefully remove the medium and add 100 microliters of DMSO to dissolve formazan crystals.Measure absorbence with the microplate spectrophotometer at a wavelength of 570 nanometers and a reference wavelength of 670 nanometers. Finally, calculate the cell viability as described in the text protocol. The synthesis scheme of DOX-PA is shown here.Palmitic acid is conjugated to doxorubicin through a pH sensitive hydrozine linker. The structure of DOX-PA was further confirmed with a proton NMR spectrum. Characteristic NMR peaks from both doxorubicin and palmitic acid are observed.Letters indicate peaks, and their corresponding moiety within the structure. Blank DSPE-PEG micelles without drug appear as a transparent liquid, whereas DOX-PA DSPE-PEG micelles appear as a red liquid, which is due to the DOX-PA loaded in the micelles. The mean particle size for blank DSPE-PEG micelles is around 17.0 nanometers.The loading of DOX-PA increased micell particle size slightly, with a mean particle size for DOX-PA DSPE-PEG micelles of around 25.7 nanometers. A concentration dependent reduction in cell viability was achieved by treating DU145 cells with free doxorubicin, free DOX-PA, or DOX-PA micelles. Although free doxorubicin is more effective than free DOX-PA or DOX-PA micelles at lower concentrations, DOX-PA groups showed greater reduction in cell viability than free doxorubicin at higher concentrations.Also, there appear to be no significant difference between DOX-PA and DOX-PA micelles at both higher and lower concentrations. After watching this video, you should have a good understanding of how to prepare the micell formulations with a film dispersion method. It's important to remember that the method described can be used to prepare micell with different drugs and carrier materials.The design of carrier material is critical to improve the formulation performance. Following this procedure, the micell formulation can be further evaluated in vivo with animal tumor models to test the anticancer activity.

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

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.

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

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.

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

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.

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

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.

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

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.

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

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

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.

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

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.

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

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.

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

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.

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

Learn to prepare and characterize lipophilic doxorubicin prodrug micelles using film dispersion methods. This protocol enhances drug loading, stability, and tumor targeting for improved cancer nanomedicine applications.

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

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