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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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 border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">DSPE-PEG2K</td>
			<td style="width:191px;">Cordenpharm</td>
			<td style="width:119px;">LP-R4-039</td>
			<td style="width:167px;">&#62;95%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Doxorubicin</td>
			<td style="width:191px;">LC Laboratories</td>
			<td style="width:119px;">D-4000</td>
			<td>&#62;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Palmitic Acid Hydrazide</td>
			<td style="width:191px;">TCI AMERICA&#160;&#160;</td>
			<td style="width:119px;">P000425G</td>
			<td>&#62;98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">610981000</td>
			<td>Anhydrous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene chloride&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">D151-4</td>
			<td>99.90%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl sulfoxide-d6</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC320760075</td>
			<td>NMR solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic Acid&#160;</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC293811000</td>
			<td>99.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silica Gel</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">L-7446</td>
			<td>230-400 mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BAKER FLEX TLC PLATES&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">NC9990129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPBS</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DU 145&#160; Prostate Cancer Cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:119px;">HTB-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">158990050</td>
			<td>98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium</td>
			<td style="width:191px;">MEDIATECH INC&#160;</td>
			<td style="width:119px;">10041CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic-Antimycotic&#160;</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">15240062</td>
			<td>100x stock solution</td>
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			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
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			<td style="width:191px;">Varian, Inc</td>
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			<td>300 NMR&#160;</td>
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			<td height="21" style="height:21px;width:216px;">B&#252;chi R-3 Rotavapor</td>
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			<td style="width:191px;">BRANSON ULTRASONICS CORPORATION&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Zetasizer Nano ZS90&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Microplate Spectrophotometer&#160;</td>
			<td style="width:191px;">Rio-Rad</td>
			<td style="width:119px;">Benchmark Plus&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
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			<td style="width:119px;">CO2 6000</td>
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			<td style="width:191px;">Systat Software, Inc.</td>
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			<td>Analytical Software</td>
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			<td height="21" style="height:21px;width:216px;">96-Well Cell Culture Plate</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">353072</td>
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			<td style="width:119px;">25-053-CI</td>
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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...

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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

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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

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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 ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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 ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...