Name: Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles (Video) | JoVE Journal
Uploaded: 2015-08-28T13:00:00.000+00:00
Duration: 7 min 32 s
Description: Learn to prepare and characterize drug-loaded polymeric micelles using the solvent casting method for enhanced drug delivery applications. This protocol enables reproducible micelle generation with stable loading and controlled release characteristics.

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5 minutes

This study was supported by the grant from AACP New Pharmacy Faculty Research Award Program, Medical Research Foundation of Oregon New Investigator Grant, Oregon State University-Startup fund, and Pacific University, School of Pharmacy Start-up fund.

1. Preparation of Individual and Multi-drug Loaded Micelles by Solvent Casting Method
<ol>
	<li>Weigh out DTX 1 mg or EVR 1 mg or both drugs at 1 mg each for the dual drug micelles (DDM).</li>
	<li>Weigh out 15 mg of PEG2000-b-PLA1800 or PEG4000-b-PLA2200 for either individual or DDM.</li>
	<li>Dissolve the drug/ drugs and the polymer in 0.5 ml of acetonitrile and place in a 5 ml round bottom flask.</li>
	<li>Form a thin drug distributed polymer film by evaporating the drug(s)-polymer acetonitrile solution under reduced pressure using a rotary evaporator. Set the rotary evaporator to 100 rpm, the water bath temperature of 40 oC and a vacuum pressure of 260 mbar for 5 min followed by a reduction to 100 mbar for 3 more min.</li>
	<li>Rehydrate the drug-polymer film with 0.5 ml of deionized water at 50 oC and gently shake the flask to form the micelles.</li>
	<li>Filter the resulting micellar solution through a 0.2 &#181;m nylon filter to remove any un-dissolved drug or contaminants into a 1.5 ml centrifuge tube.</li>
</ol>
2. Assessment of Drug Loading and Stability in Micelles Using Reverse-phase High Performance Liquid Chromatography (RP-HPLC)
<ol>
	<li>Perform RP-HPLC analysis with a C8 column equilibriated at 40 &#176;C in an isocratic mode with a mobile phase of acetonitrile/water (62/38) containing 0.1% phosphoric acid and 1% methanol at a flow rate of 1 ml/min and an injection volume of 10 &#181;l.</li>
	<li>Dilute freshly prepared micelles (section 1) 1:100 in mobile phase prior to analyzing by RP-HPLC to determine initial drug loading. Store undiluted individual micelles and DDM at room temperature (25 oC) for 48 hr and prepare fresh 1:100 diluted samples in mobile phase to re-assess by RP-HPLC and determine drug(s) stability in micelles over 24 hr.</li>
	<li>Monitor DTX and EVR peaks at 227 and 279 nm respectively with retention times of 1.7 and 5.7 min respectively. Perform all measurements in triplicate. Present data as Mean &#177; SD drug loading.</li>
</ol>
3. Assessment of micelle Particle Size by Dynamic Light Scattering (DLS)
<ol>
	<li>Dilute freshly prepared micelles (as described in section 1) in deionized water at a ratio of 1:20 to yield a final polymer concentration of 1.5 mg/ml.</li>
	<li>Measure the intensity of He-Ne laser (633 nm) at 173&#176; to determine scattering. Perform all measurements at 25 &#176;C following pre-equilibration for 2 min.</li>
	<li>Perform all measurements in triplicate. Present data as the Mean Z-average size &#177; SD along with the polydispersity index (PDI) of the distribution.</li>
</ol>
4. Assessment of In Vitro Drug Release from Individual Micelles and DDM
<ol>
	<li>Prepare individual micelles and DDM as described in section 1. Load 2.5 ml of the micelles into a 3 ml dialysis cassette with a molecular weight cut-off (MWCO) of 7,000 g/mol. 
	NOTE: This MWCO was chosen to enable the free drug(s) along with the unassociated polymer molecules to diffuse freely out of the cassette and thereby ensure sink conditions.</li>
	<li>Place the cassettes in 2.5 L of 10 mM pH 7.4 phosphate buffer (prepared by diluting stock 200 mM solution) and change the buffer every 3 hr to ensure sink conditions. Maintain the temperature of the buffer at 37 oC throughout the duration of the experiment.</li>
	<li>At 0, 0.5, 1, 2, 3, 6, 9, 12, 24, and 48 hr, withdraw 150 &#181;l of the solution in the cassettes and replace with 150 &#181;l of fresh buffer.</li>
	<li>Analyze the samples using RP-HPLC as established in section 2 to determine the drug concentration. Curve-fit the drug(s) release data based on a simple diffusion model with a one phase exponential association using stastitical software.</li>
	<li>Calculate the time needed to reach 50% of drug release (t1/2) of each drug in individual micelles or DDM based on the curve fitting. Perform all measurements in quadruplet.</li>
</ol>

<ol><li>Yokoyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymeric+micelles+as+a+new+drug+carrier+system+and+their+required+considerations+for+clinical+trials%5BTitle%5D)+AND+%22Expert+Opin+Drug+Deliv%22%5BJournal%5D)">Polymeric micelles as a new drug carrier system and their required considerations for clinical trials.</a> Expert Opin Drug Deliv. 7, 145-158 (2010).</li>
<li>Shin, H. C., Alani, A. W., Rao, D. A., Rockich, N. C., Kwon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-drug+loaded+polymeric+micelles+for+simultaneous+delivery+of+poorly+soluble+anticancer+drugs%5BTitle%5D)+AND+%22J+Control+Release%22%5BJournal%5D)">Multi-drug loaded polymeric micelles for simultaneous delivery of poorly soluble anticancer drugs.</a> J Control Release. 140, 294-300 (2009).</li>
<li>Adams, M. L., Lavasanifar, A., Kwon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amphiphilic+block+copolymers+for+drug+delivery%5BTitle%5D)+AND+%22J+Pharm+Sci%22%5BJournal%5D)">Amphiphilic block copolymers for drug delivery.</a> J Pharm Sci. 92, 1343-1355 (2003).</li>
<li>Oerlemans, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymeric+micelles+in+anticancer+therapy%3A+targeting%2C+imaging+and+triggered+release%5BTitle%5D)+AND+%22Pharm+Res%22%5BJournal%5D)">Polymeric micelles in anticancer therapy: targeting, imaging and triggered release.</a> Pharm Res. 27, 2569-2589 (2010).</li>
<li>Shin, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+3-in-1+polymeric+micelle+nanocontainer+for+poorly+water-soluble+drugs%5BTitle%5D)+AND+%22Mol+Pharm%22%5BJournal%5D)">A 3-in-1 polymeric micelle nanocontainer for poorly water-soluble drugs.</a> Mol Pharm. 8, 1257-1265 (2011).</li>
<li>Hasenstein, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antitumor+activity+of+Triolimus%3A+a+novel+multidrug-loaded+micelle+containing+Paclitaxel+Rapamycin%2C+and+17-AAG%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">Antitumor activity of Triolimus: a novel multidrug-loaded micelle containing Paclitaxel Rapamycin, and 17-AAG.</a> Mol Cancer Ther. 11, 2233-2242 (2012).</li>
<li>Mazzaferro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bivalent+sequential+binding+of+docetaxel+to+methyl-beta-cyclodextrin%5BTitle%5D)+AND+%22Int+J+Pharm%22%5BJournal%5D)">Bivalent sequential binding of docetaxel to methyl-beta-cyclodextrin.</a> Int J Pharm. 416, 171-180 (2011).</li>
<li>Iwase, Y., Maitani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+and+in+vivo+evaluation+of+liposomal+everolimus+for+lung+carcinoma+and+thyroid+carcinoma%5BTitle%5D)+AND+%22Biol+Pharm+Bull%22%5BJournal%5D)">Preparation and in vivo evaluation of liposomal everolimus for lung carcinoma and thyroid carcinoma.</a> Biol Pharm Bull. 35, 975-979 (2012).</li>
<li>Mishra, G. P., Doddapaneni, B. S., Nguyen, D., Alani, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antiangiogenic+effect+of+docetaxel+and+everolimus+as+individual+and+dual-drug-loaded+micellar+nanocarriers%5BTitle%5D)+AND+%22Pharm+Res%22%5BJournal%5D)">Antiangiogenic effect of docetaxel and everolimus as individual and dual-drug-loaded micellar nanocarriers.</a> Pharm Res. 31, 660-669 (2014).</li>
<li>Xu, W., Ling, P., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymeric+micelles%2C+a+promising+drug+delivery+system+to+enhance+bioavailability+of+poorly+water-soluble+drugs%5BTitle%5D)+AND+%22J+Drug+Deliv%22%5BJournal%5D)">Polymeric micelles, a promising drug delivery system to enhance bioavailability of poorly water-soluble drugs.</a> J Drug Deliv. 2013, 340315(2013).</li>
<li>Lavasanifar, A., Samuel, J., Kwon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poly%28ethylene+oxide%29-block-poly%28L-amino+acid%29+micelles+for+drug+delivery%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Poly(ethylene oxide)-block-poly(L-amino acid) micelles for drug delivery.</a> Adv Drug Deliv Rev. 54, 169-190 (2002).</li>
</ol>

The authors have nothing to disclose.

The use of polymeric micelles for drug delivery continues to expand due to their versatility and ability to deliver hydrophobic drugs for various disease states. Therefore, the techniques needed to prepare and characterize these formulations prior to use in cell culture or animals is a critical first step to determine the best pairing between the drug and the polymer. PEG-b-PLA are excellent amphiphilic block copolymers for drug delivery purposes. However, the block length of the hydrophilic and hydrophobic s...

Amphiphilic block copolymers with repeating structure composed of hydrophilic and hydrophobic domains can spontaneously self-assemble to form three dimensional macromolecular assemblies known as polymeric micelles. These structures have an inner hydrophobic core surrounded by a hydrophilic shell. The hydrophobic core has the ability to incorporate hydrophobic drugs either by physical entrapment through hydrophobic interactions or by chemical conjugation on to the polymer backbone.1 Many advantages exist to using these block copolymers to form micelles for drug delivery. These include incorporation of poorly soluble drugs, improving pharmacokinetics of the incorporated drugs, and the biocompatibility and/or biodegradability of the polymers makes them a safe alternate to conventional solubilizers.2 Another advantage of using polymeric micelles is their colloidal particle size, between 15&#8211;150 nm3, making them attractive for parenteral delivery. Therefore, over the last 20 years polymeric micelles have emerged as viable drug delivery systems for poorly water-soluble drugs especially for cancer therapy.3,4
Currently there are five polymeric micellar formulations for cancer therapy undergoing clinical trials.4 Four of the micelles in the clinical trials are PEG-based diblock copolymers while the last is a triblock copolymer containing polyethyleneoxide. The size of these micelles varied from 20 nm to 85 nm. The advantage of using PEG based polymers is their biocompatibility and depending on the second block can also be biodegradable. Recently new drug delivery systems based on polyethyleneglycol-block-polylactic acid (PEG-b-PLA) polymeric micelles have been developed for the concurrent delivery of multiple anticancer drugs. The PEG-b-PLA micelles are both biocompatible and biodegradable. These multi-drug loaded micelles have shown a synergistic inhibition of different cancers models in vitro and in vivo2,5,6 and fit into the current paradigm of utilizing multiple drugs in chemotherapy to prevent resistance and lowering toxicity. Therefore, there is a great deal of interest in preparing and characterizing these micellar drug delivery systems for use in cancer and other disease states.
In the work below we have outlined a step-by-step process by which such micelles can be prepared and characterized before evaluating them in disease states of interest. For the purpose of this work two poorly-soluble anti-cancer agents, docetaxel (DTX) and everolimus (EVR) have been chosen. Both DTX and EVR are poorly water-soluble compounds with intrinsic water solubilities at 1.9 and 9.6 &#181;g/ml respectively.7,8 Two PEG-b-PLA polymers with different molecular weights were used in this protocol as the building blocks for the formulated polymeric micelles, these polymers are PEG2000-b-PLA1800 (3,800 Da) and PEG4000-b-PLA2200 (6,200 Da). PEG-b-PLA micelles can therefore provide a unique platform as a nanocarrier for DTX and EVR individually and in combination. The required Reagents/Materials and Equipment needed to prepare and characterize these micelles are listed in Table 1.

<table><tbody><tr><td>PEG2000-b-PLA1800</td><td>Advanced Polymer Materials, Inc</td><td>6-01- PLA/2000</td><td>PLA MW can be specified on ordering</td></tr><tr><td>PEG4000-b-PLA2200</td><td>Advanced Polymer Materials, Inc</td><td>6-01- PLA/4000</td><td>PLA MW can be specified on ordering</td></tr><tr><td>Docetaxel</td><td>LC Laboratories</td><td>D-1000</td><td>100 mg</td></tr><tr><td>Everolimus</td><td>LC Laboratories</td><td>E-4040</td><td>100 mg</td></tr><tr><td>Acetonitrile</td><td>EMD/VWR</td><td>EM-AX0145-1</td><td>HPLC grade; 4 L</td></tr><tr><td>Round bottom flask&amp;nbsp;</td><td>Glassco/VWR</td><td>89426-496</td><td>5 ml</td></tr><tr><td>RV 10 Control Rotary Evaporators</td><td>IKA Works</td><td>8025001</td><td>Rotoevaporator</td></tr><tr><td>Shimadzhu HPLC with DAD detector</td><td>Shimadzhu</td><td></td><td>RP-HPLC</td></tr><tr><td>Slide-a-lyzer dialysis casette MWCO 7000</td><td>Thermo Scientific, Inc</td><td>66370</td><td>3 ml</td></tr><tr><td>Phosphate buffer pH 7.4, 200 mM</td><td>VWR</td><td>100190-870</td><td>500 ml</td></tr><tr><td>Malvern NanoZS</td><td>Malvern Instruments, UK</td><td></td><td>DLS</td></tr><tr><td>Nylon filter</td><td>Acrodisc/VWR</td><td>28143-242</td><td>13 mm; 0.2&amp;micro;M</td></tr><tr><td>Phosphoric acid, NF</td><td>Spectrum Chemical/VWR</td><td>700000-626</td><td>100 ml</td></tr><tr><td>GraphPad Prism</td><td>&lt;a href=&quot;http://www.graphpad.com/&quot;&gt;www.graphpad.com&lt;/a&gt;</td><td></td><td>Analysis software</td></tr><tr><td>Zorbax SB-C8 Rapid Resolution cartridge&amp;nbsp;</td><td>Agilent Technologies</td><td>866953-906</td><td>4.6 &amp;times;75 mm, 3.5 &amp;#956;m</td></tr></tbody></table>

preparation and characterization of individual and multi-drug loaded physically entrapped polymeric micelles

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.

Individual DTX or EVR micelles and DTX and EVR DDM in PEG-b-PLA micelles are successfully formulated in either PEG4000-b-PLA2200 or PEG2000-b-PLA1800 (Figure 1).
DTX, EVR, and the DDM showed similar stability in PEG4000-b-PLA2200 or PEG2000-b-PLA1800 over 48 hr (Figure 2). Initial drug loading of EVR in PEG4000-b</...

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 ...

The overall goal of the following experiment is to outline a step-by-step process of preparing and characterizing drug loaded my cells before evaluating them in disease states of interest. This is achieved by preparing the My cells by solvent casting method to generate reproducible my cells for the study. As a second step, my cell loading stability and size are assessed, which determines the ability of the my cells to load and retain clinically relevant concentrations.Next, the rate of drug release from the My cells is determined in order to ensure my cells have appropriate in vitro release characteristics before assessing in vivo stability. The results show that reproducibly generated stable my cells can provide a platform to pursue drug delivery in various disease states based on the assessment techniques presented here. The main advantage of this technique or existing methods like equilibrium loading or dialysis methods, is that the myosins can be prepared much faster with reproducible drug loading and size.The implications of this technique extend toward therapy or diagnosis of various disease states because nanoscale medicines offer opportunities beyond conventional forms, Generally individuals who are just beginning might struggle with these techniques even though they're simple because still you get to do the release studies, you can't really evaluate the burst effect. Demonstrating these procedure will be Shaman Duke, my graduate student, and Deepa my collaborator First weigh out one milligram of DTX, one milligram of EVR and 15 milligrams of Peg 4, 000 block PLA 2200 for the dual drug my cells or DDM. After weighing out, dissolve the drugs and the polymer in 0.5 milliliters of aceto nitrile, then transfer the solution to a five milliliter round bottom flask form a thin drug distributed polymer film by evaporating the drug polymer aceto solution under reduced pressure using a rotary evaporator.Following this rehydrate the drug polymer film with 0.5 milliliters of deionized water preheated at 50 degrees Celsius and gently shake the flask in a 50 degrees Celsius water bath to form the mice cells. Add the solution to a centrifuge tube and spin at 7, 200 times G for three minutes. Draw up the solution with a needle and filter the resulting Micella solution into a new 1.5 milliliter centrifuge tube using a 0.2 micron nylon filter to remove any unsolved drug or contaminants At this point, perform reverse-phase HPLC analysis with a C eight column equilibrated at 40 degrees Celsius in an is Socratic mode with a mobile phase of acetyl I trial and water dilute the freshly prepared mycells in ace eye trial at a one to 100 ratio prior to analyzing by reverse phase HPLC to determine the initial drug loading.After storing undiluted my cells at room temperature for 48 hours, prepare fresh diluted samples to reassess by reverse phase HPLC and determine the drug stability in my cells over 48 hours. Monitor the DTX and DVR peaks at 227 and 279 nanometers with retention times of 1.7 and 5.7 minutes Respectively. Present the data as the mean plus or minus standard deviation of the drug loading.Next, dilute the freshly prepared my cells and deionized water at a one to 20 ratio te a final polymer concentration of 1.5 milligrams per milliliter. Transfer the solution to a vet for dynamic light scattering measurement. Measure the intensity of helium neon laser at 173 degrees to determine the scattering.Present the data as the mean Z average size plus or minus standard deviation along with the poly dispersity index or PDI of the distribution. After preparing DDM as previously described, load 2.5 milliliters of the my cells into a three milliliter dialysis cassette with a molecular weight cutoff of 7, 000 grams per mole. Place the cassette in 2.5 liters of 10 millimolar pH 7.4 phosphate buffer during the following intervals.Withdraw 150 microliters of the solution and replace with 150 microliters of fresh buffer. Change the buffer every three hours to ensure sync conditions. Analyze the samples using reverse phase HPLC to determine the drug concentration curve.Fit the drug release data based on a simple diffusion model with a one phase exponential association using statistical software D-T-X-E-V-R and the DDM showed similar stability in PEG 2000 block PLA 1800 or PEG 2000 block PLA 2200 over 48 hours. All the, my cells retained 97%or more of the initial loading at 48 hours at room temperature. Based on the dynamic light scattering results, all my cells showed a unimodal distribution with PDI values of less than 0.2.Based on the data DTX release from individual mycells and DDM was approximately 60%over 48 hours. EVR release from individual my cells and DDM was 60%and 50%respectively. The time needed for 50%drug release from individual my cells and DDM and the goodness of fit data are presented here.The goodness of curve fitting for all my cells except EVR individual, my cells is above 0.950, which means that the assumption of first order release is a good approximation to explain drug release from individual my cells and DDM Once mastered, these techniques can be done routinely on a library of drugs and polymers as my cells in a matter of hours to week. While attending this procedure, it's important to remember to completely characterize the my cells before accessing the cell culture or the animal models following These procedures. Other delivery system like liposomal delivery system can also be evaluated or characterized in order to answer the the question about the role of drug delivery system in the efficacy of treatment.After watching this video, you should have a good understanding of how to prepare and characterize drug loading my cells before evaluating them in disease. State of interest.

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

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