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

Summary

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.

Abstract

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 µg/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.

Introduction

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 15–150 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 vivo^2,5,6 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 µg/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.

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Protocol

1. Preparation of Individual and Multi-drug Loaded Micelles by Solvent Casting Method

1. Weigh out DTX 1 mg or EVR 1 mg or both drugs at 1 mg each for the dual drug micelles (DDM).
2. Weigh out 15 mg of PEG₂₀₀₀-b-PLA₁₈₀₀ or PEG₄₀₀₀-b-PLA₂₂₀₀ for either individual or DDM.
3. Dissolve the drug/ drugs and the polymer in 0.5 ml of acetonitrile and place in a 5 ml round bottom flask.
4. 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.
5. Rehydrate the drug-polymer film with 0.5 ml of deionized water at 50 ^oC and gently shake the flask to form the micelles.
6. Filter the resulting micellar solution through a 0.2 µm nylon filter to remove any un-dissolved drug or contaminants into a 1.5 ml centrifuge tube.

2. Assessment of Drug Loading and Stability in Micelles Using Reverse-phase High Performance Liquid Chromatography (RP-HPLC)

1. Perform RP-HPLC analysis with a C8 column equilibriated at 40 °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 µl.
2. 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.
3. 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 ± SD drug loading.

3. Assessment of micelle Particle Size by Dynamic Light Scattering (DLS)

1. 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.
2. Measure the intensity of He-Ne laser (633 nm) at 173° to determine scattering. Perform all measurements at 25 °C following pre-equilibration for 2 min.
3. Perform all measurements in triplicate. Present data as the Mean Z-average size ± SD along with the polydispersity index (PDI) of the distribution.

4. Assessment of In Vitro Drug Release from Individual Micelles and DDM

1. 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.
2. 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.
3. At 0, 0.5, 1, 2, 3, 6, 9, 12, 24, and 48 hr, withdraw 150 µl of the solution in the cassettes and replace with 150 µl of fresh buffer.
4. 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.
5. Calculate the time needed to reach 50% of drug release (t_1/2) of each drug in individual micelles or DDM based on the curve fitting. Perform all measurements in quadruplet.

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Results

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

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Discussion

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...

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Materials

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