Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

1. Synthesis of DOX-PA

1. Weigh 390 mg of doxorubicin and 243 mg of palmitic acid hydrazide, and transfer to a round bottom flask.
2. Add 150 ml of anhydrous methanol to the flask with a glass syringe. Add 39 µl 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.
3. Purification of DOX-PA using a silica gel column.¹⁷
   1. 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.
   2. 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.
   3. Elute the column with a mixture of dichloromethane and methanol, while gradually increasing the percentage of methanol, thereby increasing solvent polarity (Table 1).
   4. Collect fractions of eluent in test tubes (25 ml/tube) and monitor the progress by thin layer chromatography (TLC).
   5. 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.
4. Analysis of DOX-PA by TLC.
   1. 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.
   2. 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.
   3. 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.
5. Analysis of DOX-PA with ¹H-Nuclear Magnetic Resonance Spectroscopy (¹H-NMR).¹⁸
   1. Dissolve 15 mg of DOX-PA in 1 ml of methyl sulfoxide-d6 (DMSO) and transfer the sample into an NMR tube.
   2. 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.

2. Preparation of DOX-PA Micelles by Film-dispersion Method

1. Dissolve DSPE-PEG (40 mg) and DOX-PA (4 mg) with 2 ml of methanol in a 10 ml glass vial.
2. 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.
3. Transfer 2 ml of Dulbecco's phosphate buffered saline (pH 7.4, DPBS) to the glass vial.
4. 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.
5. Keep micelles at 4 °C for short-term storage and -20 °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.

3. Characterization of DOX-PA Micelles

1. Determination of DOX-PA concentration in micelles and drug encapsulation efficiency
   1. Dissolve DOX-PA synthesized in previous steps in DMSO to prepare DOX-PA solutions of five different concentrations: 1 µg/ml, 5 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/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.
   2. Dilute 25 µl of drug-loaded micelle with 500 µl 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.
   3. Calculate encapsulation efficiency using the following equation:
      Drug Encapsulation Efficiency (%) = (amount of drugs in micelles)/(amount of added drug) × 100%
2. Characterization of particle size with dynamic light scattering (DLS)
   1. 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).
3. Evaluation of in vitro anticancer activity
   NOTE: Use appropriate sterile technique and operate inside a biosafety cabinet.
   1. 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).
   2. Aspirate DPBS, add 1 ml of trypsin solution (0.25%), and incubate for 2 min at 37 °C to detach the cells.
   3. 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.
   4. 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 µl/well). Incubate the cells in a cell culture incubator (37 °C, 5% CO₂) for 18 hr to allow cell attachment.
   5. Dilute DOX dimethyl sulfoxide (DMSO) solution and DOX-PA DMSO solution with cell culture medium to obtain final drug concentrations of 0.1 µM, 0.5 µM, 2 µM, 5 µM, and 10 µM, 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 µM, 0.5 µM, 2 µM, 5 µM, and 10 µM, respectively. Use the blank cell culture medium a control.
   6. Remove the 96-well cell culture plate from the incubator and replace the cell culture medium with 100 µl 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 °C, 5% CO₂) for an additional 72 hr.
   7. Aspirate the medium and add 100 µl of medium containing 0.5 mg/ml of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).
   8. Incubate the cells in the cell culture incubator for additional 2 hr. Carefully remove the medium and add 100 µl of DMSO to dissolve formazan crystals.
   9. Measure absorbance with the microplate spectrophotometer at a wavelength of 570 nm and a reference wavelength of 670 nm.
   10. Calculate the cell viability using the following equation:
       Cell Viability (%) = (A_Test  ⁄  A_control) × 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.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
DSPE-PEG2K	Cordenpharm	LP-R4-039	>95%
Doxorubicin	LC Laboratories	D-4000	>99%
Palmitic Acid Hydrazide	TCI AMERICA	P000425G	>98.0%
Methanol	ACROS Organics	610981000	Anhydrous
Methylene chloride	FISHER	D151-4	99.90%
Methyl sulfoxide-d6	ACROS Organics	AC320760075	NMR solvent
Trifluoroacetic Acid	ACROS Organics	AC293811000	99.50%
Silica Gel	FISHER	L-7446	230-400 mesh
BAKER FLEX TLC PLATES	FISHER	NC9990129
DPBS	Sigma-Aldrich	D8537
DU 145  Prostate Cancer Cells	ATCC	HTB-81
MTT	ACROS Organics	158990050	98%
RPMI 1640 Medium	MEDIATECH INC	10041CV
Antibiotic-Antimycotic	LIFE TECHNOLOGIES	15240062	100x stock solution
Fetal Bovine Serum	LIFE TECHNOLOGIES	10437077
Nuclear Magnetic Resonance Spectroscopy	Varian, Inc		300 NMR
Büchi R-3 Rotavapor	Buchi	1103022V1	Rotary evaporator
Ultrasonic Bath	BRANSON ULTRASONICS CORPORATION	CPX952318R
UV-VIS spectrometer Biomate 3	Thermo Spectronic
Zetasizer Nano ZS90	Malvern Instruments		Particle Size Analyer
Microplate Spectrophotometer	Rio-Rad	Benchmark Plus
Cell Culture Incubator	Napco	CO2 6000
Biological Safety Cabinet	Nuaire
SigmaPlot	Systat Software, Inc.		Analytical Software
96-Well Cell Culture Plate	Becton Dickinson	353072
Trypsin  0.25%	Corning Cellgro	25-053-CI