NANO LETTERS
Diacyllipid-Polymer Micelles as Nanocarriers for Poorly Soluble Anticancer Drugs
2002 Vol. 2, No. 9 979-982
Zhonggao Gao,† Anatoly N. Lukyanov,† Anurag Singhal, and Vladimir P. Torchilin* Department of Pharmaceutical Sciences, BouVe College of Health Sciences, Northeastern UniVersity, Boston, Massachusetts 02115 Received May 8, 2002; Revised Manuscript Received July 29, 2002
ABSTRACT Highly stable micelles with the size of 10 to 40 nm can be prepared from poly(ethylene glycol)/phosphatidyl ethanolamine (PEG−PE) conjugates. These micelles easily solubilize and firmly retain substantial quantities of various poorly soluble anticancer drugs (m-porphyrin, tamoxifen, taxol). The micelles with encapsulated drugs have the size and size distribution very close to that of original “empty” micelles. Stable drugloaded polymeric micelles may represent a convenient drug delivery system into tumors utilizing the enhanced permeability and retention (EPR) effect.
Micelles are colloidal particles with the size in a nanometer range, into which many amphiphilic molecules self-assemble spontaneously. In an aqueous environment, hydrophobic fragments of amphiphilic molecules form the core of a micelle, which is segregated from the environment by hydrophilic parts of the molecules forming the micelle corona. It was clearly shown on multiple occasions that micelles possess a number of unbeatable advantages as potential drug delivery systems for poorly soluble pharmaceuticals.1,2 The hydrophobic core of micelles may be used as a cargo space for encapsulation of a variety of sparingly soluble therapeutic and diagnostic agents. Such encapsulation substantially increases their bioavailability, protects them from destructive factors upon parenteral administration, and beneficially modifies their pharmacokinetics and biodistribution.1,2 The size of micelles permits their extravasation and accumulation in a variety of pathological sites, where the permeability of the vascular endothelium is increased, such as infarct zones3 and tumors.4-6 This fact provides a unique opportunity of physiology-based targeting of drugs and/or drug-loaded pharmaceutical carriers, such as micelles, to these pathological areas via the EPR (or “passive” targeting) effect.7,8 An additional advantage of micelles as drug carriers from the practical point of view is that they are easy to prepare on a large scale. * Corresponding author: Dr. Vladimir P. Torchilin, Department of Pharmaceutical Sciences, Bouve College of Health Sciences, 312 Mugar Bldg, Northeastern University, Boston, MA 02115. Tel: 617-373-3206; Fax: 617-373-8886; E-mail:
[email protected]. † ZG and ANL contributed equally to this work. 10.1021/nl025604a CCC: $22.00 Published on Web 08/17/2002
© 2002 American Chemical Society
A number of pharmaceutical micelle-forming compounds with low toxicity and high solubilization power are currently available. Conventional surfactants, however, have critical micelle concentration (CMC) values in a millimolar range9 and may dissociate upon being diluted to therapeutically acceptable concentrations. In vivo, this may result in micelle collapse in a large blood volume with a subsequent precipitation of the encapsulated drug, i.e., sharp decrease in its bioavailability and ability to penetrate biological barriers.2 Earlier, we have reported that conjugates of poly(ethylene glycol) and diacyllipids, such as phosphatidyl ethanolamine (PEG-PE, where PE may contain various acyl chains, such as palmityl, stearyl, etc.),10 are able to form micelles that are much more stable compared to the micelles prepared from conventional detergents. Initially, PEG-PE conjugates were introduced into the drug delivery area as liposomal surface modifiers.11-13 Phospholipid parts of these amphiphilic conjugates incorporate into the lipid bilayer and anchor the PEG blocks on the surface of liposomal (and other nanoparticular drug carriers), thus protecting them from the fast removal from the blood stream by the reticulo-endothelial system (RES).11-13 On the other hand, when placed into an aqueous media, PEG-PE conjugates themselves form micelles.10 CMC values of PEG-PE conjugates are very low (in a high nanomolar to low micromolar range) because of very strong hydrophobic interactions between double acyl chains of phospholipid residues. Earlier, we have described some preliminary investigations clearly demonstrating that PEG-PE micelles could be loaded with some model hydrophobic or hydrophobized compounds
Figure 2. Incorporation of m-porphyrin into PEG2000-PE micelles and stability of drug-containing micelles.
Figure 1. Chemical structures of chlorin e6 trimethyl ester (mporphyrin, I), tamoxifen (II), and paclitaxel (taxol, III).
and diagnostic agents10,14 and are capable to deliver their load into a poorly permeable Lewis lung carcinoma tumor in mice with a higher efficiency than even PEG-modified liposomes.15 To further explore the potential of PEG-PE micelles as drug carriers, we have performed detailed studies on the possibility to load these micelles with poorly soluble anticancer agents of different types. Here, we report the results on preparation and some properties of PEG-PE micelles loaded with a sparingly soluble agent for photodynamic therapy (PDT) of cancer, chlorin e6 trimethyl ester (modified porphyrin, m-porphyrin), and two poorly soluble anticancer drugs, tamoxifen and paclitaxel (taxol) (Figure 1). The PDT treatment approach utilizes a combination of light irradiation and chemicals for the in situ production of toxic free radicals and is used in the treatment of cutaneous T-cell lymphoma and some other tumors.16,17 The use of PDT is complicated by some undesirable side effects caused by the accumulation of PDT agents in nontarget organs.16 Poor solubility of some porphyrin derivatives is also an issue18 and requires increased quantities of the drug to be used to achieve a therapeutic effect, which, in turn, increases side effects. The incorporation of porphyrin into PEG-PE micelles may increase its bioavailability and reduce certain side effects by increasing its accumulation in tumors via the EPR effect. Poly(ethylene glycol) with molecular weight of 2000 Da modified with distearoyl-PE (PEG2000-PE, Avanti Polar Lipid, Inc., Alabaster, AL) was used to prepare m-porphyrinloaded micelles. m-Porphyrin was purchased from Porphyrin Products, Inc. (Logan, UT). m-Porphyrin solution in methanol was added to the solution of PEG2000-PE in chloroform to obtain 1:10 to 1:2 drug/polymer w/w ratio. Organic 980
solvents were removed under vacuum to produce a film consisting of m-porphyrin/PEG2000-PE mixture. Micelles were formed by an extensive vortexing of the m-porphyrin/ PEG2000-PE film in an aqueous buffer. Nonincorporated m-porphyrin was separated by filtration of the micelle suspension through a 0.2 µm filter (m-porphyrin crystals as well as crystals of other insoluble drugs under normal circumstances cannot pass through the filter unless the drug is solubilized by nanoscopic micelles). The m-porphyrin concentration in filtrates was estimated by measuring its fluorescence at the excitation wavelength of 635 nm and the emission wavelength of 674 nm (F635/674) after 100- to 200fold dilution of the samples in methanol. In the absence of PEG-PE, less than 1% of m-porphyrin dispersed at the same concentration as in PEG-PE-containing samples was found in the filtrate, indicating that the drug that passes the filter in the presence of PEG-PE is solubilized by micelles. Figure 2 shows the results on encapsulation of m-porphyrin into PEG-PE micelles. At an initial m-porphyrin/PEG2000PE w/w ratio of up to 1:5, the drug incorporates into micelles with an efficiency close to 100%. The efficiency decreases to about 80% at the initial w/w ratio of 1:2. The results obtained demonstrate that PEG2000-PE micelles may be loaded with the poorly soluble PDT agent, m-porphyrin, to about 30% of micelle weight. The storage experiments demonstrated that micelles completely retain an encapsulated m-porphyrin for at least a month at room temperature (Figure 2). Tamoxifen is another marginally water soluble drug for cancer chemotherapy.19 This drug has been used to treat a variety of estrogen receptor positive carcinomas such as breast cancer, prostate carcinoma, ovarian carcinoma, renal carcinoma, melanoma, colorectal tumors, desmoid tumors, pancreatic carcinoma, and pituitary tumors.20 Long-term tamoxifen therapy, however, causes such side effects as endometrial cancer and drug resistance.21 Drug drawbacks may be overcome by incorporation of tamoxifen into micelles, which should increase its bioavailability and tumor accumulation. The preparation of tamoxifen-loaded micelles followed the pattern described for m-porphyrin. Micelles prepared from PEG-PE with a molecular weight of PEG block of 5000 Da (PEG5000-PE, Avanti Polar Lipid, Inc., Alabaster, AL), Nano Lett., Vol. 2, No. 9, 2002
Figure 3. Incorporation of tamoxifen into PEG5000-PE micelles at 1:5 initial drug/polymer w/w ratio. Bar 1 represents relative drug concentration in the sample before filtration through 0.2 µm filter; bar 2 represents drug concentration in the filtered solution of drugcontaining micelles; bar 3 represents drug concentration in the filtered suspension of free drug.
were used in this case. Weight ratios of tamoxifen/PEG5000PE in 1:50 to 1:5 intervals have been studied. The concentration of tamoxifen in each experiment was determined by fluorescence measurements (F245/385) after dissolving the preparations in methanol and UV irradiation for 30 min. Figure 3 shows that in the presence of micelles, at the highest drug-to-polymer ratio studied, more than 95% of tamoxifen was incorporated into PEG-PE micelles and became capable of passing through the 0.2 µm filter. The results obtained demonstrate that PEG-PE micelles may be loaded with tamoxifen up to 1:5 drug/polymer weight ratio, i.e., micelles can be prepared to contain up to 20 wt % of the drug. The third drug used in our experiments, paclitaxel (taxol), is an anticancer drug that interferes with the cellular propagation by arresting the cell replication process. An important problem complicating the clinical use of taxol is its poor solubility in water and in most pharmaceutically acceptable solvents. In addition, taxol is a toxic drug and therefore, large doses may cause severe side effects.22 Similar to other anticancer drugs discussed above, the incorporation of taxol into PEG-PE micelles may substantially enhance the efficiency of the treatment. Taxol-loaded PEG2000-PE and PEG5000-PE micelles were prepared by an extensive vortexing of mixed drug/PEGPE films in an aqueous buffer followed by removal of excess free drug crystals by filtration as described above for other anticancer agents. The concentration of taxol was determined by reversed phase HPLC with taxol detection by optical density at 227 nm. The incorporation of taxol is shown in Figure 4. The concentration of an encapsulated taxol linearly depended on the concentration of PEG-PE. The encapsulation efficiency was higher in the case of PEG2000-PE obviously because the hydrocarbon chain-to-polymer corona ratio is higher in the case of this conjugate, and these micelles provide more hydrophobic cargo space per mg of material compared to PEG5000-PE micelles. Though the taxol/PEG-PE incorporation ratio was relatively low (about 1.5 wt %), the results reported are still therapeutically significant, because taxol is active at very low concentrations. It is currently supplied as a 6 mg/mL solution in a 1:1 v/v mixture of Cremaphor EL and dehydrated alcohol. This carrier has been reported to induce an anaphylactic reaction in a number of patients.23 It is easy Nano Lett., Vol. 2, No. 9, 2002
Figure 4. Encapsulation of paclitaxel (taxol) by PEG-PE micelles as a function of PEG-PE concentration. Table 1: Typical Size Distributions of PE-PEG Micelles Loaded with Various Anticancer Agents PEG2000-PE-based size distribution (%) micelle composition
5.5-7 7-10 10-13 13-17 17-23 23-30 nm nm nm nm nm nm
PEG2000-PE m-porphyrin/PEG2000-PE taxol/PEG2000-PE
