Abstract: This review describes our recent efforts to focus on important function of intelligent polymeric micelles from different block copolymers as novel carrier for delivery of most anticancer drugs and nucleic acids. The polymeric micelles feature a spherical sub-100 nm core-shell structure in which anticancer drugs are loaded avoiding undesirable interactions in vivo. Chemical modification allows the polymeric micelles to release drugs selectively according to the type of chemical modification and nature of polymers. Installation of folic acids on the micelle surface improves cancer cell-specific drug delivery efficiency along with pH controlled drug release. These intelligent micelles appear to be superior over classical micelles that physically incorporate drugs. Studies showed both controlled drug release and targeted delivery features of the micelles reduced toxicity and improved efficacy significantly. Further developments potentiate combination delivery of multiple drugs using mixed micelles. Micelle-forming block copolymer-drug conjugates, micellar nanocontainers and polyion complex micelles have been obtained that mimic functional aspects of biological carriers, namely, lipoproteins and viruses. Therefore, clinically relevant performance of the polymeric micelles provides a promising approach for more efficient and patient-friendly cancer therapy. Intelligent polymeric micelles may be advantageous in terms of safety, stability and scale-up.
INTRODUCTION
The concept of selective delivery of drugs to site of action was first introduced by Paul Erhlich early. He proposed a Magic Bullet, i.e., carriers with specific affinity for certain organs, tissues or cells for drug targeting. Since, then delivery systems such as liposomes, microspheres and nanoparticles have been developed. They have been able to widen the gap between the efficacy and toxicity of drugs. In recent years, numerous drugs designed to target various cellular processes have emerged, creating a demand for the development of intelligent Drug Delivery Systems (DDS) that can sense and respond directly to pathophysiogical conditions and can maximize the efficacy of therapeutic treatments, sparing physiologically healthy cells and tissues and thereby improving a patient’s quality of life. This new class of intelligent therapeutics refers to intelligent and responsive delivery systems that are designed to perform various functions like detection, isolation and/or release of therapeutic agent for the treatment of diseased conditions (Peppas et al., 2006). The greatest challenge in drug targeting is to achieve higher organ or tissue selectivity. Another so far unsolved problem is the delivery of nucleic acid drugs. Most of the available anti cancer agents are not able to differ between healthy and cancerous cells and thus lead to systemic toxicity. The basic idea for the solution of this problem is the application of polymeric micells (nanoparticles) equipped with targeting units for tumor-specific delivery. The Polymeric micelle is a nano-supramolecular assembly with three-dimensional spherical micelles with a hydrophilic corona and a hydrophobic core and its surface and core were modified with piloting molecules for cancer cells binding for controlled drug release. A hydrophilic shell helps them to stay unrecognized, during blood circulation these nanosized particles with a typical size of 10-100 nm are able to accommodate lipophilic drugs in their interior and alter their kinetics in vitro and in vivo. The new type of amphiphilic block Copolymers are build up of a hydrophobic synthetic polymer component in the core and single-stranded DNA forming the corona of these micelles. As new targets for specific localization of chemotherapeutics incorporated into nanoparticle Folate Receptors (FRs) are considered because they are highly expressed on the surface of various cancer cells. In theory the targeted therapies can achieve the maximum therapy with the minimum side effects.
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| Fig. 1: | Amphiphilic block copolymers and polymeric micelles |
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| Fig. 2: | Aketch of a polymeric micelles loaded with drug
in core |
In reality, delivering potent drugs to where they are necessary is still challenging for clinicians and pharmaceutical scientists. It is because most of potent drugs are small molecules that can freely diffuse in both benign and malignant diseased cells. This induces non-specific drug distribution in the body. Even if the drugs are highly specific to certain molecular targets, they often suffer from low solubility, rapid metabolism or antagonistic interaction with other drugs. Therefore, it is almost impossible to simultaneously control the bioactivities and physicochemical properties of multiple drugs, overcoming aforementioned pharmacokinetic barriers. Drug Delivery Systems (DDS) appeared to be a promising and reliable approach to deliver potent drugs to the site of action precisely and timely. Currently available macromolecular drug carrier systems may include water-soluble polymers, dendrimers, polymeric micelles and liposomes (Kataoka et al., 2001). Each carrier has advantageous features to provide structural flexibility, multiple functional moieties, a sequestered nano depot and robust stability, respectively. Among these carriers, only the polymeric micelles undergo dynamic physicochemical changes during drug entrapment and release in terms of molecular assembly and dissociation between block copolymer components. The polymeric micelles are spherical supramolecular nanoassemblies prepared from self-assembling amphiphilic block copolymers (Fig. 1, 2).
They feature a sub-100 nm core-shell structure which provides a nano depot for hydrophobic drugs enveloped with a hydrophilic shell, improving drug solubility (Chu, 1995). The hydrophilic shell suppressing protein adsorption allows the polymeric micelles to avoid foreign body reaction while improving drug solubility. This property is called stealth functionality. Because of their characteristic structure and stealth functionality, the polymeric micelles can stably transport bioactive molecules to the tumor tissues suppressing the immune response and non-specific drug distribution to the normal tissues.
CLASSIFICATION DEPENDING ON DRUG LOADING METHOD
In physical drug entrapment type micelles, they incorporate drug payloads through the hydrophobic interaction in the micelle core (Chu, 1995). Drugs can be entrapped also in gel-like amorphous core. In either case, the equilibrium rates determine the physicochemical stability and drug release patterns of the polymeric micelles, which are controlled time-dependently. In contrast, covalent drug conjugation type micelles have drug-binding linkers that stably tether drugs in the micelle core until the polymeric micelles accumulate in the site of action and are exposed to the in vivo stimuli such as ions, endogenous signal peptides, enzymes and pH that trigger drug release. Covalent drug conjugation type micelles appear to be more stable than physical drug entrapment type micelles as long as the linkage remains intact. Since, their drug release patterns can be modified according to chemical stability of drug-binding linkers, covalent drug conjugation type micelles provide environment-responsive controlled drug release systems, or also called intelligent systems.If the core of the micelles is modified for drug entrapment, the surface of the micelles can be functionalized with targeting molecules that can specifically interact with certain molecular targets on the lesions. Receptors, intracellular organelles and signal peptides are such examples. Antitumor activity and bioavailability of polymeric micelles can be improved further by installing targeting molecules that are specific to malignant cell membrane transporters or intracellular proteins. Receptor recognition and its accompanying cellular interaction and response are crucial issues on the design of effective drug carriers. Indeed cellular response and drug efficacy increase significantly by simply changing drug delivery approaches because targeting molecules can enhance interactions between the polymeric micelles and the cells (Holland et al., 1995). This widens the therapeutic window of drug payloads, eventually improving their bioavailability.
Classical polymeric micelles with physical drug entrapment: Amphiphilic block copolymers consist of multiple segments with distinct solubility against certain solvents. Generally two or three segments are conjugated linearly to prepare amphiphilic block copolymers. Depending on the thermodynamic conditions, amphiphilic block copolymers may form nano structures such as lamellas, globules, cylinders, vesicles and micelles. In particular amphiphilic block copolymers from hydrophilic and hydrophobic segments undergo spontaneous self-assembling in the aqueous solutions, sequestering hydrophobic segments from aqueous environments by hydrophilic segments. This phenomenon is useful to dissolve hydrophobic and fatty materials as likely seen in Low Molecular Weight Surfactants (LMWS) forming micelles. Compared to LMWS micelles, polymeric micelles prepared from amphiphilic block copolymers are superior in stability and most noticeably in high capacity for the incorporation of guest molecules in the core.
Micelle formation and physicochemical properties: Micelle formation of amphiphilic block copolymers is accompanied with minimizing free energy and entropy change is generally considered the most important factor to form stable polymeric micelles. When it comes to preparing drug-loaded polymeric micelles, influence of enthalpy becomes also important to micelle formation. Our early studies have shown the importance of providing the most thermodynamically favorable conditions on micelle formation, adjusting concentrations, temperature and solvents along with block copolymer compositions. During the process of the entropy-driven micelle formation, the concentrations of polymers in solutions would be the most important factor. Indeed a critical micelle concentration, or CMC, varies depending on block copolymer types and compositions. It generally ranges between 10-6 and 10-7 M which is 1000 times lower than that of LMWS micelles. Interestingly, although it is obvious that the polymeric micelles are stable above the CMC, chain exchange can occur irrespective of the CMC level (Moffitt et al., 1996). This intriguing phenomenon demonstrates that polymeric micelle formation is thermodynamically stabilized yet not completely frozen. Nevertheless, it must be noted that the kinetic of chain exchange between the polymeric micelles is extremely slow compared to LMWS micelles. Also, the chain exchange between the polymeric micelles is suppressed as the micelle core becomes hydrophobic by drug entrapment. It maintains the polymeric micelles stable until they are used for the treatments. Polymeric micelles can be prepared mainly by two methods:
| • | The most general method is through dialysis |
| • | Second method is combination of emulsification, evaporation and sonication |
Another possible method is solution spray process. Amphiphilic block copolymers may be dissolved in aqueous solutions directly to form micelles, yet particle size would become larger with a broad distribution, compared to the polymeric micelles prepared through the dialysis method. This reduces drug-loading efficiency.
Covalent drug conjugation type micelles
pH sensitive polymeric micelles: A new approach of drug loading to the micelles has been made recently by conjugating drugs to the micelle-forming block copolymers through in vivo stimuli-responsive linkers (Alexandridis and Yang, 2000). In vivo stimuli may include enzymes, oxygen and proton. The polymeric micelles appeared to effectively protect biological drugs from enzymatical degradation (Price, 1983). Whereas, protons, other ions and small molecules in vivo that can penetrate into the micelle core easily are considered effective triggers to initiate drug release from the micelles. An approach of pH-controlled drug release is also useful to target other in vivo acidic environments.
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| Fig. 3: | Fluorescence quenching effects in the micelle
core and intracellular distribution |
Compared to physiological conditions at pH 7.4, intratumoral space and intracellular compartments, such as late endosomes and lysosomes are known acidic between pH 6.8–7.2 and pH 4–6, respectively. Thus, if drug carriers could incorporate drugs stably at pH 7.4 and release the drugs at pH below 6, these carriers would selectively deliver drugs to the intracellular regions. Schiff base, or azomethine, is considered the most facile and appropriate linkage to design such pH-sensitive drug release systems. Generally imine bond is stable at pH 7.4 while cleavable at pH below 6 (Quintana et al., 1993a). However, the imine bond is reversible even at pH 7.4, it is concerned that the conjugation between drugs and polymers are not stable enough to retain drugs during blood circulation. To the contrary, hydrazone bond appears to be more stable than the imine bond while it shows excellent pH-sensitivity.
Figure 3 shows the images of cells exposed to the polymeric micelles as well as free drugs. As seen in physical drug entrapment type micelles, fluorescent drugs undergo fluorescence quenching in the polymeric micelles core while showing strong fluorescence again when drugs are liberated. Compared to the cells where free drugs accumulate in the nuclei quickly after 1 h incubation, the cells treated with the polymeric micelles still show the low fluorescence level after the same incubation time. However, the cells with the polymeric micelles show drug distributions in both cytoplasm and cell nuclei after 24 h, while free drugs mainly accumulate in cell nuclei irrespective of incubation time. It is intriguing that relatively large amount of drugs remain in the cytoplasm when the cells are treated with the polymeric micelles. This demonstrates the controlled and sustained release of drugs from the polymeric micelles.
Folate-conjugated polymeric micelles
Folate-conjugated block copolymer for active cancer targeting intracellular
delivery of anticancer drugs: As for cancer targeting methods, active targeting
is more advanced than passive targeting. it is of significant importance to
select appropriate interactions and molecules for active targeting, without
hampering physicochemical properties of original drug carriers. Large targeting
molecules tend to result in undesirable intermolecular interaction or aggregation.
Small molecules and oligopeptides have drawn a particular attention because
they would have the least influence on the physicochemical properties of macromolecular
drug carriers Therefore, drug delivery systems with small targeting molecules
have become popular yet challenging to design active drug targeting. Among small
targeting molecules, folic acid is known as a promising candidate because of
its low molecular weight (MW = 441.40) and excellent binding/recognition affinity
against the receptors called Folate-Binding Proteins (FBP). Cancer cells overexpress
FBPs on the cell membrane which can be targeted by delivery systems using folate.
Folic acid is conjugated to drug molecules and nanocarriers to promote their
cellular uptake through folate receptor mediated endocytosis. Hetero bifunctional
PEG is used for the surface modification of the polymeric micelles. In order
to confirm receptor recognition, Surface Plasmon Resonance (SPR) measurements
are widely used.
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| Fig. 4: | Receptor recognition and cellular interaction
of folate-conjugated micelles |
Micelles without folate were used as control yet did not show any instant cellular interaction. The results are corresponding well with the SPR results, potentiating cancer cell targeting of the folate conjugated polymeric micelles. Noticeably complete suppression of non-specific interaction of the polymeric micelles with cancer cells is unlikely because the cells with no receptors can engulf the polymeric micelles through macropinocytosis. The installation of targeting molecules is currently at a stage to improve cellular uptake. Cancer cell-specific uptake of nanoparticles still remains a challenging issue. Nevertheless, through active targeting, the polymeric micelles can recognize cancer cells more efficiently than normal tissue and cells.
In order to confirm general effects of folate installation on antitumor activity of the polymeric micelles, various human cancer cell lines have been screened using the folate-conjugated polymeric micelles. As shown in Fig. 5, most cancer cell lines become more sensitive to folate-conjugated polymeric micelles than the polymeric micelles without folate. In some cancer cells, bioactivity of folate conjugated polymeric micelles was even greater than free drugs. Particularly the drug resistant HCT-15 cell line showed marked sensitivity to the chemotherapy. Considering Multidrug Resistance (MDR) is mainly induced by overexpression of P-glycoproteins on the cancer cell surface, these results potentiate the treatment of other MDR phenotype cancer cells using the polymeric micelles for active drug targeting.
Antitumor activity and bioavailability of drug through micelles In vitro cytotoxicity: Most anticancer drugs are limited in their clinical applications because of low water solubility and high toxicity due to narrow therapeutic window and non-specific distribution of anticancer drugs in the body results in rapid clearance of therapeutic agents from the body leads to repetitive drug infusion in order to maintain drug concentrations in the blood which may induce either chronic toxicity or resistance to chemotherapy. Pioneers in 1970s suggested that drug delivery systems based on polymer science and supramolecular chemistry could overcome these limitations (Quintana et al., 1993b). Aforementioned approaches also have demonstrated that drug efficacy is indeed enhanced by using drug carriers including the polymeric micelles. One can control the cytotoxicity level against the targeted cells simply by selecting drug entrapment methods. Thus, the correlation between drug release control and cytotoxicity is very important particularly when designing drug carriers for either systemic or local drug delivery. For these reasons, in contrast to free drugs, in vitro cytotoxicity of the polymeric micelles cannot be interpreted directly to estimate the in vivo antitumor activity.
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| Fig. 5: | Cytotoxicity response screening in various human
cancers |
Biodistribution: In order to develop the polymeric micelles optimized for in vivo use, the fate of polymer components and their assemblies in the body should be studied. This enables us to design the polymeric micelles that safely protect and deliver therapeutic materials in vivo avoiding the host defense system. Generally, distribution of external materials in the body is influenced by the surface properties. In comparison to low molecular weight materials, polymers which hardly penetrate blood vessel walls circulate in the vascular space after infusion. If polymers are cationic they aggregate with anionic proteins in the body or directly interact with cells, inducing pulmonary embolism for instance. If polymers are sufficiently water soluble, they start circulating in the blood stream and reach the first gate to pass, the kidneys. The kidneys excrete external materials with molecular weight and size of less than 50,000 and 6 nm, respectively, into the urine. Since, protein adsorption occurs also by hydrophobic interactions, bioavailability of drug carriers relies on how effectively the polymeric micelles can sequester hydrophobic drug payloads from the in vivo environments. Folate-conjugated polymeric micelles show an intriguing correlation between ligand content and tissue accumulation. It is surprising that folate conjugation shows almost no change in tumor tissue accumulation of the polymeric micelles.
Despite SPR measurements that showed a marked decrease in receptor recognition with substitution below 10%, there was no noticeable change in the biodistribution results. In contrast, the polymeric micelles with higher folate content appeared to accumulate in the liver and spleen more preferentially. Folate conjugation seems to change the surface property of the polymeric micelles, resulting in the increase accumulation in the liver and spleen. These results postulated that active drug targeting may mainly improve the cellular interaction rather than alter the characteristic biodistribution of the polymeric micelles, although this might be specific to folate-conjugated systems.
When it comes to biodistribution data analysis, it is important to understand that accumulation of the polymeric micelles does not always mean the intracellular drug accumulation and also that it is not the amount of drug carriers that influence toxicity but that of free drugs released within the tissues. Normally drug concentrations are proportional to the accumulation of prodrugs. This fact results in some discrepancy between biodistribution and toxicity data of the polymeric micelles. For instance, although accumulation in the liver and spleen increased in proportion to prolonged blood circulation, the polymeric micelles generally show less toxicity than free drugs. This result emphasizes again the importance of drug release control along with cancer targeting approaches. For these reasons, biodistribution studies should be conducted by considering that accumulation of the polymeric micelles is not always the same with the actual amount of active small molecules (Fig. 6).
The polymeric micelles would have another fundamental problem which is related to the total amount of polymers used for the delivery of drugs enough for the treatments.
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| Fig. 6: | Biodistribution of polymeric micelles (a) Blood,
(b) Tumor and (c) Liver |
The low drug-loading content requires a large amount of polymers to deliver active drug molecules sufficient to antitumor activity.
In vivo antitumor activity: Reduced toxicity and effective activity of physical drug entrapment micelles were observed previously in animal models against all cancers tested such as mouse colon carcinoma (C26), mouse sarcoma (M5076), human lung cancer (Lu-24), human breast cancer (MX-1) and mouse leukemia (P388) (Triolo et al., 2000a). The pH-sensitive and folate conjugated micelles also showed an improved activity and reduced toxicity. In Fig. 7, time-dependent changes in tumor volume and body weight are summarized.
Free drugs show effective tumor growth suppression, yet they are accompanied with serious body weight decrease of the animals. In comparison, the polymeric micelles showed less toxicity, retaining antitumor activity. Previously, physical drug entrapment type micelles are as potent as free drugs in terms of the dosage used for the treatment (Triolo et al., 2000b). The pH-sensitive micelles were also beneficial in suppressing tumor growth, yet the dose was slightly higher than that of physical drug entrapment type micelles as well as free drugs (Allen et al., 1999).
APPLICATIONS
Application in combination chemotherapy using polymeric micelles Modification of delivery environment: It is obvious that the polymeric micelles significantly improve the efficacy and reduce toxicity, compared to the conventional drug formulations. As one of the possible approaches to treat cancers, combination chemotherapy using the pH-sensitive micelles and intracellular signal inhibitors has been recently investigated (Nishiyama et al., 2005).
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| Fig. 7(a-b): | Antitumor activity of polymeric micelles |
The concept is to modify delivery environment to be more suitable to drug carriers and thereby therapeutic efficacy can be improved. This new class of approach would be promising in cancer combination therapy using macromolecular drug carriers.
Concurrent delivery of multiple therapeutic agents: From the pharmaceutical point of view, concurrent infusion of multiple drugs is effective for the treatments yet extremely difficult to realize because every drug shows distinct pharmacokinetics and comes with various risks accompanying co-action of another drug molecule. Solely determining a formulation for co-solubilization of multiple drugs requires enormous efforts because each drug has different solubility parameters and miscibility. DMSO, Cremophor and other surfactants are generally used to dissolve poorly water soluble drugs. However, drug formulations using these vehicles have inherent toxicity, and therefore, only a limited amount is allowed for infusion. More seriously drug molecules can interact with each other inducing unfavorable side effects in this formulation. Recent efforts have revealed that the polymeric micelles would be a promising drug formulation for the simultaneous delivery of multiple drugs. By incorporating multiple drug molecules in the same micelle core, one can concurrently deliver various therapeutic agents to the tumor tissue.
Application of block copolymer micelles for delivery of gene and related compounds: Delivery systems are required to efficiently transport the administered DNA into the target cells in vivo. So far, viral vectors are the most efficient gene delivery systems and most widely used in clinical trials. Nevertheless, despite its high efficacy, the use of viral vectors is limited by safety problems. Therefore, the success of gene therapy and antisense therapy critically depends on the development of safe synthetic gene delivery systems. For this purpose, a formidable effort has been devoted to developing non-viral vectors such as cationic lipids and cationic polymers. Block copolymer micelles entrapping plasmid DNA and oligonucleotides have been developed as non-viral DNA delivery systems.
CONCLUSION
Finally the dawning of combination therapy using polymeric micelles is of significance. It might be an approach to install target specific molecules on the micelle, to modify drug delivery environments, or to incorporate different types of bioactives concurrently. In every case, it is obvious that intelligent drug delivery using the polymeric micelles will possibly bring the most facile, versatile and efficient methodology in chemotherapy treatments for human diseases.