Polymeric micelles and nanoemulsions as tumor-targeted drug carriers: Insight through intravital imaging
Graphical abstract
Introduction
Among various suggested drug carriers, two types, namely liposomes and polymeric micelles, are the most extensively studied and developed. Both are spherical nanoparticles with a core-shell structure. Liposomes have an internal aqueous compartment sequestered by a phospholipid shell, which allows encapsulation of water-soluble drugs in the inner core. In contrast, polymeric micelles are characterized by hydrophobic cores and hydrophilic shells. The hydrophobic micelle cores serve for encapsulation of lipophilic drugs. Micelle formation is thermodynamically driven; micelles are formed by self-assembly of individual molecules (unimers) of amphiphilic block copolymers.
While liposomes entered the market at the end of the last century and have been extensively used in medical practice, only a few polymeric micelles have progressed to clinical trials [1], [2], [3], [4] and only one type of micelle has reached the market [3], [4] despite more than twenty years of extensive research and development and sometimes excellent preclinical results. Micellar systems have been mostly tested for the delivery of doxorubicin (DOX) [1], [2] and paclitaxel (PTX) [3], [4], [5], [6], [7], [8]. Micellar encapsulation enhances aqueous drug solubility, prevents drug degradation under the action of body fluids and, most importantly, provides for some extent of tumor targeting. A comprehensive review of micelle application in drug delivery was recently published [9].
In clinical trials, micellar formulations of DOX NK911 and SP 1049C [2] manifested the same spectrum of side effects as free DOX, suggesting a lack of effective tumor targeting. However for Pluronic® micelle encapsulated DOX, this disadvantage has been surpassed by the invaluable benefit of suppressing the development of drug resistance [10], [11], [12], [13], [14], [15], [16], [17], [18].
For PTX, no micellar formulation besides NK105 [5] and Genexol PM® [4], [6], [7], [19], [20] has progressed to clinical trials due to rapid loss of drug from the carrier. Still, despite unfavorable pharmacokinetic parameters [4], the PTX-loaded poly(ethylene oxide)-co-poly(d,l-lactide) (PEO2000-co-PDLA1750) micelles (Genexol®-PM) have shown promising therapeutic results for breast, lung, and pancreatic cancer [3], [8], [20].
There may be a number of reasons for the conflicting outcomes of preclinical and clinical trials of polymeric micelles. One may be related to the lower permeability of human tumor blood vessels when compared to subcutaneous mouse tumor xenografts. Another might be premature micelle degradation in circulation leading to a premature drug release. As mentioned above, block copolymer micelles are formed by self-assembly of amphiphilic block copolymer molecules in aqueous milieu. The self-assembly occurs when copolymer concentration reaches some critical value called the critical micelle concentration (CMC). Below the CMC, block copolymer molecules exist in solution in the form of individual molecules (unimers). When the copolymer concentration drops below the CMC, micelles dissociate into unimers. Because systemic injections and especially infusion of micellar formulations are associated with a very substantial dilution in the circulatory system, micelles may dissociate into unimers upon injection. To prevent premature micelle dissociation, micellar systems should either have very low CMC (i.e., manifest thermodynamic stability), or have slowly dissociating cores (i.e., manifest kinetic stability).
Unstable micelles may be strengthened by the introduction of strong hydrophobic interactions into micelles cores. This may be achieved, for instance, by the introduction of oil [21] or mixtures of hydrophobic drugs [22]. Recently, one of us (NR) suggested micelle stabilization by the introduction of perfluorocarbon (PFC) compounds [23], [24], [25], [26], [27], [28]. Besides stabilization against degradation upon dilution, an important advantage of using perfluorocarbon cores is the opportunity of acoustic droplet vaporization (ADV) [29], [30], [31] that instigates drug release from nanodroplets under the action of ultrasound.
With this approach, drug-loaded polymeric micelles serve as starting points for developing drug-loaded nanoemulsions. The stability and drug release properties of the latter depend on the type and structure of the copolymer that forms nanodroplet shells [24], [26], [28]. Nanoemulsions as well as micelles manifested strong therapeutic effects (dramatically extended life span, suppression of metastases and ascites) in preclinical studies of breast, ovarian, and pancreatic cancer [25], [26], [27], [28], [32]. However side-by-side comparison of micelles and nanoemulsions as drug carriers and vital information on their stability in circulation, extravasation rates and drug retention in vivo has been lacking in the literature.
In the current study, we used intravital laser fluorescence microscopy and fluorescently labeled drug nanocarriers or fluorescently labeled drug to monitor carrier and drug stability in circulation and extravasation rates upon intravenous injections to pancreatic tumor bearing mice. Two sets of experiments were performed. In the first set, we compared extravasation and interstitial diffusion rates of PEG-PDLA unimers, micelles and PEG-PDLA-stabilized perfluorocarbon nanodroplets. In the second set, extravasation of nanodroplets was compared with that of the nanodroplet encapsulated drug, green-fluorescent paclitaxel (F-PTX).
Section snippets
Block copolymer
Water soluble, biodegradable block copolymer poly(ethylene oxide)-co-poly(d,l-lactide) (PEG-PDLA) with molecular weight of either block of 2000 Da was obtained from Akina, Inc., (West Lafayette, IN, USA).
Micelle preparation
The empty or PTX-loaded PEG-PDLA micellar solutions were prepared by a solid dispersion technique as described in refs. [26], [27].
To prepare fluorescently labeled PEG-PDLA micelles, a 3.76 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)2000-N′-carboxyfluorescein]
Preliminary experiments: unimers and nanodroplets in normal tissue
Preliminary experiments were performed with the microscope focused on mouse thigh subcutaneous muscle and adipose tissue (Fig. 1A). Blood vessels were visualized by injection of fluorescently labeled RBCs as explained in Methods. The initial formulation used in these studies comprised 99.5% (vol.) nanodroplets, however, traces of micelles were also present (see Supplemental materials, Table S1). Nanodroplets were separated by centrifugation and re-dispersed in the PBS. Both the nanodroplets and
Micelle stability in circulation and possible mechanisms of extravasation
The kinetic stability of micelles depends on the physical state of the micelle cores and is highest for micelles with solid cores formed by hydrophobic blocks with a glass transition temperature Tg above physiological temperature, such as poly(l-lactide) (PLLA), for which Tg = 60–65 °C; in addition, the regular structure of PLLA blocks favors their crystallization. However the excessive strength of solid cores results in a very strong drug retention and slow rate of the biodegradation, which does
Conclusions
This study of nanoparticle extravasation and tumor accumulation revealed, via intravital imaging, intimate details of carrier and drug behavior in circulation and in tissue. This information is vital for future clinical application of drug nanocarriers. The results pave the way for new directions in optimization of drug nanocarriers. Of a paramount importance is optimization of relative rates of carrier internalization, drug release, and drug and carrier diffusion in the tumor tissue. In
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Current address: Fox Chase Cancer Center, P0103, Department of Radiation Oncology, 333 Cottman Avenue, Philadelphia, PA 19111.