Article pubs.acs.org/Langmuir
Polymeric Premicelles as Efficient Lipophilic Nanocarriers: Extending Drug Uptake to the Submicellar Regime María Méndez-Pérez,† Belén Vaz,‡ Luis García-Río,*,† and Moisés Pérez-Lorenzo*,§ †
Center for Research in Biological Chemistry and Molecular Materials, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain ‡ Department of Organic Chemistry and §Department of Physical Chemistry, Universidade de Vigo, 36310 Vigo, Spain S Supporting Information *
ABSTRACT: A multitechnique investigation on the self-assembly behavior of a biocompatible polymer in the high dilution regime is reported herein. The obtained results unambiguously reveal the existence of premicellar structures that may further extend the efficiency of traditional polymeric micelles as drug-delivery vehicles. Such an expansion in the excipient capacity arises from (i) the increased drug retention of submicellar assemblies due to their higher resistance to dilution and therefore to their improved circulation time and (ii) the superior carrier permeability of these premicellar aggregates as a result of their smaller size, which makes these drug vehicles more effectively targeted to the tumors through the socalled enhanced permeability and retention effect. The uptake ability of the polymeric premicelles described in this work has been tested through the use of Nile Red as drug model given its intermediate lipophilicity (log P ≈ 3−5) similar to that of potent chemotherapy agents and its microenvironment-sensitive fluorescence properties relevant for localization purposes. Thus, it has been found that an efficient drug encapsulation can be achieved under conditions well below the normally required critical micelle concentration. These results may constitute a promising strategy in order to develop new and more efficient polymeric formulations in drug delivery technology.
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INTRODUCTION Chemotherapeutic agents are widely used for the treatment of cancer diseases. However, many anticancer drugs are poorly soluble in water, which can cause serious drawbacks for their clinical application. To overcome this issue, the most commonly used techniques to prepare liquid dosage formulations of lipophilic drugs are pH adjustment, cosolvency, complexation, and micellization.1−4 Micellar drug carriers have emerged over the past years as a promising strategy for achieving an improved delivery of hydrophobic agents.5 Thus, their cores serve as excipient to load the active pharmaceutical ingredient (API), while their hydrophilic crowns allow the suspension of the system in an aqueous medium. Among the different materials for preparing these carriers, polymeric micelles constitute a valuable tool for the vehiculization of poorly water-soluble drugs.6−12 These aggregates, compared with traditional surfactant-based micelles, are considerably more stable due to their remarkably lowered critical micelle concentration (CMC).13 In this regard, low molecular weight surfactant-based micelles show high CMC values, which gives rise to a lack of stability of the colloidal carrier after its systemic administration and results in the precipitation of the solubilized drug. Conversely, polymer-based micelles exhibit slower rates of dissociation, allowing the retention of the loaded drug for a longer period of time and, in the end, achieving a higher accumulation of the drug at the target site. However, despite the great body of literature on this issue, not much interest has © 2013 American Chemical Society
been devoted to the characterization and application of these polymer-based self-assembled systems below their CMC. This lack of attention contrasts with the numerous experimental and theoretical papers reporting the appearance of submicellar structures for low molecular weight surfactants.14−23 In fact, many unusual phenomena in surfactant solutions occurring at much lower concentrations than CMC have been frequently attributed to premicellar aggregation. The term premicelle is applied to submicellar assemblies that are formed spontaneously or in some cases generated by interaction with other reagents. In the latter case, it may not be easy to distinguish between an induced micellization and the formation of premicelles.24 In the case that the drug-carrier functionality could remain unaffected in the less-structured submicellar domain, polymeric submicellar aggregates may constitute an extension of the encapsulation capability of their micellar analogues given the lower value of critical premicelle concentration (CPC) compared to that of CMC. Thus, the higher resistance of premicelles to dilution would lead to an improved circulation time of the drug carrier after its application and therefore to a superior performance. Most importantly, if these submicellar structures are smaller than their micellar counterparts, these drug vehicles may be more Received: June 12, 2013 Revised: July 11, 2013 Published: August 8, 2013 11251
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Figure 1. Schematic of the permeation of API-loaded premicellar polymeric carriers through the leaky endothelial junctions of a blood vessel. the probe into a 1-cm pathlength quartz cuvette, subsequently allowing the solvent to evaporate. Then, 3 mL of the appropriate polymer solution was added to the cuvette, mixed thoroughly, and allowed to equilibrate. In all cases the final probe concentration was 2.0 × 10−4 M. Steady-state fluorescence experiments were recorded using a Cary Eclipse spectrophotometer and collected from a 1-cm pathlength quartz cuvette at 25 °C. All emission spectra were corrected for emission monochromator response and were background-subtracted using appropriate blanks. Slits and rate of acquisition were chosen for a convenient signal-to-noise ratio. Pyrene and Nile Red were acquired from Aldrich and were of the highest available purity. For pyrene, the fluorescence spectra were measured with an excitation wavelength of 334 nm. In this case, the intensities of the first (I1) and third (I3) vibronic bands located at 373 and 384 nm, respectively, were measured in order to determine the variation of the I1/I3 ratio. For Nile Red, the fluorescence spectra were obtained with an excitation wavelength of 550 nm, and the change in the wavelength of maximum emission was monitored. Samples for fluorescence studies were prepared by using the method previously described for UV−vis studies. In all cases the final probe concentration was 4.1 × 10−7 M. 1 H NMR measurements were performed on a 400 MHz Varian INOVA spectrometer equipped with a magnetic field gradient probe. Signal postprocessing was performed with MNova software. The samples were prepared using D2O (99.9%) supplied by Aldrich as solvent. The DOSY spectra were acquired with the standard stimulated echo pulse sequence using LED and bipolar gradient pulses. The gradient strengths (G) were changed from 2.1 to 64.3 G cm−1 in 20 steps, and the gradient pulse duration (δ) was kept constant at 2 ms. To obtain reliable results for the polymer selfdiffusion coefficient (D), the diffusion time (between leading edges of the field gradient pulses, Δ) was optimized for each sample to a value typically between 20 and 100 ms. The self-diffusion coefficient was calculated according to the Stejskal−Tanner formula:26
effectively targeted to the tumors through the so-called “enhanced permeability and retention (EPR) effect”,25 which consists of the penetration of the drug carriers into the tumor tissues through the leaky vasculature associated with them (Figure 1). This may then result in a higher drug concentration at the tumor site compared to that obtained through micelles with an inadequate size. It is for this reason that exploring the nature and encapsulating ability of self-assembled carriers in the submicellar regime may constitute a promising strategy in order to develop new and more efficient polymeric formulations. As a proof-of-concept of the ideas put forward above, a study on the aggregation dynamics of PEG-40 stearate (PEG40S) in the submicellar domain and its efficiency as a lipophilic drugtrapping system was carried out. PEG40S is a synthetic polymer composed of PEG (poly(ethylene glycol)) and stearic acid, a naturally occurring fatty acid. The fact that PEG40S may be used for oral applications establishes it as an ideal candidate in order to carry out this analysis. Once the self-assembly behavior region of PEG40S had been investigated, Nile Red (NR) fluorophore was selected as a drug model molecule so as to test the potential encapsulation ability of the aggregates found. NR constitutes an ideal candidate for this study given (i) its intermediate lipophilicity (log P ≈ 3−5) similar to that of potent chemotherapy drugs such as Paclitaxel, (ii) its microenvironment-sensitive fluorescence properties relevant for localization purposes, and (iii) its low cost compared to that of active pharmaceutical ingredients, which made it possible to gain insight into the encapsulation ability of the polymer aggregates before properly optimizing the system with the desired API.
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⎡ ⎛ δ⎞ ⎤ I = I0 exp⎢− γ 2G2δ 2⎜Δ − ⎟D⎥ ⎝ ⎣ 3⎠ ⎦
EXPERIMENTAL SECTION
Synthetic polymer PEG-40 stearate (Figure 2) was provided by Seppic and used without further purification.
(1)
where I and I0 are the signal intensities obtained for a given gradient strength and 0, respectively, and γ is the gyromagnetic constant. The experimental data were collected by observing the evolution of the signal intensity of the ethylene oxide protons. Compared to that, other signals from the polymer were too weak to be efficiently monitored. The obtained data were analyzed using Origin to estimate D from the observed monoexponential decay. The sizes of the PEG40S-based aggregates in aqueous solution were measured using a dynamic light scattering instrument (NanoZS, Malvern Instruments) with a 633 nm “red” laser. For each sample, 3 measurements with 10 runs were performed, and the average size was calculated. Polymer concentrations ranged across 4 orders of magnitude (10−6 to 10−2 M).
Figure 2. Structure of poly(oxyethylene) (40) stearate, PEG40S (MW 2046 g mol−1). UV−vis absorbance spectra were recorded on a Cary 500 scan UV− vis−NIR spectrophotometer fitted with a thermostatted holder and collected from a 1-cm pathlength quartz cuvette at 25 °C. The solvatochromic probe 2,6-dichloro-4-(2,4,6-triphenyl-1-pyridinio)phenolate, also known as Reichardt’s dye 33, was obtained from Sigma-Aldrich and used as received. The change in its wavelength of maximum absorbance was monitored in order to express the polarity in the ET scale. Typically, samples for UV−vis studies were prepared by transferring an appropriate aliquot of an ethanolic stock solution of
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RESULTS AND DISCUSSION In a first approach the self-assembly behavior of PEG40S has been examined through the use of 2,6-dichloro-4-(2,4,611252
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triphenyl-N-pyridino)phenolate (from now referred to as ET(33)) as colorimetric probe. The high sensitivity of ET(33) absorption spectrum to small changes in medium polarity makes this dye a valuable tool in order to study polymer micellization processes. Due to the remarkable blue shift experienced by this betaine as solvent polarity is increased, this dye can be employed so as to build an empirical polarity scale (ET).27 The ET scale allows detecting the formation of microenvironments where local polarity differs from that of the bulk aqueous solution. Its value is defined as the transition energy of the dissolved betaine dye in the appropriate solvent measured in kcal/mol according to eq 2:
ET =
hcNA 28591 = λmax λmax
(2)
where h is Plank’s constant, c is the speed of light, NA is Avogadro’s number, and λmax is the longest wavelength intramolecular charge-transfer π−π* absorption band of this zwitterionic betaine. Another advantage of using this probe lies in its low pKa value (4.78), which allows ET(33) to exist as a phenolate at near neutral acidity, not requiring pH values above 10 to be applicable to aqueous solutions.28 As shown in Figure 3 (top), there is a marked solvatochromic effect in the presence of increasing PEG40S concentrations. This noticeable shift points out to the formation of low polarity microenvironments in the bulk aqueous solution when this polymer is added. In view of the results shown in Supplementary Table S-1, the local polarity of these confined locations would roughly correspond to that found within a CTAB micelle in aqueous solution. Figure 3 (bottom) shows the variation of the ET parameter at increasing PEG40S concentrations. As depicted, up to 2.0 × 10−4 M the ET value remains constant (∼70 kcal/mol) and equal to the value established for water (see Supplementary Table S-1). Above this concentration the ET parameter decreases, reaching a value of ∼62 kcal/mol. The polymer concentration at which this decline starts perfectly matches the CMC value of several ethoxylated fatty acids closely similar to PEG40S.29−31 The magnitude of this value is further supported by kinetic measurements of the influence of PEG40S concentration on the observed rate constant for the solvolysis reaction of 4-methoxybenzenesulfonyl chloride (data not shown). Having reached this point, it must be noted that for a given polymer the critical concentration for the formation of submicellar structures may be 1−2 orders of magnitude lower than that of their corresponding micelles. Therefore, taking into account that the probe concentrations in the experiments previously described are rather similar to the estimated CMC (∼10−4 M in all cases), it seems evident that in order to perform an efficient exploration of the submicellar domain for PEG40S an alternative spectroscopic technique requiring a rather lower probe concentration is necessary. Accordingly, the self-assembly behavior of PEG40S was examined through the use of pyrene (Py) as fluorescence probe since this method involves the use of probe concentrations of ∼10−7 M, which will give rise to a higher sensitivity toward the detection of potential submicellar aggregates. The emission spectrum of pyrene exhibits five peaks in the range of 370−395 nm (denoted as I1−I5). Among these, the ratio of intensity of the first (I1 at 373 nm) and third vibronic band (I3 at 384 nm) constitutes a parameter sensitive to the polarity of pyrene’s
Figure 3. Change of the UV−vis spectra of Reichardt’s dye 33 in the presence of increasing PEG40S concentrations (some data omitted for clarity) (top) and evolution of the ET parameter (eq 2) as a function of the polymer content (bottom). [ET(33)] = 2.0 × 10−4 M; T = 25.0 °C.
environment.32 Thus, I1/I3 shows a value close to 0.6 in hydrocarbon solvents, 1.1 in ethanol, and 1.8 in water. The sensitivity of pyrene to the solubilizing medium has been widely used to determine CMCs as well as other micelle characteristics since the aggregation of surfactant monomers provides a less polar environment sensed by the probe after its corresponding uptake. Hence, when studying the self-assembly behavior of PEG40S in aqueous solution, a decrease of the I1/I3 ratio is expected as a result of the formation of polymer aggregates and the subsequent incorporation of pyrene to the these hydrophobic cores. Figure 4 (top) shows the evolution of the fluorescence emission spectrum of pyrene with PEG40S concentration. From these measurements the evolution of the I1/I3 ratio as a function of the polymer concentration can be monitored. This dependence is illustrated in Figure 4 (bottom). Figure 4 (bottom) shows a gradual decrease of the I1/I3 ratio at PEG40S concentrations above ∼2.0 × 10−6 M. This result points out the formation of hydrophobic microenvironments in the bulk aqueous solution well below the CMC value (2.0 × 10−4 M), which seems to reveal the existence of PEG40S-based premicellar assemblies. It is also worth noting that the 11253
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derived from the interaction of a ground-state and an excitedstate pyrene monomer when both are found in close proximity.35 Such an interaction gives rise to an excimer state that fluoresces at a substantially longer wavelength compared to that of the monomer emission, which results in the band finally observed. In general, the appearance of this signal is not observed in isotropic pyrene solutions. This is the case, for example, of pyrene-saturated aqueous solvents ([Py] = 5.0 × 10−7 M) or ethanolic solutions ([Py] = 2.0 × 10−6 M) where no formation of the (PyPy)* species takes place. On the other hand, micelle-like solutions can facilitate the formation of the excimer even in rather diluted Py solutions, since high local concentrations may be achieved within the colloids. Therefore, the probability of (PyPy)* formation will depend on a situation where an aggregate contains, at least, double occupancy by pyrene molecules. Thus, while a polymeric assembly with two or more pyrene molecules will readily form excimers due to the micellar proximity effect, a structure enclosing a single pyrene molecule will not give rise to (PyPy)* species. In the latter case, this is due to the fact that the movement of the probe molecules from one aggregate to another is rather long compared to the lifetime of their excited state.36 Accordingly, the appearance of a band around 480 nm at concentrations well below the CMC seems to evidence the existence of premicellar aggregation, while its subsequent disappearance suggests a redistribution of the Py monomers among the increasing number of premicellar (or micellar) aggregates as PEG40S concentration is raised. This will be, in the end, the controlling factor in the formation of the excimers. In line with these observations, Figure 5 shows the dependence of the excimer-to-monomer intensity ratio (Iex/
Figure 4. Evolution of the fluorescence emission spectra of pyrene with PEG-40 stearate concentration (some data omitted for clarity) (top) and evolution of the I1/I3 ratio as a function of the polymer content (bottom). λexc = 334 nm; [Py] = 4.1 × 10−7 M; T = 25.0 °C.
reduction in the I1/I3 parameter covers a wide range of polymer concentrations after its initial drop is reached. These gradual declines have been commonly interpreted as a concurrent gentle increase in the aggregation number of the polymer.17 This tendency contrasts with the sharp decreases observed for low molecular weight surfactants, which is explained on the basis of a rapid conversion from the monomer to a selfassembled structure. Figure 4 (bottom) also shows how the relationship between the first and third vibronic band of pyrene remains constant for high polymer concentrations (I1/I3 ≈ 1.25). This limit value agrees with that found by other authors when studying the location and number of solubilized pyrene molecules in heptaethyleneoxide monoalkyl ether CnE7 (n = 10, 12, 14, and 16) surfactant micelles.33 The I1/I3 ratio has been also estimated in ethylene glycol and tri(ethylene glycol) as 1.64 and 1.57, respectively.34 The magnitude of these values compared to that found in this work seems to place pyrene molecules in the most hydrophobic inner core of the PEG40Sbased aggregates. Figure 4 (top) shows that under a certain range of polymer concentrations the emission fluorescence spectra exhibit a band centered at 480 nm. This signal, from now referred to as Iex, must be ascribed to the formation of a (PyPy)* excimer species
Figure 5. Evolution of the excimer-to-monomer intensity ratio with PEG-40 stearate concentration. λexc = 334 nm; [Py] = 4.1 × 10−7 M; T = 25.0 °C.
I1) on polymer concentration. As depicted in this figure, at a PEG40S concentration of ∼2.0 × 10−6 M the value of Iex/I1 remarkably increases compared to that found in bulk water. Such an increment confirms the formation of the (PyPy)* excimer and, therefore, the existence of a confined microenvironment below the CMC where the interaction between a ground-state and an excited-state pyrene monomer can be facilitated. At a PEG40S concentration of ∼2.0 × 10−5 M a maximum value for Iex/I1 is reached followed by a decline of the 11254
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Figure 6. Typical example of the signal intensity decay for a series of 1H NMR 1D acquisitions on PEG-40 stearate (δ = 3.59 ppm) and water (δ = 4.64 ppm) at an increasing gradient strength (top) and fit to the Stejskal−Tanner equation at δ = 3.59 ppm (bottom). [PEG40S] = 6.00 × 10−4 M; T = 25.0 °C.
aggregation.38,39 Such changes may consist of either the variation of chemical shifts, line widths, line shapes, or signal intensities of the 1H NMR spectrum. Likewise, one major advantage of using this spectroscopic technique is the absence of any probe species, since as mentioned before the presence of additional reagents may raise doubts about the events observed in the submicellar region. Hence, 1H NMR DOSY (diffusionordered spectroscopy) has been employed in order to quantify the size of PEG40S aggregates in aqueous solution, since this property can reflect the state in which the molecules are organized. DOSY provides an accurate and noninvasive measurement of molecular diffusion in media such biofluids, complex chemical mixtures, and multicomponent solutions. The experiments are performed by recording consecutive 1H NMR spectra in which the gradient strengths are increased in a series of 1D proton acquisitions. By analyzing the evolution of signal intensity vs gradient for a given chemical shift, an exponential decay arises (Figure 6). This correlation allows us
excimer-to-monomer intensity ratio. This marked decline is justified on the basis of the increasing number of premicelles (below the CMC) or micelles (above the CMC) as polymer concentration is increased. In a crude approximation, it may be assumed that there is a direct proportionality between the Iex/I1 ratio and the local concentration of pyrene monomers. Such a relationship will depend on the rate constants of excimer fluorescence, lifetimes of monomers and excimers, diffusibility of pyrene in the host matrix, and geometrical factors.37 However and, as stated previously, in some cases it may not be easy to distinguish between the formation of premicelles and an induced micellization. Therefore, in view of all the results shown above, the self-assembly behavior of PEG40S has been re-analyzed through 1H NMR in order to find further evidence regarding the existence of this premicellar aggregation. Nuclear magnetic resonance has been exploited as a powerful method on studying surfactant aggregation since changes due to the alteration of the chemical environment of amphiphile molecules can provide a direct and strong evidence of premicellar 11255
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pyrene as fluorescence probe (Figure 4 and 5). In the same way, a value for CMC of 2.0 × 10−4 M can be achieved. This result perfectly matches the CMC value obtained through UV− vis and kinetic measurements as well as literature values for similar polymers.29−31 By means of the PMC and CMC values and the expressions for the mole fraction of PEG40S in its free, premicellar, and micellar state (eq 3, 4 and 5), the amount of polymer that can be found free and/or self-assembled can be determined as a function of PEG40S concentration.
to obtain the self-diffusion coefficient of PEG40S at increasing concentrations (see Experimental Section). Figure 7 shows the influence of the polymer concentration on its observed self-diffusion coefficient (Dobs). As shown, Dobs
⎧ ⎪1 if [PEG40S]T < PMC χfree = ⎨ ⎪ ⎩ PMC/[PEG40S]T if [PEG40S]T > PMC
χpmic
(3)
⎧ 0 if [PEG40S]T < PMC ⎪ ⎪1 − PMC/[PEG40S] if PMC < [PEG40S] T T ⎪ = ⎨ < CMC ⎪ ⎪(CMC − PMC)/[PEG40S]T if [PEG40S]T ⎪ ⎩ > CMC (4)
Figure 7. Influence of PEG-40 stearate concentration on its observed self-diffusion coefficient showing a biphasic inhibition pattern. T = 25.0 °C.
⎧ ⎪ 0 if [PEG40S]T < CMC χmic = ⎨ ⎪ ⎩1 − CMC/[PEG40S]T if [PEG40S]T > CMC
decreases as the polymer concentration is increased. This reduction takes place in two different segments. First, Dobs decreases to subsequently remain constant over a significant range. With further raising of the polymer concentration a second drop in the value of Dobs is found. In either case, the lowering of Dobs cannot be attributed to a change in the viscosity of the samples since the water diffusion coefficient has been found to remain constant over the whole range of PEG40S concentrations. Hence, the initial decrease in the value of Dobs should be ascribed to the formation of premicellar aggregates as PEG40S concentration is raised while the subsequent drop in the self-diffusion coefficient (starting at 2.0 × 10−4 M) must be due to the appearance of larger and more structured aggregates such as micelles. Accordingly, the progressive reduction of Dobs as PEG40S concentration is raised may be justified by the slower diffusion of premicelles and micelles compared to that of the free polymer. At extremely low concentrations, PEG40S is exclusively found as a free polymer (i.e., nonaggregated) and the observed self-diffusion coefficient corresponds to that of this form (Dobs = Dfree). However, a NMR sensitivity drawback arises given the reduced range of concentrations at which the polymer remains solely in this state (ca. CMC
As shown below, by applying eqs 3, 4 and 5, eqs 6 and 7 can be rewritten as a function of polymer concentration and critical concentration values (eq 8 and 9): Dobs =
PMC (Dfree − Dpmic) + Dpmic if PMC [PEG40S]T
< [PEG40S]T < CMC
Dobs
(8)
Figure 9. Dynamic light scattering measurements showing the size distribution profile at increasing PEG-40 stearate concentrations (some data omitted for clarity). T = 25.0 °C.
PMC CMC (Dfree − Dpmic) + = [PEG40S]T [PEG40S]T
(Dpmic − Dmic) + Dmic if [PEG40S]T > CMC
(9)
this case, the simultaneous presence of premicelles and micelles is masked by the lack of resolution of the DLS measurements for the given sizes (see Table 1). Slight differences between the results measured by DLS (in H2O) and those obtained by NMR (in D2O) may be ascribed to the variation produced by the well-known solvent effect. Once these submicellar aggregates were unambiguously identified, their uptake ability as lipophilic carrier needed to be tested. With this aim, Nile Red has been used as a drug model molecule given that its intermediate lipophilicity (log P ≈ 3−5) is similar to that of potent chemotherapy agents. NR is a highly fluorescent solvatochromic dye that has been extensively applied to elucidate the structure and dynamics in microheterogeneous and biological systems.40−42 The fluorescence lifetime of NR markedly decreases with the increase of the hydrogen bond donating ability of the medium, since vibrations associated with H-bonding are involved in its emission deactivation process. Therefore, it appears reasonable to expect that an effective encapsulation of NR in either the premicellar or micellar PEG-based aggregates will lead to an increase of the fluorescence intensity of this probe. This assumption is justified on the basis of the fact that NR would lack the ability to engage in hydrogen bonding in the waterexcluded cores in which it would be located. Figure 10 shows the emission fluorescence spectra for NR at increasing PEG40S concentrations. As can be observed, there is a remarkable enhancement in the fluorescence intensity of NR as PEG40S concentration is raised. Such a behavior evidences the inclusion of this fluorophore in a polymer hydrophobic core. This finding is further supported by the solvatochromic effect observed in this figure. It must be noted that the emission spectra of this dye shifts to shorter wavelengths when decreasing the environment polarity. Accordingly, when following the evolution of the maximum of the NR emission peak (λmax) as a function of PEG40S concentration, one should expect a blue shift if an effective encapsulation of the drug model molecule was taking place. The spectral change shown in Figure 10 (where λmax shifts from 660 to 635 nm as PEG40S is increased) reflects the presence of NR in a hydrophobic environment and confirms
The self-diffusion coefficients for premicelles (Dpmic) and micelles (Dmic) can be calculated by eqs 8 and 9, respectively (see Supporting Information). Furthermore, given the direct relationship between the self-diffusion coefficients and the aggregate radii, the size of the different self-assembled structures present in solution can be determined through the use of the Stokes−Einstein equation (eq 10):
D=
kT 6πηrH
(10)
where rH is the hydrodynamic radius and η is the viscosity of the solvent. All of these results are displayed in Table 1. Table 1. Self-Diffusion Coefficients and Diameter Sizes Calculated in This Work for the Self-Assembled Polymer Structures of PEG-40 Stearate free polymer premicelle micelle
D (m2 s−1)
ϕ (nm)
(1.6 ± 0.1) × 10−10 (6.9 ± 0.2) × 10−11 (2.7 ± 0.1) × 10−11
5.8 ± 0.2 14.6 ± 0.4
As stated previously, the smaller aggregate size in the submicellar regime compared to that found for the micelles may constitute an advantage in order to achieve a higher degree of tissue permeation in polymer formulations. The existence of the premicellar aggregates reported herein is further supported by dynamic light scattering measurements (DLS). In this regard, the results shown in Figure 9 evidence the existence of self-assembled structures well below the CMC and the absence of them below the calculated PMC. These observations confirm the presence of premicelles in solution. Likewise, a slight increase in the mean diameter of the polymeric assemblies is observed as PEG40S concentration is raised. This variation is consistent with the transition from premicelles to micelles as CMC is surpassed. It must be also noted that the coexistence of these two types of aggregates in the range ∼2.0 × 10−4 to ∼2.0 × 10−3 M (denoted in Figure 8) is not perceived in Figure 9. In 11257
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stresses the importance of submicellar structures as vehicles of poorly water-soluble drugs.
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CONCLUSIONS A multitechnique analysis of the self-assembly behavior of PEG40S in its high dilution regime was carried out. The obtained results underline the promising role of submicellar structures in drug uptake by polymers. The great potential of these systems as drug-delivery vehicles compared to their corresponding micellar carriers is given by their higher resistance to dilution and, therefore, to their improved circulation time, as well as their smaller size that allows them to achieve a higher degree of permeation at the target site through the EPR effect while keeping their ability to act as nanocarriers hosting poorly soluble drugs. Thus, polymeric premicelles may further extend the efficiency of traditional polymeric micelles as lipophilic vehicles. The results attained in this work suggest that premicellar drug uptake may constitute a promising approach in order to develop new and more efficient polymeric formulations in drug delivery technology.
Figure 10. Influence of PEG-40 stearate concentration on the fluorescence intensity of Nile Red (some data omitted for clarity). λexc = 550 nm; [NR] = 4.1 × 10−7 M; T = 25.0 °C.
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ASSOCIATED CONTENT
S Supporting Information *
the encapsulating ability of the polymer-based aggregates. Furthermore, considering that this shift is initiated at a PEG40S concentration of ∼2.0 × 10−6 M it can be concluded that the encapsulation of the lipophilic drug model can easily take place in the submicellar region by means of polymeric premicellar carriers. This statement is more clearly illustrated in Figure 11
Wavelength of maximum absorbance and transition energy for ET(33) in different media and calculations of the self-diffusion coefficients for premicelles and micelles. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS B.V. and M.P.-L. acknowledge financial support from Isidro Parga Pondal Program (Xunta de Galicia, Spain). This work was funded by the Spanish Ministry of Economy and Competitiveness (Project CTQ2011-22436) and Xunta de Galicia (PGIDIT07-PXIB209041PR, PGIDIT10PXIB209113PR, 2007/085 and INBIOMED-FEDER "Unha maneira de facer Europa").
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REFERENCES
(1) He, Y.; Yalkowsky, S. H. Solubilization of Monovalent Weak Electrolytes by Micellization or Complexation. Int. J. Pharm. 2006, 314, 15−20. (2) Sweetana, S.; Akers, M. J. Solubility Principles and Practices for Parenteral Drug Dosage Form Development. PDA J. Pharm. Sci. Technol. 1996, 50, 330−342. (3) Li, P.; Tabibi, S. E.; Yalkowsky, S. H. Solubilization of Flavopiridol by pH Control Combined with Cosolvents, Surfactants, or Complexants. J. Pharm. Sci. 1999, 88, 945−947. (4) Florence, A. T.; Attwood, D. Physicochemical Principles of Pharmacy, 5th ed.; Pharmaceutical Press: London, 2011. (5) Kim, S.; Shi, Y.; Kim, J. Y.; Park, K.; Cheng, J.-X. Overcoming the Barriers in Micellar Drug Delivery: Loading Efficiency, in Vivo Stability, and Micelle−Cell Interaction. Expert Opin. Drug Delivery 2009, 7, 49−62 and references therein. (6) Kataoka, K.; Matsumoto, T.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Fukushima, S.; Okamoto, K.; Kwon, G. S. Doxorubicin-Loaded Poly(ethylene glycol)-poly(β-benzyl-l-aspartate) Copolymer Micelles: Their Pharmaceutical Characteristics and Biological Significance. J. Controlled Release 2000, 64, 143−153.
Figure 11. Nile Red uptake as a function PEG-40 stearate concentration determined according to the magnitude of NR blueshift (some data omitted for clarity). [NR] = 4.1 × 10−7 M; T = 25.0 °C.
where the drug uptake as a function of polymer concentration is calculated according to the magnitude of NR blue-shift. In this respect, it can be assumed that (i) no uptake occurs at exceptionally low PEG40S concentrations since the wavelength of NR maximum emission corresponds to that found in water (λmax = 660 nm),43 and (ii) a complete encapsulation of the probe is achieved at PEG40S concentration of ∼1 × 10−4 M as no further blue-shift above this concentration is observed. As depicted in Figure 11 a complete encapsulation of NR is attained even before the CMC is reached. This observation 11258
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dx.doi.org/10.1021/la4022273 | Langmuir 2013, 29, 11251−11259
