Drug Encapsulation within Self-assembled Microglobules Formed by Thermoresponsive Supramolecules

Abstract

An 8-(phenyl)-2′-deoxyguanosine derivative self-assembles in aqueous media into discrete hexadecamers that further self-assemble above 32 °C into microglobules that encapsulate the drug doxorubicin.

Advances in supramolecular chemistry have led to the design of a variety of biomimetic materials that are suitable for the development of stimuli responsive nanocarrier systems.^1–3 Light, pH, magnetic fields and temperature are among the most frequently used stimuli.⁴ Nano- and microscopic globular assemblies made from thermoresponsive polymers are likewise promising systems for responsive drug carriers.^{5, 6} Besides the Elastin-Like Polypeptides,⁷ most of the work in this area has relied on the poly(N-isopropylacrylamide)⁸ scaffold, and its copolymers, as environmentally responsive materials due to their sharp coil-to-globule transition at biocompatible temperatures of around 32 °C.⁹

Supramolecular self-assembly offers a complement ary strategy to the use of polymers for the development of functional nanostructures.¹⁰ To circumvent some of the limitations shown by polymers (e.g., polydispersity, lack of self-correcting synthesis) and to obtain new thermoresponsives scaffolds, recently, our lab developed an 8-(meta-acetylphenyl)-2′-deoxyguanosine (mAG) derivative that self-assembles in aqueous media into discrete supramolecular hexadecamers that exhibit the Lower Critical Solution Temperature (LCST) phenomenon.¹¹ Such LCST phenomenon occurs with a transition temperature (T_t) of 58 °C, above which the supramolecular hexadecamers engage in a temperature induced assembly to form solid nanoscopic globules of low polydispersity.¹¹ We hypothesized that these globules could provide a versatile scaffold for host-guest recognition in aqueous media. Furthermore, if the T_t were reduced to a value closer to and below body temperature, these systems may become suitable to prepare thermoresponsive nano- or microcarriers for bioactive materials such as drugs. Herein, we report our initial attempts towards achieving these goals.

Controlling the T_t via intrinsic parameters (i.e., structural information in the building blocks of supramolecules) enables the reliable construction of nanostructures of well-defined size and composition. In recent years we have developed a family of 8-aryl-2′-deoxyguanosine (8ArG) derivatives as versatile recognition motifs for the construction of supramolecular nanostructures in both organic^12,13 and aqueous media¹⁴ (Scheme 1). Our studies suggest that properly placed functional groups can increase the stability and specificity of the resulting G-quadruplex supramolecules by enhancing non-covalent interactions such as hydrogen bonds and pi-stacking.¹⁵ To this end, we synthesized the 8-(meta-ethoxycarbonylphenyl)-2′-deoxyguanosine (mECGD2OH, 1) derivative, which shows a lower T_t than our previous mAG-based system. This new 8-(meta-carbonylphenyl)-2′-deoxyguanosine derivative (1) was synthesized using a methodology similar to that previously reported by us.¹¹ The pure derivative 1 was obtained after column chromatography as confirmed by NMR, IR, and mass spectrometry (for details see ESI^†, Fig. S3-S4). Monomer 1 is poorly soluble at at room temperature (c.a., 23 °C) in an aqueous phosphate-buffered solution (pH 7.4), but goes in solution upon addition of KI (2 M). ¹H NMR experiments of homogeneous solutions of 1 (10 mM, pH 7.4, 2 M KI) in H₂O-D₂O (9:1, potassium buffer) reveal the signatures for its self-assembly. The ¹H NMR spectrum shows the characteristic double set of signals corresponding to two pairs of tetrads in different chemical environments (Fig. 1). The 2D NOESY spectrum further supports the formation of 1₁₆, by showing the signature cross peaks characteristic of a hexadecamer in water (ESI, Fig. S6).¹⁴

Scheme 1.

(a) Representation for the (a) formation of a hexadecamer by mECGD2OH (1) in aqueous media; and (b) stimuli-responsive behavior of 1₁₆ that enables the encapsulation of Doxorubicin·HCl (DOX) within the resulting microglobule.

Fig. 1.

Partial ¹H NMR spectrum showing the region of the N1-H and aromatic signals of 1₁₆(500 MHz, 10 % D₂O in phosphate-buffered solution, pH 7.4, 2 M KI). The red cartoon represents 1₁₆.

The thermoresponsiveness of 1₁₆ was determined by transmittance experiments. Aqueous phosphate-buffered solutions (pH 7.4, 2 M KI) of 1₁₆ were completely homogeneous and highly soluble at room temperature. The turbidity for solutions of 1₁₆ increased upon heating, becoming cloudy at ~ 32 °C. Transmittance measurements at 500 nm confirm these observations (Fig. 2a). Upon cooling it back to 25 °C, the solution again becomes transparent. This process is reversible and can be repeated at least ten times with no signs of fatigue (e.g., decomposition, irreversible aggregation) (Fig. 2b). Dynamic light scattering (DLS) studies provide essential information regarding the sizes of the various supramolecules as a function of temperature (25–76 °C). Below the T_t, the average hydrodynamic diameter (D_H) for 1₁₆ is 5.0 nm (Fig. 2c). Upon reaching T_t, the D_H of the newly formed species in solution increased by four orders of magnitude to >10 μm. Continued heating of the solution from 40 to 60 °C leads to a gradual decrease of the D_H until the values stabilized at ~ 9 μm. This behavior is consistent with a steady dehydration of the globules. Conversely, the sudden increase in size above 70 °C and the concomitant increase in polydispersity are likely due to the formation of amorphous aggregates upon the melting of 1₁₆ (Fig. S9, ESI^†). Optical microcopy (OM), with temperature control (37 °C), showed that these aggregates are discrete microglobules with a relatively uniform distribution of sizes (Fig. 2d).

Fig. 2.

(a) Turbidity curve (measured at 500 nm) for 1₁₆ (red circles). (b) Change in transmittance at 500 nm when alternating the temperature between 25 °C and 35 °C. (c) Average hydrodynamic diameters (D_H) of 1₁₆ as a function of temperature measured by DLS. (d) OM micrograph of 1₁₆ at 37 °C. All measurements were performed using an aqueous phosphate-buffered solution (pH 7.4, 2 M KI) of 1 (10 mM). The white scale bar represents 20 μm.

We next assessed the influence of binding a guest molecule on the thermoresponsive properties from 1₁₆. Doxorubicin hydrochloride (DOX) was chosen as the guest molecule because its current use as anticancer agent and its inherent fluorescence properties.¹⁶ Previously, DOX has been conjugated to different carrier molecules such as peptides¹⁷ and others macromolecules.¹⁸ Its fluorescence properties has enabled monitoring its distribution, or that of its polymer conjugates, in micelles and even in cells.¹⁹ As shown in Fig. 3a, in the absence of DOX, 1₁₆ exhibited a T_t of around 32 °C (pH 7.4). However, in the presence of the drug (2 equivalents of DOX per hexadecamer) the T_t increases modestly by up to 2 °C. DOX, which possesses an anthraquinone moiety, could potentially interact with the core of 1₁₆ in a manner reminiscent to that of daunomycin, a similar anthraquinone drug that interacts with G-quadruplex DNA structures.²⁰ This behavior agrees with our previous report using a hexadecamer related to 1₁₆, in which we showed the tuning of the T_t to higher values by its co-assembly with a more hydrophilic derivative.¹¹ Additional DOX, however, does not seem to further increase the T_t, underscoring the potential versatility of this system to encapsulate other drugs with little effect in the thermoresponsive properties.

Fig. 3.

(a) Effect of increasing the amount of DOX in the T_t of 1₁₆. (b) Absorption spectra for DOX alone (3.13 mM) and 1₁₆ (10 mM) with 5 equivalents of DOX below (25 °C) and above (40 °C) T_t. The solutions of 1₁₆ with DOX were incubated (45 min) below or above the T_t and then centrifuged. The supernatant was diluted (800×) with the same buffer (phosphate buffer, pH 7.4, 2 M KI) and the absorption spectra of the diluted solutions were measured. (c) Fluorescence microscopy images of DOX encapsulated in the globules at 37 °C. The white scale bar represents 20 μm.

Sedimentation experiments provide further evidence for the encapsulation of DOX within the globules. Host 1₁₆ (0.625 mM) was mixed with DOX (5 equivalents) in a phosphate solution (pH 7.4, 2 M KI) and incubated (45 min) either below or above the T_t. Afterwards, the solution was centrifuged and the absorbance of the supernatant was measured. The spectra of the solutions of DOX alone and with 1₁₆, after incubation below the T_t followed by centrifugation, were practically identical (Fig. 3b). In contrast, after incubation above the T_t (40 °C) followed by centrifugation, the supernatant registered a decrease in the absorption from DOX. This indicates that the sedimented globules retain the molecules of DOX. About 75% of DOX was encapsulated within the globules as inferred by the diminished absorbance at 491 nm. Fluorescence microscopy studies are also consistent with the encapsulation of DOX within the microglobules (Fig. 3c).

In summary, we have demonstrated that discrete thermosensitive supramolecules assembled from 8ArG derivatives are an attractive and complementary strategy to polymer based systems for drug encapsulation at biocompatible temperatures. The scope and limitations of related systems for the encapsulation of other bioactive molecules are currently underway.