Anticancer drug delivery systems based on specific interactions between albumin and polyglycerol

Abstract

Here, we report for the first time a facile method for the preparation of novel drug delivery systems based on supramolecular interactions between β-cyclodextrin-polyglycerol conjugates (β-CD-g-PG) and human serum albumin (HSA). The results obtained by combined experimental/modeling studies showed that the main drivers to the formation of HSA/β-CD-g-PG supramolecular entities are host–guest interactions between β-CD-g-PG and the aromatic side-chains of HSA residues. Due to these interactions, HSA undergoes a confined yet fundamental conformational transition, leading to greater exposure of the hydrophobic protein domains available to hydrophobic drug binding. Next, the binding affinity and loading capacity of the HSA/β-CD-g-PG supramolecular nanovector for doxorubicin (DOX) and paclitaxel (PTX) were investigated. Both drug/HSA binding constants (K_b,HSA/DOX = 3.24 × 10³ M⁻¹ and K_b,HSA/PTX = 5.65 × 10¹ M⁻¹) sensibly increased in the presence of β-CD-g-PG (K_{b,HSA/β-CD-g-PG/DOX} = 2.78 × 10⁴ M⁻¹ and K_{b,HSA/β-CD-g-PG/PTX} = 2.82 × 10² M⁻¹). In line, both protein drug loadings increased by about 20% upon HSA/β-CD-g-PG interaction (80% and 71% for DOX and PTX, respectively) with respect to the loading capacity of the bare HSA (60% and 50%). Due to the improved loading capacity with minimal changes in the structure of HSA, this system is a promising vector for future cancer therapy.

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Introduction

Conventional drug delivery systems (CDDs) are becoming less efficient due to the frequent emergence of immunogenicity effects upon administration. To avoid this and other problems associated with CDDs, in recent years proteins or their conjugated structures have been proposed as carriers for therapeutic agents.¹ Biohybrid materials consisting of proteins and synthetic polymers have received much attention, since these conjugates present attractive features such as efficient transport and improved pharmacokinetic properties.² Among possible proteins, albumin, the main transporter and major protein component of blood, has been found to be a versatile, nontoxic, non-immunogenic, biocompatible and biodegradable carrier for drug delivery.³

Human serum albumin (HSA) plays a key role in maintaining blood osmotic pressure and in acting as one of the main transporter of both endogenous and exogenous ligands, such as bilirubin, fatty acids, ions, and drugs.⁴ The tertiary structure of this mainly-helical protein resembles a heart shape, with approximate dimensions of 8 × 8 × 3 nm, and consists of three domains: domain I (residues 1–195), II (residues 196–383), and III (384–585). Each domain, in turn, features two subdomains (A and B), and 17 disulphide bridges stabilize the whole protein structure. As a transport protein, HSA possesses several binding sites for its ligands.⁵ Specifically, aromatic and heterocyclic ligands are found to bind within two hydrophobic pockets in subdomains IIA and IIIA, namely site I and site II.^5,6 Contextually, seven binding sites for fatty acids are localized in subdomains IB, IIIA, IIIB and on the subdomain interfaces.⁵ Taken together, HSA possesses a unique set of properties, which make it well suited for development as drug nanocarrier.

On the other hand, hyperbranched polyglycerols are water soluble, biocompatible, and multivalent materials with many potential applications ranging from drug delivery to catalysis.⁷ Specifically, the protein-resistance property of polyglycerol is one of the most important feature of this polymer in biomedical applications. Due to this property, nonspecific interactions between polyglycerol and proteins and, consequently, its immunogenicity are very low.⁸ Cyclodextrins (CDs) are torus-shaped cyclic oligosaccharides made of six, seven, or eight glycosidic units linked by α(1 → 4) bonds and are called α-, β-, and γ-CD, respectively. This molecular structure has the capability to make inclusions (i.e., guest–host) compounds with aliphatic and especially aromatic molecules. Cyclodextrins are used in pharmaceutical applications for numerous purposes, including improving the stability, bioavailability, and delivery of drugs.

Accordingly, in this work we conceived the idea of exploiting all beneficial properties of both polyglycerol and β-cyclodextrin by creating cyclodextrin-polyglycerol conjugates (β-CD-g-PG) having special site for specific interactions.⁹ In fact, due to the particular supramolecular chemistry for each subdomain of albumin, it interacts with different objects specifically.¹⁰ As a result, we expect that β-CD-g-PG conjugates will interact with albumin mainly via cyclodextrin and host–guest interactions. Under this hypothesis, these specific interactions between albumin and β-CD-g-PG should lead to a hybrid system with useful properties originating from both materials.

Thus, aim of the present study is to verify these assertions and demonstrate the potentialities of these novel HSA/β-CD-g-PG host–guest systems for delivery and release of anticancer drugs. Our molecular design is indeed oriented toward the development of low-cost, easy to fabricate, easy to use and robust drug delivery systems. Under this perspective, we designed the present biohybrid-nanomaterials, which can be even produced by in situ administration of the precursors. All supramolecular anticancer drug delivery systems synthesized in this work were also characterized and investigated by various spectroscopic techniques including fluorescence, dynamic light scattering (DLS), ultraviolet-visible (UV-vis), circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Molecular simulations were also employed to shed light on the possible mechanism of interactions between all components of these novel supramolecular drug delivery systems. Further, we examined the interaction of two hydrophobic anticancer drugs – doxorubicin and paclitaxel – to gain further information on the effects of drug molecular structure on the HSA/β-CD-g-PG/drug complex formation. Based on the overall experimental and simulation evidences obtained, the present results may provide new insights on the rational design of natural supramolecular drug carriers. To the best of our knowledge, this is the first report of host–guest interactions between albumin and β-CD-g-PG to produce a novel and effective anticancer drug delivery system.

Experimental

Full information concerning the used materials, instruments, synthesis procedures and methods for simulation studies are available on the experimental section of the ESI.†

Results and discussion

Characterization of HSA/β-CD-g-PG supramolecular entities

β-CD-g-PG macromolecules with different molecular weights and functionality were prepared according to the procedure already reported in literature¹¹ (Scheme 1). Based on our previous works and on further experiments (data not shown), the optimal polyglycerol/β-cyclodextrin conjugate was obtained by using 10 mol glycidol per mole of the hydroxyl functional group of cyclodextrin, following the synthetic route summarized in Scheme 1.

Scheme 1 Preparation of β-CD-g-PG conjugates.

For the preparation of the HSA/β-CD-g-PG supramolecular entities (Fig. 1), β-CD-g-PG was added to a phosphate buffered saline (PBS) solution of HSA, and the resulting mixture was gently shaken for 1 h (Fig. 1). Host–guest interactions between HSA and β-CD-g-PG were investigated using a combination of different spectroscopy methods and molecular simulation techniques.

Fig. 1 Host–guest interactions between HSA and β-CD-g-PG leading to HSA/β-CD-g-PG supramolecular entities.

Circular dichroism (CD) spectroscopy analysis. Circular dichroism (CD) spectroscopy is a widely used method to determine protein conformation, structure and stability in solution, and to monitor interactions between proteins and other molecules. Accordingly, CD spectra of HSA alone and in the presence of β-CD-g-PG at increasing concentrations were recorded to decipher the structural and conformational changes, if any, exerted by β-CD-g-PG on the secondary structure of the protein (Fig. 2). As seen in this figure, the CD spectrum of native albumin exhibits two negative absorption bands in the far-UV region, peaking at 208 nm (π–π*) and 222 nm (n–π*), characteristic of the α-helix portions of the protein.¹² The addition of β-CD-g-PG to the HSA solution results in minimal reduction in band intensity at all wavelengths of the relevant CD spectra, without any discernible shift of the band maxima. This increase in ellipticity suggests a stabilization of the complex with respect to free HSA. In other words, the intermolecular interactions between β-CD-g-PG and HSA do not substantially perturb the β-helical motifs of the protein to an extent to leave a signature on the relevant CD spectra. This, in turn, implies that the β-CD-g-PG molecules cannot penetrate inside the structure of HSA but, rather, they bind to the protein surface. This somewhat expected result can simply rationalized considering that HSA is a single polypeptide chain characterized by the presence of 17 disulfide bridges, located are regular intervals along the protein structure. Accordingly, in addition to its general compact folded nature, HSA flexibility is considerably restricted by virtue of this network of S–S links. Hence, when β-CD-g-PG interacts with HSA in solution, the protein native structure is not expected to unfold to a considerable extent due to β-CD-g-PG surface binding.

Fig. 2 Far-UV CD spectra of HSA (2 × 10⁻⁶ M) in the absence (a) and presence of different amounts of β-CD-g-PG (b–e). β-CD-g-PG concentrations from (b) to (e): 0.26 × 10⁻³, 0.52 × 10⁻³, 1.05 × 10⁻³, and 2.1 × 10⁻³ M. T = 37 °C.

In agreement with these confined spectral perturbations, the calculated α-helical content of HSA decreases only slightly, i.e., from 59.2% for pristine HSA to 52.5% upon formation of the HSA/β-CD-g-PG supramolecular entity (Table 1). Correspondingly, also the other motifs characterizing the secondary structure of HSA manifest only minor changes in their values. Notably, HSA is a dynamic, rapidly moving, “breathing” molecule having a rotational diffusion coefficient of 20 ns (ref. 4) and it is known to have some degree of flexibility, expansion, and contraction, which enables significant conformational transitions; hence, the precise, average shape that HSA takes on in aqueous solution is still under debate.^13,14 Therefore, the structural perturbations exerted on HSA by β-CD-g-PG upon binding are indeed negligible.

Table 1 Amount of different secondary structural motifs of free HSA and in the HSA/β-CD-g-PG complexes as derived from the corresponding CD spectra. [β-CD-g-PG] = 2.1 × 10⁻³ M

Structure	HSA	HSA-β-CD-g-PG
α-Helix	59.2%	52.5%
β-Antiparallel	3.8%	4.7%
β-Sheet	4.6%	5.4%
β-Turn	12.9%	13.8%
Random coil	19.5%	23.5%

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Steady-state fluorescence spectroscopy analysis. Since circular dichroism does not provide details regarding the precise structure of proteins, fluorescence techniques – which are more sensitive in detecting small changes at molecular level – were used to gain further structural information about these supramolecular systems.¹⁵

Generally, fluorescence of proteins originates from three residues namely tryptophan, tyrosine, and phenylalanine. Actually, the intrinsic fluorescence of HSA is mainly contributed by tryptophan 214 alone (located in the protein subdomain IIA), whereas the emission of phenylalanine and tyrosine is negligible due to their very low quantum yield.¹⁶ The fluorescence of Trp-214 may change when HSA interacts with other molecules, which could be reflected in its fluorescence spectra in the UV region. Here, the fluorescence quenching of HSA in the presence of various concentrations of β-CD-g-PG was measured to shed some light in the quenching mechanism and the relevant, underlying intermolecular interactions.

As shown in Fig. 3, the intensity of the characteristic broad emission band of HSA at 345 nm regularly decreases with increasing β-CD-g-PG, concentration, with a detectable bathochromic shift of the emission maximum of HSA by 5 nm. Accordingly, the quenching and the red shift in the HSA emission maxima not only confirm the onset of intramolecular interactions between β-CD-g-PG and the protein, but also imply that β-CD-g-PG is introducing an increase in the polarity of the micro-environment around the Trp-214 moiety. Eventual dilution effect upon addition of β-CD-g-PG solutions can be ruled out, because the volume of solution containing HSA (4 mL) is far larger than the β-CD-g-PG solution (≤200 μL).

Fig. 3 Quenching of HSA fluorescence (a) upon increasing β-CD-g-PG concentration (b–k) at 37 °C and pH 7.4. β-CD-g-PG concentrations from (b) to (k): 0.26, 0.52, 0.79, 1.05, 1.316, 1.58, 1.84, 2.10 and 2.37 × 10⁻³ M. HSA concentration is 2 × 10⁻⁶ M.

Fluorescence quenching is usually classified as dynamic (DQ) or static (SQ). Generally, the two mechanisms can be distinguished by their different dependence on temperature. Higher temperatures result in faster diffusion and larger amounts of collision quenching, and will typically lead to the dissociation of weakly bound complexes and smaller amount of static quenching.¹⁷ Accordingly, the value of the quenching constant k_q should increase for DQ while it should decrease for SQ with increasing temperature.¹⁸ To demonstrate the quenching mechanism, the fluorescence quenching data were analyzed using the well-known Stern–Volmer equation:¹⁹

F₀/F = 1 + k_qτ₀[Q] = 1 + K_SV[Q] (1)

in which F and F₀ are the fluorescence peak intensities of the fluorophore (i.e., Trp-214 in HSA) in the absence and presence of quencher, respectively, k_q is quenching rate constant of HSA, τ₀ is the average life time of the protein (5.6 ns for HSA),²⁰ and [Q] is the concentration of the quencher (β-CD-g-PG in this case). The product of k_q and τ₀ is also known as the Stern–Volmer quenching constant K_SV. The Stern–Volmer plot for the quenching of HSA by β-CD-g-PG at 37 °C is shown in Fig. 4.

Fig. 4 Stern–Volmer plots for the quenching of HSA by β-CD-g-PG in PBS solution at 37 °C (A) and at different temperatures (B). [HSA] = 2 × 10⁻⁶ M.

As can be seen in Fig. 4A, the value for k_q calculated using eqn (1) is equal to 7.65 × 10¹² M⁻¹ s⁻¹. Since this value is much larger than the maximum collisional quenching constant (2.06 × 10¹⁰ M⁻¹ s⁻¹), SQ is the dominant quenching mechanism for the HSA/β-CD-g-PG supramolecular complex.^21,22 To further verify this assertion, the dependence of k_q on temperature was investigated. Accordingly, the Stern–Volmer plots for the quenching of HSA by β-CD-g-PG at three different temperatures (i.e., 20, 30, and 37 °C) are displayed in Fig. 4B. The calculated values of the quenching constant at 20 and 30 °C are 1.35 × 10¹³ M⁻¹ s⁻¹ and 8.91 × 10¹² M⁻¹ s⁻¹, respectively. The inverse correlation of k_q with temperature supports the evidence that the fluorescence quenching of HSA by β-CD-g-PG was initiated by complex formation between HSA and β-CD-g-PG rather than by dynamic collision between the two macromolecules.^23,24

For stating quenching and under the assumption that there are n equivalent and independent binding sites for the quencher (Q) on the protein, the binding constant K_b for β-CD-g-PG onto HSA can be calculated according to the modified Stern–Volmer equation:^16,25

log[(F₀ − F)/F] = log K_b + n log[Q] (2)

where F and F₀ denote steady-state fluorescence of HSA with and without quencher, respectively, K_b is the binding constant, and [Q] is the concentration of the quencher (β-CD-g-PG in this case) (see Fig. 5). The results obtained are listed in Table 2.

Fig. 5 Modified Stern–Volmer plot for the quenching of HSA by β-CD-g-PG in PBS solution at different temperatures. [HSA] = 2 × 10⁻⁶ M.

Table 2 Binding parameters of β-CD-g-PG and HSA at different temperature. The values of K_b and n were obtained from data fitting in Fig. 5. The corresponding ΔG_b values were obtained via eqn (3)

T (°C)	Kb (M^−1)	n	R^2	ΔGb (kcal mol^−1)
20	879	1.14	0.997	−3.95
30	331	1.05	0.975	−3.49
37	244	1.02	0.997	−3.39

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It can be seen from Table 2 that the supramolecular complexes between HSA and β-CD-g-PG can be easily formed at all temperatures; also, the number of binding sites n is always close to 1, indicating that there is only 1 β-CD-g-PG binding site on the protein surface. Interestingly, the calculated K_b values in Table 2 are in excellent agreement with the K_b of 185 M⁻¹ recently reported by Gosh et al.²⁶ for the binding of unmodified β-cyclodextrin (β-CD) to HSA at room temperature. Moreover, the highest affinity of β-CD-g-PG towards the protein with respect to the unmodified β-CD suggests a beneficial effect of the polyglycerol substituent in promoting the formation of the HSA/β-CD-g-PG supramolecular assembly.

Nature of binding interactions between HSA and β-CD-g-PG. The intermolecular binding forces between a protein and its endogenous or exogenous ligands, contributing to favorable, and hence negative values of the corresponding free energy of binding ΔG_b, defined as:

ΔG_b = ΔH_b − TΔS_b = −RT ln K_b (3)

include van der Waals and hydrophobic interactions, electrostatic forces, hydrogen bonds. Also, solvation and entropic effects play a major role in determining the binding affinity in a protein/ligand complex formation. According to Ross and Subramanian,²⁷ for intermolecular complexes dominated mainly by hydrophobic interactions, both the enthalpic (ΔH_b) and the entropic (TΔS_b) components of ΔG_b should be positive, whereas negative values of ΔH_b and TΔS_b should characterize binding governed by hydrogen bonds and/or van der Waals interactions. Lastly, small-positive or negative values of ΔH_b and positive TΔS_b values should accompany protein/ligand binding driven by Coulombic forces although, in all cases, the concomitant albeit differential contribution of all intermolecular interactions cannot be entirely ruled out. In the limited range of temperature values such as those considered in this work (i.e., ΔT = 17 °C), the variation of enthalpy with temperature can be neglected, so that the enthalpy variation for β-CD-g-PG binding to HSA can be calculated by applying van't Hoff equation:in which R is the gas constant (1.987 cal mol⁻¹ K⁻¹), T₁ and T₂ are two absolute temperature values (K), and K_b,1 and K_b,2 are the binding constant at T₁ and T₂, respectively.

Thus, choosing T₁ = 293 K (20 °C) and T₂ = 310 K (37 °C), and inserting the corresponding values of K_b,1 and K_b,2 (Table 2) into eqn (4), the relevant value of ΔH_b can be estimated. Also, using the corresponding ΔG_b values in Table 2, the entropic component TΔS_b can be readily obtained from eqn (3) as:

TΔS_b = ΔH_b − ΔG_b (5)

The first three columns of Table 3 show the results of this analysis. As can be seen, both the enthalpic and the entropic component of the free energy of binding ΔG_b of β-CD-g-PG onto HSA are negative, implying that the binding process between the modified β-CD and the protein is predominantly enthalpic, while the entropic variation opposes binding, as most often seen for ligand/protein complexation.²⁸ Also, the equally negative sign of ΔH_b and TΔS_b suggests that van der Waals, hydrophobic, and hydrogen bond intermolecular interactions are prevalently leading the HSA/β-CD-g-PG binding process, as it could be expected given the nature of the receptor and its ligand (vide infra).

Table 3 Enthalpy (ΔH_b), entropy (TΔS_b), and free energy of binding (ΔG_b) for the HSA/β-CD-g-PG supramolecular entity at different temperatures. ΔH_b and TΔS_b values were obtained via eqn (4) and (5) (see text for details). The values of ΔH_b,sim, TΔS_b,sim, and ΔG_b,sim obtained from molecular dynamics (MD) simulations of the HSA/β-CD-g-PG complex at 37 °C are reported for comparison

T (°C)	ΔHb (kcal mol^−1)	TΔSb (kcal mol^−1)	ΔGb (kcal mol^−1)
20	−13.61	−9.66	−3.95
30		−10.12	−3.49
37		−10.22	−3.39

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Ultraviolet-visible (UV-vis) absorption analysis. The structure of the HSA/β-CD-g-PG supramolecular assembly was further investigated by UV-vis measurements. The strong absorption peak at 218 nm originates from the absorption of π → π* transition of the >C=O group of HSA residues, whereas the weak absorption peak at 278 nm results from the adsorption of the protein aromatic residues (mainly Trp, and eventually Tyr, and Phe) (curve a in Fig. 6).^18,29 With the addition of β-CD-g-PG, the intensity of the sharp peak at 278 nm moderately and gradually increases but no shift in its position is observed.

Fig. 6 UV-visible spectra of free HSA (a) and in the presence of different concentrations of β-CD-g-PG (b–k). β-CD-g-PG concentrations from (b) to (j): 0.26, 0.52, 0.79, 1.05, 1.31, 1.58, 1.84, 2.1, 2.37, and 2.6 × 10⁻³ M respectively. [HSA] = 2 × 10⁻⁶ M. T = 37 °C.

This is in harmony with the small structural perturbation on the overall secondary structure of HSA exerted by β-CD-g-PG upon binding. Contextually, the second, broad peak also increases with a progressive slight shift towards lower wavelengths, suggesting a moderate change in the microenvironment around Trp, in keeping with the results obtained from fluorescence quenching evidences. The entity of these spectral variations is another confirmation of the non-covalent nature of intermolecular interactions underlying the complex formation between β-CD-g-PG and HSA.

One additional method to distinguish static and dynamic quenching is by careful examination of the absorption spectra of the fluorophore. Collisional quenching (or DQ) only affects the excited states of the fluorophore and thus no changes in the absorption spectra are expected. In contrast, ground-state complex formation (i.e., SQ) will frequently result in perturbation of the absorption spectrum of the fluorophore.²³ To reconfirm that quenching mechanism of fluorescence of HSA by β-CD-g-PG is initiated by ground-state complex formation, we performed UV-visibile (UV-vis) adsorption experiments of HSA, β-CD-g-PG, and HSA/β-CD-g-PG system (Fig. 7). As seen in Fig. 7, the intensity of both HSA peaks (at 218 nm and 278 nm) slightly increases upon addition of β-CD-g-PG, suggesting the existence of an HSA/β-CD-g-PG ground state complex and, therefore, the static mechanism of HSA fluorescence quenching by the polyglycerol-modified β-CD. Concomitantly, the invariance of the maximum absorption wavelengths implies that the interaction between β-CD-g-PG and HSA is non-covalent in nature.

Fig. 7 Absorption spectra of (a) β-CD-g-PG (6.0 μM), (b) HSA (3.0 μM), and (c) HSA/β-CD-g-PG (6.0 μM) at 37 °C. The absorbance spectra for the wavelength range from 250 to 300 nm are depicted in the inset.

The value of the binding constant K_b for the formation of the supramolecular complex between HSA and β-CD-g-PG obtained from fluorescence quenching experiments (eqn (2)) can be validated using UV-vis data in Fig. 6 (see ESI and Fig. 6S† for details). Considering the different sensitivity and approximations underlying the two spectroscopic techniques, the corresponding K_b value of 419 M⁻¹ is in agreement with the one obtained by fluorescence spectroscopy (K_b = 244 M⁻¹, Table 2), thereby confirming the moderate strength of the intermolecular, non-covalent interactions between HSA and β-CD-g-PG.

Dynamic light scattering (DLS) and zeta-potential (ζ) analysis. Dynamic light scattering (aka photon correlation spectroscopy or quasi-elastic light scattering) is yet another spectroscopic technique that can be used to determine the size distribution profile of small particles in suspension or (macro)molecules in solution.³⁰ Therefore, in the present work DLS measurements were employed to estimate the average hydrodynamic diameter d_h of HSA, β-CD-g-PG and their supramolecular assembly in solution, as shown in Table 4 and Fig. 7S.†

Table 4 DLS average hydrodynamic diameter d_h (nm) and zeta-potential ζ (mV) of HSA, β-CD-g-PG, and their supermolecular assembly in solution. pH = 7.4, T = 37 °C

Compound	dh (nm)	ζ (mV)
HSA	6	−7.27
β-CD-g-PG	3	−1.23
HSA-β-CD-g-PG	11	−9.40

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As seen, the hydrodynamic diameter of native HSA was found to be 6 nm at pH = 7.4 and at 37 °C, in good agreement with previous literature reports.³¹ With the addition of β-CD-g-PG, the d_h value of the resulting complex increases to 11 nm, a value somewhat larger than the sum of the hydrodynamic diameters of the pure components. Given the small structural change of HSA induced by β-CD-g-PG upon binding, the increase in the HSA/β-CD-g-PG supramolecular complex dimension cannot therefore be ascribe to a substantial unfolding of HSA but, rather, to an extended conformation of the polyglycerol chains of β-CD-g-PG into the solvated environment. This result, as discussed later in detail, could be an expected result since the polyglycerol derivative of β-CD can be though of as composed of two molecular moieties: the relatively hydrophobic cyclodextrin cavity and a the polar outer shell, whose hydrophilicity is enhanced by the presence of the PG chains. Accordingly, the former tend to interact with HSA, thereby forming the necessary stabilizing network of hydrophobic, van der Waals and hydrogen bond interactions with the residues lining the binding pocket of HSA. At the same time, the latter can potentiate the binding by shielding these interactions from water.

The zeta potential of a protein is correlated to its electric surface properties, which, in turn, are generated from presence of ionized residues (e.g., aspartic and glutamic acid, arginine and lysine). As such, the effective charge and, hence, the value of ζ on a given protein is affected by pH, ionic strength, and the eventual accumulation of ligand on the protein surface. At pH 7.4, the three homologous, globular domains of native HSA in solution carry a net charge of −9e (domain I, N-terminal), −8e (domain II), and +2e (domain III, C-terminal), giving a total net charge for the protein of −15e (when calculated from the amino acid composition) or −19e if measured experimentally (due to additional bound ions) at pH 7.4.³² In line, the present value of ζ measured for HSA is negative, and equal to −7.27 mV (Table 4). The addition of β-CD-g-PG (ζ = −1.23 mV) results in a ζ value for the supramolecular complex of −9.40 mV. The decrease in the ζ-value upon complex formation supports the main dispersive nature (i.e., hydrophobic, hydrogen bond and van der Waals interactions) of the intermolecular forces leading to the HSA-β-CD-g-PG supramolecular assembly.

Molecular modeling of the HSA/β-CD-g-PG supramolecular assembly. In order to support the experimental data and to gain a better understanding of the interaction between HSA and β-CD-g-PG at the molecular level, the HSA/β-CD-g-PG supramolecular assembly was further investigated by molecular modeling (see ESI† for detailed information). Docking calculations followed by atomistic molecular dynamics (MD) simulations showed that β-CD-g-PG binds within a loop in the subdomain IIA of HSA (residues Q228–E232), the hydrophobic aromatic side chain of F230 being perfectly encased in the cavity of the β-CD moiety (Fig. 8A).

Fig. 8 (A) Structure of the HSA/β-CD-g-PG supramolecular assembly as obtained by molecular simulations. The protein is depicted as golden ribbons while the β-CD-g-PG molecule is shown as atom-colored sticks-and-balls and encased in its van der Waals surface (color legend: gray, C; red, O). Oxygen water atoms, chlorine and sodium ions are shown as cyan, green, and purple spheres, respectively. Hydrogen atoms are omitted for clarity. Comparison of equilibrated MD snapshots of the HSA/β-CD-g-PG (B) and HSA/β-CD (C) complexes using the same representation style and color scheme of panel (A). (D) Binding free energy (ΔG_bind) and its deconvolution in its contributing components for HSA/β-CD-g-PG and HSA/β-CD complexes.

Next, by applying the well-validated MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area) approach³³ the free energy of binding (ΔG_b) between the protein and the cyclodextrin derivative was calculated (see ESI†). The relevant ΔG_b value of −3.57 kcal mol⁻¹ at T = 37 °C, in excellent agreement with the corresponding experimental value (Table 3) confirms that β-CD-g-PG binds HSA with a moderate affinity. The deconvolution of ΔG_b into its components (see ESI† for details) further reveals that the net electrostatic contribution to the affinity of the complex is significantly unfavorable. Specifically, the mean value of the total electrostatic energy contribution (ΔG_ele = ΔE_ele + ΔG_PB) is positive and amounts to 22.63 kcal mol⁻¹, while the corresponding value of the dispersive forces (i.e., van der Waals and hydrophobic interaction energies, ΔG_disp = ΔE_vdW + ΔG_NP) is remarkably favorable, being equal to −36.55 kcal mol⁻¹.

Accordingly, we can conclude that the overall enthalpic contribution governing the spontaneous binding of β-CD-g-PG onto HSA is dominated by dispersive interactions, in agreement with experiments. Performing a per residue decomposition of the enthalpic component of ΔG_b shows that the β-CD moiety of β-CD-g-PG affords the most effective contribution to HSA binding, while the polyglycerol branches seem to be almost ineffective. To dissect this aspect in more detail, we performed MD simulation of the complex between the unmodified β-CD and the HSA under the same conditions (Fig. 8B and C). The calculated free energy of binding value for the HSA/β-CD complex, ΔG_b = −2.04 kcal mol⁻¹, confirms that the unmodified CD has less affinity for HSA than its PG-modified counterpart (ΔG_b = −3.57 kcal mol⁻¹). As can be further inferred from the results displayed in Fig. 14D, the entropic component of the ΔG_b, being negative, opposes binding in both HSA/β-CD-g-PG and HSA/β-CD complexes (TΔS_b HSA/β-CD-g-PG = −10.35 kcal mol⁻¹ and TΔS_b and HSA/β-CD = −6.56 kcal mol⁻¹, respectively). At the same time, however, the unmodified cyclodextrin establishes weaker intermolecular interactions with the HSA since, for this system, non only the overall dispersive component of ΔG_b is less favorable (ΔG_disp = −33.77 kcal mol⁻¹) but, at the same time, the β-CD/HSA complex pays higher molecular desolvation penalty upon binding (ΔG_PB = 42.16 kcal mol⁻¹) with respect to the polyglycerol-modified β-CD counterpart. In other words, the contribution of the PG branches translates in two meaningful positive effect for the binding with HSA by: (1) rendering the β-CD cavity more hydrophobic thereby improving the interactions with the HSA residues; and (2) providing a hydrophilic shielding of the binding region by exposing the polar moieties to the solvent with a subsequent lower desolvation energy paid by β-CD-g-PG upon binding with HSA.

Characterization of HSA/β-CD-g-PG/drug supramolecular entities

After the preparation and characterization of the polyglycerol modified β-CD/HSA supramolecular entities, the two anticancer drugs doxorubicin (DOX) and paclitaxel (PTX) were added to the HSA/β-CD-g-PC solutions to investigate the structural and binding/release properties of the resulting nanocarrier/drug systems.

Circular dichroism (CD) spectroscopy analysis. Initially, the conformational state of HSA in the resulting nanovector/drug complexes was analyzed. The CD spectra of HSA/β-CD-g-PG/drug complexes show almost the same pattern of HSA, as native or in complex with β-CD-g-PG (Fig. 9).

Fig. 9 Far-UV CD spectra of HSA/β-CD-g-PG/DOX and HSA/β-CD-g-PG/PTX (right). Concentrations of DOX and PTX from (a) to (c): 1.4, 2.8, and 4.2 × 10⁻⁴ M. All spectra were recorded in ethanol/water solution at 37 °C.

However, the ellipticity of the protein is significantly reduced compared to that of the pristine HSA in the 200–260 nm wavelength range. Elaboration of CD data indeed reveals that, in the presence of either DOX or PTX, the α-helical content of HSA plummets from 52.5% for HSA/β-CD-g-PG/HSA to 44.2% for HSA/β-CD-g-PG/DOX and 42.1% for HSA/β-CD-g-PG/PTX, respectively (Table 5). The decrease in α-helix content was accompanied by an increase in the β-sheet, β-turn and β-antiparallel motifs; similarly, the unstructured (random coil) portion of the protein increases in both drug complexes (Table 5). This may suggest that both DOX and PTX are able to interact with the residues of the polypeptide chain of HSA, partially destroying the hydrogen bonding networks, thereby evoking some degree of protein destabilization, the adoption of a more open conformation ultimately resulting in better exposure of the hydrophobic cavity available to DOX/PTX binding.

Table 5 Amount of different secondary structural motifs in the HSA/β-CD-g-PG/DOX and HSA/β-CD-g-PG/PTX complexes as derived from the corresponding CD spectra. [DOX] = [PTX] = 4.2 × 10⁻⁴ M

Structure	HSA/β-CD-g-PG/DOX	HSA/β-CD-g-PG/PTX
α-Helix	44.2%	42.1%
β-Antiparallel	6.9%	6.7%
β-Sheet	6.7%	6.9%
β-Turn	15.4%	15.5%
Random coil	26.8%	28.8%

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Steady-state fluorescence spectroscopy analysis. The interaction between the two anticancer drugs (DOX and PTX) and the HSA/β-CD-g-PG supramolecular assembly was next investigated by fluorescence spectroscopy. As expected, the fluorescence of the protein/CD derivative complex is gradually quenched upon addition of DOX or PTX, confirming the onset of intermolecular interactions between both drugs and the HSA/β-CD-g-PG assembly (see Fig. 3S and 4S†). Furthermore, a small blue shift was observed with increasing drug concentration, which suggests that the fluorescence chromophore of HSA was placed in a more hydrophobic environment after the addition of either DOX or PTX. The quenching mechanism was then determined to confirm the complexation between the drugs and the HSA/β-CD-g-PG assembly. The static nature of the quenching mechanism was confirmed by the analysis of fluorescence data according to eqn (1). The relevant Stern–Volmer plots are shown in Fig. 10A and B. It can be seen that, as for the case of HSA/β-CD-g-PG complexation, the calculated values of the quenching constant k_q (2.58 × 10¹² M⁻¹ s⁻¹ for HSA/β-CD-g-PG/DOX and 7.72 × 10¹¹ M⁻¹ s⁻¹ for HSA/β-CD-g-PG/PTX, respectively) are also greater than the limiting diffusion constant for HSA (2.0 × 10¹⁰ M⁻¹ s⁻¹); accordingly, fluorescence quenching was not initiated by dynamic collision but from the formation of the relevant complexes. Also, the values of k_q reveal that DOX acts as a stronger fluorescence quencher upon binding to the HSA/β-CD-g-PG supramolecular assembly than PTX. Panels C and D in Fig. 10 show the Stern–Volmer plots for the HSA/DOX and HSA/PTX complexes for comparison. As inferred from these figures, the values of the k_q for the protein/drug systems – both in excellent agreement with previous reports^34,35 – are very similar to those of the drug bound to HSA/β-CD-g-PG complex. The minimum effect of the presence of the polyglycerol-modified β-CD on the k_q value of both drugs supports the idea that β-CD-g-PG binds in a different region of the HSA surface with respect to DOX and PTX binding sites (see the modeling section for further discussion on this point).

Fig. 10 Stern–Volmer plots for the quenching of HSA/β-CD-g-PG at 37 °C by DOX (A) and PTX (B). [DOX] = 3.4, 6.7, 13, 17, 20, 24, 27 and 30 μM; [PTX] = 12, 23, 35, 47, 59, 70, and 82 μM. [HSA] = 2 μM. An ethanol/water (50 : 50) mixture was used as solvent in both systems. Stern–Volmer plots for the quenching of HSA at 37 °C by DOX (C) and PTX (D). [DOX] = 6.7, 13, 17, 20, 24, and 34 μM; [PTX] = 12, 23, 35, 47, 59, 70, and 82 μM. [HSA] = 2 μM in ethanol/water solution.

Next, the modified Stern–Volmer eqn (2) was used for the determination of the binding constant K_b and number of binding sites n of DOX and PTX onto the HSA/β-CD-g-PG complex. The same calculations were performed also in the case of pristine HSA/drug binding, for comparison. Fig. 11 shows the relevant results, which are numerically summarized in Table 6.

Fig. 11 Modified Stern–Volmer plot for the quenching of HSA/β-CD-g-PG by DOX (A) and PTX (B) in ethanol/water solution at 37 °C. Modified Stern–Volmer plot for the quenching of HSA by DOX (C) and PTX (D) in ethanol/water solution at 37 °C, [HSA] = 2 × 10⁻⁶ M.

Table 6 Binding parameters for DOX and PTX with HSA/β-CD-g-PG and with pristine HSA. The values of K_b and n were obtained from data fitting in Fig. 11. The corresponding ΔG_b values were obtained via eqn (3)

System	Kb (M^−1)	n	R^2	ΔGb (kcal mol^−1)
HSA/β-CD-g-PG/DOX	18 980	1.03	0.993	−6.07
HSA/β-CD-g-PG/PTX	812	0.83	0.984	−4.13
HSA/DOX	3236	0.83	0.993	−4.98
HSA/PTX	176	0.76	0.998	−3.18

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The values of K_b listed in Table 6 clearly show the beneficial effect of the presence of β-CD-g-PG on the affinity of both anticancer drugs for HSA. Moreover, DOX is endowed with a more favorable free energy of binding ΔG_b with respect to PTX, both towards native HSA and its β-CD-g-PG supramolecular complex, by virtue of a more efficient network of hydrophobic and hydrogen bond intermolecular interactions, as discussed in details in the molecular modeling section below. Of note, the K_b values for HSA/DOX (3256 M⁻¹) and HSA/PTX (176 M⁻¹) derived from the present fluorescence quenching data are in good agreement with previous reports.^19,36

Dynamic light scattering (DLS) and zeta-potential (ζ) analysis. DLS measurements were performed on the HSA/DOX, HSA/PTX, and the HSA/β-CD-g-PG/drug complexes to derive their average molecular dimensions listed in Table 7.

Table 7 DLS average hydrodynamic diameter d_h (nm) and zeta-potential ζ (mV) of HSA/DOX, HSA/PTX, HSA/β-CD-g-PG/DOX and HSA/β-CD-g-PG/PTX in solution. pH = 7.4, T = 37 °C

Compound	dh (nm)	ζ (mV)
HSA/DOX	22	−22.47
HSA/PTX	25	−18.53
HSA/β-CD-g-PG/DOX	16	−26.96
HSA/β-CD-g-PG/PTX	20	−24.56

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These data reveal that the hydrodynamic diameter of HSA increased from 6 to 22 and 25 nm in the presence of DOX and PTX, respectively. On this basis, it can be inferred that, upon drug binding, the resulting HSA/drug complexes aggregate to a certain extent. This inference is confirmed through studies of the size distribution of HSA (Fig. 7S†) and the HSA/drug complexes (Fig. 8S†).

Whilst an almost monodisperse solution of HSA in present in the PSB buffer (pH = 7.4 and 37 °C) (6 nm), the addition of either DOX or PTX causes the formation of aggregates with size increasing to 22 and 25 nm, respectively. These results imply that the binding between HSA and DOX and PTX leads to a conformational change in HSA (Table 5), which contributes to the aggregation of the resulting HSA/drug complexes. The presence of β-CD-g-PG bound on the surface of HSA, on the other hand, seems to disfavor the formation of higher aggregates, likely by hampering optimal protein–protein and/or drug–drug interactions.

The zeta-potential values at physiological pH of the HSA/β-CD-g-PG/DOX and HSA/β-CD-g-PG/PTX complexes are highly negative and approximately equal to −27 and −25 mV, respectively (Table 7). These ζ values confirm the notion that either drug, upon binding onto the HSA/β-CD-g-PG assembly, provokes a perturbation in the secondary structure of the protein (Table 5), which ultimately results in the exposition of negatively charged amino acid side chains on the protein surface. The increased negative protein surface charge – and hence the related enhanced Coulombic repulsions – accounts for the smaller dimensions of the HSA/β-CD-g-PG/DOX and HSA/β-CD-g-PG/PTX bigger aggregates with respect to those of the binary (i.e., drug/protein) complexes (Table 7). This property, coupled with the likely further beneficial effect of intrinsic protein resistance and steric impediment to aggregation exerted by the presence of β-CD-g-PG on the surface of HSA, can be extremely advantageous when considering the polyglycerol-modified β-cyclodextrin–human serum albumin supramolecular assembly as a possible nanovector for drug delivery. In addition, negative surface charge of drug delivery systems will affect their uptake which is a significant factor and should be investigated experimentally.

Drug loading efficacy (DLE) and in vitro drug release analysis. The elaboration of furter UV-vis data (see ESI† for details) allowed to estimate the drug loading efficacy (DLE) of DOX and PTX by native HSA and its β-CD-g-PG adduct. Interestingly, the DLE values for DOX and PTX show an increase of 20% for both drugs with respect to the native protein when binding to the HSA/β-CD-g-PG supramolecular assembly (from 60% to 80% in the case of DOX and from 50% to 71% in the case of PTX, respectively, Fig. 13S and 14S†). This result shows that the polyglycerol-derivative of β-CD plays a key role in potentiating the drug loading capacity of the protein (Fig. 13S and 14S†) and, as such, in rendering HSA a more efficient nanocarrier.

Finally, the in vitro release of DOX and PTX from the HSA/β-CD-g-PG supramolecular assembly was investigated; the relevant profiles are shown in Fig. 12. It can be seen that the rate of release of both drugs from the modified HSA strongly depends on the pH, the % of drug released being more favorable at acidic pH in both cases. This is an expected result, as it is known that HSA undergoes pH-induced further secondary conformation unfolding which, in turn, might weaken drug binding and, hence, facilitate drug release from its binding site. Contextually, this is a favorable evidence since tumor microenvironment is usually acidic;³⁷ accordingly, all other conditions being equal, the release of both anticancer drugs from the HSA/β-CD-g-PG will be at the tumor site enhanced with respect to the considerable lower one attained at neutral pH.

Fig. 12 In vitro DOX and PTX release profiles from HSA/β-CD-g-PG/DOX (A) and HSA/β-CD-g-PG/PTX (B) complexes at 37 °C.

Molecular modeling of the HSA/β-CD-g-PG supramolecular assembly. To dissect the effect of the presence of β-CD-g-PG on the surface of HSA on the binding of DOX and PTX on the protein at the molecular level we performed molecular simulation binding studies of the two drugs both on pristine and β-CD-g-PG-modified HSA. In agreement with previous works,^34,35 DOX and PTX dock within two different protein binding sites: the first, in fact, finds a good encasement in a hydrophobic pocket of the subdomain IB while PTX is nested more within the protein core in subdomain IIa (Fig. 13A and C).

Fig. 13 Binding mode of DOX (A) and PTX (C) onto HSA. Panels (B) and (D) are zoomed views of the binding site of HSA/DOX and HSA/PTX binding sites, respectively. In all panels, the protein is depicted as a golden ribbon. In panels (A) and (C) the drugs are highlighted by their firebrick (DOX) and orange red (PTX) van der Waals surfaces. In panels (B) and (D) the drugs are portrayed in atom-colored sticks-and-balls (gray, C; red, O; blue, N). H-bonds interactions are shown as dotted black lines. Hydrogen atoms, water molecules, ions and counterions are omitted for clarity. HSA residues interacting with the drugs are shown as colored sticks and labeled.

The drug/protein intermolecular interactions were quantified by carrying out MM/PBSA MD simulations. As shown in Fig. 13B and D, the two anticancer agents established a similar network of interactions with the protein. Specifically, both drug binding is driven by a combination of a weak array of hydrogen bonds (HBs) and hydrophobic forces. In particular, the side chains of the hydrophobic residues V140, M147, Y185, F189, L206, and L209 lean the binding pocket of DOX; more, we confirmed the establishment of HBs among several of the drug heteroatoms both with the polar side chains of the HSA residues R141, H170, R210, and K214 and the backbone NH group of residues R138 and L139 (Fig. 13B and Table 8). At variance, MD simulations carried out with PTX revealed the presence of hydrophobic interactions with the side chains of W238, L262, I314, H312, and A315 while some donor and acceptor atoms of the molecule found their appropriate counterpart in the polar amino acid residues K219, K223, R242, R246, and H266 (Fig. 13D and Table 4). The overall contribution of these favorable interactions calculated by MM/PBSA approach led to ΔG_bind of −4.82 kcal mol⁻¹ for HSA/DOX complex and −3.31 kcal mol⁻¹ for HSA/PTX system, respectively. These data confirm the moderate affinity of the two drugs for HSA, in agreement with previously reported results^34,35 and with our experimental analysis (see Table 6).

Table 8 HSA residues involved in H-bonds and the corresponding Average Dynamic Length (ADL) for HSA/DOX, HSA/β-CD-g-PG/DOX, HSA/PTX and β-CD-g-PG/HSA/PTX complexes. bb = backbone

Complex	HSA residue	ADL (Å)
HSA/DOX	R138-L139 (bb)	2.76 ± 0.06
	R141	2.87 ± 0.03
	H170	3.01 ± 0.03
	R210	2.67 ± 0.04
	K214	2.73 ± 0.02
HSA/β-CD-g-PG/DOX	R138-L139 (bb)	2.66 ± 0.05
	R141	2.49 ± 0.02
	H170	2.63 ± 0.04
	R210	2.38 ± 0.05
	K214	2.25 ± 0.03
HSA/PTX	K219	2.93 ± 0.02
	K223	2.99 ± 0.02
	R242	2.88 ± 0.03
	R246	3.15 ± 0.04
	H266	3.19 ± 0.04
HSA/β-CD-g-PG/PTX	K219	2.68 ± 0.04
	K223	2.70 ± 0.02
	R242	2.65 ± 0.05
	R246	2.92 ± 0.03
	H266	2.83 ± 0.04

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The deconvolution of the enthalpic component of ΔG_b was next performed for both systems with the aim of quantifying the single contribution of each amino acid involved in the drug binding. The collected results not only substantiate the fact that DOX is a stronger binding of HSA with respect to PTX, as more residues are involved in more efficient protein/drug binding, but also the fact that network of interactions between the protein and the drugs is overall rather weak. Indeed, no residues exhibited an enthalpic gain upon drug binding greater than 0.6 kcal mol⁻¹ (Fig. 14).

Fig. 14 Comparison of per-residue binding enthalpy decomposition for DOX (A) and PTX (B) in complex with HSA and HSA/β-CD-g-PG.

In the previous modeling section we described how HSA and β-CD-g-PG interact through the formation of a thermodynamically stable complex. However, to understand the effect of the presence of β-CD-g-PG onto the HSA surface on drug binding, we then carried out MD simulations of the supramolecular HSA/β-CD-g-PG system in complex with DOX and PTX, respectively. Starting from the equilibrated MD structure of the β-CD-g-PG/HSA complex, DOX and PTX were docked in their corresponding HSA binding sites (Fig. 15A and C). Next, we performed extensive MD simulation runs with the aim of checking whether the structural HSA modifications promoted by β-CD-g-PG could eventually affect the binding mode of DOX and PTX onto the protein. Finally, MM/PBSA calculations were applied to estimate the affinity (ΔG_b) of DOX and PTX for the supramolecular HSA/β-CD-g-PG complex. In agreement with the experimental results, the calculated affinity against β-CD-g-PG/HSA for both drugs increases, as the corresponding estimated ΔG_b values are −6.32 kcal mol⁻¹ for DOX and −4.35 kcal mol⁻¹ for PTX, respectively. The conformational changes induced by the presence of the polyglycerol conjugate indeed perturb both protein drug binding regions, with consequent alteration of the relevant drug binding modes (Fig. 15B and D). This rearrangement reflects into a stronger network of the binding interactions between HSA and DOX and PTX. Indeed, all detected H-bonds became shorter, as revealed by the calculated ADL in Table 8 and, hence, stronger. Furthermore, the per residue enthalpic deconvolution for the HSA/β-CD-g-PG/drug complexes clearly emphasize hydrophobic forces are enhanced for these systems since the involved HSA residues are characterized by a more favorable enthalpic gain upon the binding with the drugs (Fig. 14).

Fig. 15 Binding mode of DOX (A) and PTX (C) with HSA/β-CD-g-PG. In both panels the proteins is depicted as a gold ribbon, while β-CD-g-PG and the drugs are highlighted by their atom-colored (gray, C; red, O) and green van der Waals surfaces, respectively. (B) Superposition of equilibrated MD snapshots of the binding mode of DOX with HSA (firebrick) and HSA/β-CD-g-PG (green); (D) superposition of equilibrated MD snapshots of the binding mode of PTX with HSA (orange red) and HSA/β-CD-g-PG (chartreuse). In all panels, hydrogen atoms, water molecules, ions and counterions are omitted for clarity.

Conclusions

In this work we synthesized and characterized, by a combination of experiment and modeling, a novel human serum albumin–β-cyclodextrin derivative supramolecular system as an efficient nanocarrier for prototypical anticancer drugs such as doxorubicin and paclitaxel. The findings stemming from our combined approach confirmed and explained how the interactions between β-CD-g-PG and HSA improve the affinity of both DOX and PTX for the HSA/β-CD-g-PG adduct with respect to the native protein, and yielded a molecular rationale for these evidences. Since albumin is one of the most important carriers for multiple drugs and therapeutic agents, the properties of the this supramolecular HSA/β-CD-g-PG nanocarrier as derived from the present study, albeit rather preliminary, may have a significant impact in future pharmacological and clinical applications; indeed, further in vitro and in vivo studies are currently ongoing in our laboratories in these directions.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25463a

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