Molecular Switch For the Assembly of Lipophilic Drug Incorporated

Article pubs.acs.org/Biomac

Molecular Switch For the Assembly of Lipophilic Drug Incorporated Plasma Protein Nanoparticles and In Vivo image Guangming Gong, Yan Xu, Yuanyuan Zhou, Zhengjie Meng, Guoyan Ren, Yang Zhao, Xiang Zhang, Jinhui Wu,* and Yiqiao Hu* State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P. R. China S Supporting Information *

ABSTRACT: A strategy to manipulate the disulfide bond breaking triggered unfolding, and subsequently assembly of human serum albumin (HSA) in a lipophilic drug-dependent manner is present. In this study, the hydrophobic region, a molecular switch of the HSA, was regulated to form HSA-paclitaxel (HSA-PTX) nanoparticles by a facile route. High-resolution transmission electron microscopy and fluorescence quenching indicate that HSA coassembled with PTX, which acts as a bridge to form core−shell nanoparticles about 50−240 nm in size, and that PTX might bind to the subdomain IIA sites of HSA. Change of ultraviolet absorption and circular dichroism spectra reveal the formation of HSA-PTX nanoparticles, which is a safety, injectable pharmaceutic nanocarrier system for tumor target. This method to prepare nanocarrier systems for hydrophobic guest molecules reveals a general principle of self-assembly for other plasma proteins and other pharmacologically active substances with poor water solubility. It also provides a basis for developing nanocarrier systems for a wide range of applications in nanomedicine, from drug delivery to bioimaging systems.

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of its poor water solubility (0.3 ug mL−1), Cremophor EL (CrmEL)/ethanol is used to enhance its solubility, but the latter, a component of the formulation, is toxic to human body and can cause negative side effects. Some PTX formulations including liposomes, nanoparticles, emulsions, microspheres, micelles, PEG-PTX, and nanoshells have been widely explored to acquire improved physicochemical properties and biodistribution of PTX.24−29 Among these, Abraxane is a novel PTX formulation for the therapy of metastatic breast cancer.30 However, this nanocarrier system is fabricated by mechanical force, which is independent of the natural characteristic of HSA, that is, the amphipathicity, with low drug loading efficiency. We want to take on the challenge of fabricating a nanocarrier system by finely modulating the molecular switch of proteins. In previous work, we developed a strategy for fabrication of blank nanoparticles consisting of β-mercaptoethanol (β-ME)HSA molecules.31 In this study, HSA and PTX were arranged as two components. PTX was entrapped noncovalently in the hydrophobic cavity of HSA to form HSA-PTX nanoparticles. This was accomplished using β-ME to regulate the “exposedhidden” switch, that is, the hydrophobic region, of HSA. The morphology and physical states of the nanoparticles were explored, and the spectrum was used to characterize the structural changes and the molecular mechanism of the assembly. Furthermore, fluorescence-probe-labeled HSA-PTX nanoparticles were fabricated to explore the biodistribution and tumor targeting capability of this nanocarrier system in vivo.

INTRODUCTION Protein assemblies are ubiquitous in nature. Proteins such as silk proteins, viral capsids, and amyloid proteins have been widely used as building blocks to study the assembly of macromolecular complexes,1,2 smart biomaterials,3,4 and models of human diseases in vitro.5−8 Recently, simulation of the hydrophobic effect in protein−protein interactions9,10 and drug−protein interactions11,12 to fabricate protein nanocarrier systems for lipophilic drugs has engaged the attention of research efforts.13,14 The employment of plasma protein as building block to fabricate nanocarrier systems for lipophilic drugs and fluorescent probes has attracted much attention because of their biocompatibility, nongenetic, and safety. Among these, albumin has been widely used for nanoparticles preparation.15,16 Human serum albumin (HSA), a kind of plasma protein that has 585 amino acids, 17 disulfide bridges, three subdomain sites (I, II, III), and hydrophobic cavities,17 can accommodate and transport lots of hydrophobic and hydrophilic molecules to target organs and tissues.18 Its nanoparticles can preferentially accumulate at tumor and inflamed tissues because of passive targeting by enhanced permeable reaction (EPR) effects and the active targeting mediated by gp60 receptors expressed on these tissues.19−21 Therefore, HSA is a versatile tool to fabricate nanocarrier systems. Furthermore, the safety of HSA-based formulation has been clinically proved,22,23 and it is of great value to fabricate HSA as the vehicles of therapeutic drugs and fluorescent probes. PTX is a widely clinical used chemotherapeutical drug, which is highly efficient in treating breast and other cancers. Because © 2011 American Chemical Society

Received: June 6, 2011 Published: October 27, 2011 23

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Article

EXPERIMENT SECTION

no nanoparticles were detected by dynamic light scattering (DLS) (Figure 1, Stage I).

Materials. HSA, rhodamine-B isothiocyanate, and near-infrared (NIR)-797-isothiocyanate were bought from Sigma Chemical. β-ME was purchased from Promega (Madison, WI). Male ICR mice (8−10 weeks old and weighing 20−25 g) were purchased from Animal Center of Drum Tower Hospital (Nanjing, China). PTX was obtained from Xuanhao Biomedical (Xian, China). All reagents were used as received. Preparation of HSA Particles and HSA-PTX Nanoparticles. We dissolved 100 mg of HSA in 10 mL of Tris buffer (5 mM) at 37 °C, and 900 μL of β-ME was added to prepare blank HSA particles. HSA (100 mg) was dispersed in 100 mL of ultrapure water with constant stirring at 37 °C, and 350 μL of β-ME was added. PTX (10 mg mL−1, ethanol as solvent) was added after 10 min of stirring. The HSA nanoparticles and HSA-PTX aqueous suspension were extensively dialyzed with membrane (MW: 8K Dolton) to remove any remnant β-ME. Electron Microscopy. HSA-PTX nanoparticles were prepared as previously mentioned. Blank HSA nanoparticles (ultrapure water as solvent) were obtained without PTX, and its concentration was 1% at 50 °C. Three solutions of the HSA-PTX nanoparticles were placed on a 200-mesh copper grid coated with carbon. All solutions were examined in duplicate. After deposition, the grid was air-dried. All specimens were observed using a JEOL JEM-2100 electron microscope. Fluorescence Spectral Change of HSA before and after Binding to PTX. Fluorescence spectra were obtained on a spectrofluorometer, F-7000 (HITACHI, Japan). The excitation wavelength was 295 nm, and the emission wavelength was from 300 to 450 nm. The slit width was 2.5 nm. The measurements were performed in triplicate. A 350 μL aliquot of β-ME was added to 100 mL of HSA solution (1 mg mL−1) at 37 °C. After 10 min, 50 μL of PTX solution (10 mg mL−1) was added. Then, a certain volume of solution was taken to obtain the fluorescence spectra. The process from adding PTX to obtaining fluorescence spectra was recycled nine times. Ultraviolet Detection of the Interaction of β-ME-HSA with PTX. UV/vis spectrophotometer (UV-2450, Shimadzu, Japan) equipped with a 1.0 cm quartz cell was used for recording the UV spectra at 200 to 300 nm. A 2 μL aliquot of the PTX solution (10 mg mL−1) was added to the solution of β-ME-HSA (1 mg mL−1); then, the spectra of the solution were obtained after filtering. This process was repeated four times at 37 °C. Circular Dichroism Spectra. Circular dichroism (CD) measurements of the three substances were performed on a JASCO 810 (Japan) using a 1.0 cm quartz cell. The wavelength was from 200 to 280 nm, and the scanning speed was 50 nm min−1. The concentration of HSA, β-ME-HSA, and HSA-PTX solution was 10 mg mL−1 (quantified by Coomassie Kit). In Vivo Image. Preparation of NIR-797-labeled PTX nanoparticles: A weighed amount (30 mg) of HSA was dissolved in 3 mL of Tris buffer (0.05 M, pH 7.8); then, 3 mg NIR-797 (300 μL of DMSO as solvent) was added to the mixture. The resulting solution was continuously stirred for 10 h. The solution was dialyzed with 0.05 M Tris buffer for 12 h and was then used to prepare HSA-PTX nanoparticles. We injected 0.2 mL of NIR-797-HSA-PTX nanoparticles and 0.2 mL of NIR-797-HSA with the same fluorescent intensity into S180 tumor (left axilla)-bearing mice through the tail vein, respectively. After injection, the tumor-bearing mice were imaged using Maestro Dynamic − Lumina system (CRi, Woburn, MA). The emission wavelength at 800 nm was collected, and the exposure time was set to 2 s. Scans were carried out at 1, 24, 72, and 108 h postintravenous administration.

Figure 1. Self-assembled HSA-PTX complex within three stages. Stage I, non-nanoparticle complex; Stage II, nanoparticles, measured from 50 to 240 nm; Stage III, microparticles, 2 mg mL−1 HSA solution, denatured with β-ME in 5 mM Tris buffer at 37 °C and with gradual addition of PTX. The size of the complex was detected by DLS.

With additional PTX, the complexes self-aggregated into nanoparticles (Figure 1, Stage II). Meanwhile, the solution changed from clear to bluish, which indicated the formation of nanoparticles. When the concentration of PTX was ∼0.77 mM, HSA-PTX nanoparticles became their peak size, which was ∼240 nm. The assemblies studied here have improved PTX’s solubility (from 0.3 μg mL−1 to 3 mg mL−1). This may have resulted from complexation and nanoparticle formation with HSA. The drug loading of the nanoparticles can amount to 26.8%, according to eq 1 of the Supporting Information. However, when the volume of PTX solution (ethanol as solvent) was >7% of that of HSA solution (v/v), large particles were formed, and the solution turned turbid and gray (Figure 1, Stage III). Electron Microscopy of the Nanoparticles. Highresolution transmission electron microscopy (HRTEM) was used to explore the structure and morphology of the nanoparticles. The images reveal that the HSA-PTX nanoparticles have a spherical structure (Figure 2a, Supporting Information), which is different from the chrysanthemum-like

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Figure 2. HRTEM of (a) HSA-PTX nanoparticles; (b) sample from PTX water solution at the bluish color stage; (c) β-ME-HSA nanoparticles without PTX; (d) a slice of HSA-PTX nanoparticles stained with osmium tetroxide; (e) DLS of HSA-PTX nanoparticles; and (f) HSA-PTX nanoparticles dialyzed for 24 h.

RESULTS AND DISCUSSION Fabrication of HSA-PTX Nanoparticles. β-ME was used as a disulfide bond breaking molecule to fabricate HSA-PTX nanoparticles. When the concentration of PTX was β-ME-HSA > HSA-PTX (Figure 4B). The decrease in helicity possibly occurred because of the interactions among HSA, β-ME, and PTX that led to a conformational change in HSA.35 Dynamic Change of the Size of HSA-PTX Nanoparticles. To verify the role of PTX during the self-assembly process, we prepared a series of HSA-PTX nanoparticles. As the concentration of HSA increased from 0.5 to 5 mg mL−1 and that of PTX increased from 5 to 25% (w/w), the hydrodynamic diameter of HSA-PTX nanoparticles increased from 0 to 213 nm (Table S1, Supporting Information). All of these results confirm that HSA at low concentration could develop into a nanosystem with the synergistic effect of PTX. Mechanism for the Formation of HSA-PTX Nanoparticles. Surface hydrophobicity is indispensable for aggregation initiated by protein−protein interactions.36 Hence, it is speculated that for the hydrophobic region a molecular switch could be necessary for the assembly of HSA nanoparticles and more exposed hydrophobic regions could be present in β-MEHSA than in HSA. Three aspects are involved in the hypothesis. First, 1-(anilinon)aphthalene-8-sulfonic acid (ANS)37,38 was used to observe the change in the exposed hydrophobic region of HSA (Figure S1, Supporting Information). Results showed that the fluorescence intensity of β-ME-HSA (0.34 mg mL−1) was 878.6387 ± 42.03887 and that of native albumin (0.34 mg mL−1) was 793.058 ± 45.95123 (p = 0.075). The fluorescence of the former was 10% higher than that of the latter. When the concentration of HSA was 0.2 mg/mL, the ANS fluorescence of β-ME-HSA was 5% higher than native HSA (data not shown). These results support the hypothesis that unfolded HSA has more hydrophobic moieties than native albumin does. Second, the hydrophobic region change of blank HSA during assembly was also detected by the ANS fluorescence change in preparing blank HSA nanoparticles. Results showed that as the assembly of blank HSA nanoparticles went on, the hydrophobic region of HSA was gradually reduced, indicating the involvement of the hydrophobic region in the assembly.31 However, in the formation of HSA-PTX nanoparticles, the

Figure 3. Fluorescence spectral change of HSA after adding PTX from a to l: (a) native HSA, PTX free; (b) β-ME-modified HSA, PTX free; (c−i) PTX concentration: 1.18 × 10−5, 2.36 × 10−5, 3.54 × 10−5, 5.9 × 10−5, 7 × 10−5, 8.26 × 10−5, 9.44 × 10−5, 11.8 × 10−5, and 29.8 × 10−5 M; and (j) HSA free, PTX (29.8 × 10−5 M), ethanol/water (50:50) as solution. 25

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Figure 4. (A) UV/vis absorption spectra of (a) free PTX (10 mg mL−1, mixed solvent with the ethanol/water volume ratio of 1:1), (b) free β-MEHSA, β-ME-HSA solution (10 mg mL−1) containing different concentrations of PTX (weight fraction of HSA), (c) 2/100, (d) 4/100, (e) 6/100, and (f) 8/100. (B) CD spectra of (a) native HSA, (b) β-ME-HSA, and (c) HSA of HSA-PTX. (The spectrum of PTX was subtracted from the spectrum of HSA-PTX.) HSA content was 10 mg mL−1 in the three groups.

ANS could bind to lipophilic PTX, which made reversed results. Therefore it is only indirectly inferred that the hydrophobic region is necessary for HSA and HSA-PTX assembly. Third, it was presumed that the interaction force involved in the assembly of HSA might be a constant because when the concentration of β-ME-HSA was 0.5 mg mL−1 nanoparticles could not be formed without the synergetic effect of PTX. However, blank β-ME-HSA nanoparticles would be obtained at 10 mg mL−1 concentration without the help of PTX.31 It was speculated that at low concentration HSA molecules might have a less hydrophobic region, and the interaction force among HSA molecules was too weak to form nanoparticles. With additional PTX that acted as a bridge, HSA-PTX nanoparticles could be formed. At high concentration, the interaction force among HSA molecules, having a more hydrophobic region, was strong enough to form nanoparticles without the help of PTX (Figure 5).

Scheme 1. Schematic Representation of the Nanoparticles Formed by PTX and HSA and the Structures of HSA and PTX: ① HSA molecule, ② β-ME-HSA, ③ β-ME-HSA-PTX complex, and ④ and ⑤ HSA-PTX nanoparticles

in contact with the aqueous solution. If 1.5 mL of ethanol was added to 8.5 mL of HSA-PTX solution, in which the lyophilized HSA-PTX nanoparticles were dispersed with water or phosphate buffer solution, precipitation composed of HSA (protein quantity kit detection) and PTX (HPLC detection) was developed as well. Here two main factors might be involved in the assembly. (i) Gong et al. proposed that the hydrophobic interaction was the element for the fabrication of HSA-PTX nanoparticles.39 Ethanol can displace ANS molecules and occupy the binding sites of fatty acid interacting with HSA at the hydrophobic region.40 In this study, ethanol could disrupt the HSA-PTX nanoparticles when their volume fraction in the HSA solution was >15%. Hence, ethanol might have displaced PTX from HSA, and PTX might have had a hydrophobic interaction with albumin. (ii) Hydrogen bonds between PTX and PTX may contribute to the stabilization of the assembly.41 The stability of HSA-PTX nanoparticles could not be influenced by 1 M (final concentration) sodium chloride. Therefore, the hydrophobic interaction and hydrogen bond may have worked in the formation of the nanoparticles.

Figure 5. Schematic representation of the interaction between HSA and PTX with different concentration. (a) Low concentration of HSA and high PTX; (b) middle concentration of HSA and PTX; and (c) high concentration of HSA without PTX. Curved lines, PTX; ○, HSA; , interaction force among HSA molecules.

Accordingly, it is inferred that the more hydrophobic region of HSA is presented as its concentration increases, and the hydrophobic region, involved in the molecular switch of HSA, might play key roles in the formation of assembling. An “exposed-hidden” switch mechanism is proposed to explain the formation of HSA-PTX nanoparticles (Scheme 1). Initially, the introduction of β-ME, which destroys the disulfide bond of HSA, exposes the hydrophobic region, a molecular switch of HSA. The PTX tends to associate with the exposed hydrophobic parts of the protein to form the non-nanoparticle complex of HSA-PTX. The solubility of the complexes might exceed that of PTX because no nanoparticles was formed (Figure 1, Stage I). Additional PTX increases the size of the aggregate (Figure 1, Stage II). Because PTX is enclosed in the hydrophobic center of the nanoparticles, the system is stable. The hydrophilic regions of HSA may act as a protective sheath 26

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X-ray Powder Diffraction. X-ray powder diffraction (XRD) was applied to investigate the physical state of PTX in the nanoparticles. The PTX loaded in nanoparticles was amorphous because no crystalline peaks of PTX appeared in the HSA-PTX nanoparticles.39 However, the physical mixture of PTX and HSA presents clear crystalline peaks of PTX (Figure S4, Supporting Information). In Vivo Image. Besides cell accumulation testing, the tumor targeting capability of NIR-797-conjugated HSA-PTX nanoparticles in mice afflicted with tumors was also studied. The NIR-797-HSA-PTX nanoparticles prepared by HSA conjugated with NIR-797 were administered in vain, and blank NIR-797-HSA was set as a control. Figure 6 shows that

therapeutical drugs, data not shown), with serious side effects and short blood circulation times, and fluorescent probes, such as rhodamine B and NIR-797. Further studies are underway.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information * Drug loading efficiency of PTX, hydrophobic region of HSA, stability of HSA-PTX nanoparticles, drug releasing profiles, and size of HSA-PTX nanoparticles detected by DLS, XRD. This material is available free of charge via the Internet at http:// pubs.acs.org.

Corresponding Author *Tel: +86 13601402829. Fax: +86 25 83596143. E-mail: [email protected], [email protected], [email protected].

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ACKNOWLEDGMENTS We thank State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and all members of our research group.

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Figure 6. NIR-797 images of S180 tumor-bearing mouse in vivo and (a) NIR-797-HSA-PTX nanoparticles and (b) NIR-797-HSA at 1, 24, 72, and 108 h postintravenous injection.

REFERENCES

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NIR-797 molecules in the two groups distribute all over the body in the first 24 h, and signal intensity of NIR-797 is stronger on the abdomen than on other places and then gradually increases in the tumor region. At 108 h, the intensity of NIR-797-HSA-PTX was relatively stronger at the tumor site than that of NIR-797-HSA, which was distributed relatively all over the body. The ex vivo image of the tissues indicated that the NIR-797-HSA-PTX accumulated in the tumor, liver, and kidneys as NIR-797-HSA did (Figure S5, Table S2, Supporting Information), and the fluorescence intensity of the former was stronger than that of the latter in tumor but was weaker in kidneys. It is inferred that HSA-PTX nanoparticles possess better tumor-targeting probability in contrast with HSA protein.

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CONCLUSIONS In summary, this study presents a nonemulsion and ultrasoundfree way to fabricate nanoparticles encapsulating hydrophobic guest molecules by regulating the “exposed-hidden” switch, that is, the hydrophobic region of HSA by breaking the disulfide bond or by inter/intramolecular disulfide cross-linking42 wherein β-ME acts as a promoter. HSA-PTX nanoparticles, which were about 50−240 nm in size, with a core−shell structure and drug loading of 26.8%, were successfully fabricated because of the synergistic effect of encapsulation and the hydrophobic interaction among PTX and β-ME-HSA. β-ME-HSA was nontoxic,31 and HSA-PTX nanoparticles were stable in 5% glucose buffer before 24 h. HSA-PTX nanoparticles had tumor-targeting ability in vivo image. Therefore, HSA-PTX nanoparticles are a safe, injectable pharmaceutic nanocarrier system for tumor target. Using this facile approach, the building blocks of selfassembly can be generalized to other human and animal plasma proteins (bovine serum albumin and other proteins, data not shown), anticancer drugs (Aclacinomycin and other chemo27

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