Single-protein-based theranostic nanosystem within sub-10 nm scale for tumor imaging and therapy

Abstract

A single-protein-based theranostic nanosystem (SPTN) based on non-covalent interaction between bovine serum albumin (BSA), gadolinium (Gd) and the anticancer hydrophobic drug doxorubicin (DOX) was designed and developed via a facile, environmentally benign approach. Gd was incorporated into the structure of BSA via a simple biomineralization process, while DOX was encapsulated in Gd–BSA nanoparticles via hydrophobic interaction between DOX and BSA, as well as a coordination effect between DOX and Gd. The whole SPTN system was kept in one single BSA molecule to form a single protein based nanosystem with an overall size of around 7 nm favorable for potential renal clearance. The SPTN nanosystem showed not only robust biocompatibility, excellent T₁-weighted MR imaging effect but also fine pH-responsive drug release characteristics. Furthermore, in vivo therapeutic efficacy experiment showed that the nanoparticle DOX–Gd–BSA had an obvious therapeutic efficacy toward hepatoma tumor bearing mice.

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Introduction

With the rapid development of nanomedicine, the emerging term of nanotheranostics defined by the combination of therapeutic and diagnostic capability into a single nanoparticle, is a promising strategy that enables one to get valuable biodistribution information, monitor the real-time therapeutic process, evaluate in vivo performances and study the therapeutic mechanism. The combined technique will potentially result in improved disease management and the acceleration of drug development.^1–5 Generally, the above highly integrated, coordinated advantages necessitate the assembly of different components into a basal structure while keeping their functions not compromised. Over the past decade, a number of organic nanoplatforms including polymer micelles, vesicles, dendrimers etc. and inorganic nanoplatforms including iron oxide nanoparticles, quantum dots, gold nanoparticles, silica nanoparticles etc., have been developed to construct theranostic nanoparticles by multiple modification steps.^6,7 Currently, most of reported theranostic nanosystem needs to be synthesized in a tedious, labor intensive, multiple-step process, significantly impeding the advance and impact of this area. Some of representative works are shown to provide a more detailed description of the dilemma. Usually a core–shell structure is needed to be constructed to confer colloidal suspendability and drug carrier ability to the particles before drug loading for inorganic nanoplatforms.⁸ Taking a core–shell theranostic nanoparticle as an example,⁹ the nanoparticle is composed of a superparamagnetic iron oxide (SPIO) core which was prepared through a solvothermal reaction. A thin silica layer was coated on the surface of the SPIO core via a sol–gel reaction to stabilize the SPIO nanoparticles as well as render the surface negatively charged surface. Gold nanorods (GNRs) were then anchored onto the Fe₃O₄@SiO₂ core via electrostatic interactions for photothermal therapeutic effect. Finally, another thin silica layer was coated on the outside to render the hydrodynamic stable system. The whole process takes multiple steps resulting in a highly complex, variant system. Practical and clinical applications require more convenient yet robust strategy.¹⁰

Moreover, it is reported that the size of the nanoparticles is a critical parameter which affects their bio-distribution, metabolism pathway and finally their toxicity in vivo. Nanoparticles with a hydrodynamic diameter (HD) of less than 10 nm can lead to renal clearance of metal-containing nanoparticles which is an especially important consideration due to agent toxicity and potential for interference with other diagnostic imaging modalities.^11,12 While the size of the theranostic nanoparticles constructed by the afore-mentioned organic and inorganic nanoplatforms usually exceed this range, ranging from dozens of nanometers to hundreds of nanometers. For example, the average diameter of the afore-mentioned core–shell theranostic nanoparticles increases to about 200 nm after the multistep coating procedure.

Herein, bovine serum albumin (BSA), an inexpensive, commercially available biomacromolecule with rich amino acid residues as well as active functional groups is chosen as the scaffold to develop theranostic nanoparticles.^13–15 A facile, environmentally benign approach was designed to synthesize SPTN nanosystem. First, gadolinium-loaded BSA nanoparticles (Gd–BSA nanoparticles) were prepared via a simple biomineralization process. Then, the anticancer hydrophobic drug doxorubicin (DOX) was incorporated to form the theranostic nanoparticles DOX–Gd–BSA or Gd–BSA–DOX (abbreviated as DOX–Gd–BSA) via non-covalent interaction. Macromolecular mass spectrometry demonstrated that the chemotherapeutics DOX and the diagnostic reagent Gd are integrated on a single BSA molecule. T₁-weighted MR imaging effect and tumor therapy capability of SPTN have been investigated. The single molecule characteristic of this nanocarrier makes SPTN system with a hydrodynamic diameter (HD) of less than 10 nm which is important for the rapid clearance of the metal-containing nanoparticles from the body.

Experimental

Materials

Bovine serum albumin (BSA) from Sigma-Aldrich (Shanghai, China) and used as received. Gadolinium chloride hexahydrate (GdCl₃·6H₂O) was purchased from Sinopharm Chemical Reagent Co., Ltd (SCRC, Shanghai, China) and used as received. Doxorubicin hydrochloride (DOX·HCl) were purchased from Beijing United Technology Co., Ltd and used as received. Dulbecco's modified Eagle's medium trypsin, Dulbecco's phosphate buffered saline (DPBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), fluorescein isothiocyanate (FITC), penicillin–streptomycin were obtained from Gibco Invitrogen Corp. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Beyotime Institute of Biotechnology. Ultrapure Millipore DI water (18.2 MU cm resistivity at 25 °C) was used. Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee of Tongji University.

Synthesis of Gd–BSA nanoparticle

In a typical protocol, 100 mg BSA was dissolved in 10.0 mL DI water for 30 minutes under stirring. Then 7.5 mg GdCl₃·6H₂O was added dropwise into the BSA stock solution under vigorous stirring. 5 min later, the pH of the solution was adjusted to 8.4 with 0.1 M sodium hydroxide solution with constant stirring at room temperature. After 1 hour, the solution was dialyzed in DI water to remove the excess Gd³⁺ before freeze-drying.

Preparation of the theranostic nanoparticle

10 mg dried Gd–BSA was dispersed in 10 mL DI water. 2.5 mg DOX·HCl were dissolved in 10 mL DI water under slight stirring to prepare 250 μg mL⁻¹ stock solution, then it was added into the Gd–BSA solution slowly under constant stirring at room temperature. 20 μL triethylamine was subsequently added to the solution to desalinate DOX·HCl. After one hour, the solution was dialyzed against the DI water to remove the residual DOX with MWCO (Molecular weight cut-off) of 3500 before freeze-drying.

Drug loading capability and in vitro drug release study of the theranostic nanoparticle

To determine the drug loading amount, a certain amount of DOX–Gd–BSA nanoparticles was dispersed in 10 mL of 0.5 M hydrochloric acid solution. After all of the nanoparticles were completely dissolved, the concentration of the solution was quantitatively detected with a UV-vis spectrophotometer (UV759S, Q/YXL270) by comparing the absorbance of this solution at 480 nm with a calibration curve of aqueous DOX solution with known concentration.

In a typical drug release experiment, DOX–Gd–BSA nanoparticles were dialyzed against PBS solution at room atmosphere (pH = 7.4, 6.8, 6.3, 5.7) at 37 °C. At predetermined time, 2 mL solution was taken out from the PBS solution with 2 mL fresh buffer being added to keep the same condition. The released DOX concentration was determined by measuring the UV absorbance at 480 nm.

To determine the drug loading content, DOX–Gd–BSA (2.6 mg) was dissolved in hydrochloric acid (0.5 mol L⁻¹, 10 mL) and the mixture was measured using UV absorbance at 480 nm. The concentration of DOX was calculated according to the standard curve: c (mg mL⁻¹) = (A−0.0162)/21.93642, and A was the UV absorbance at 480 nm. The UV absorption strength of the mixture was 0.3584, thus the concentration of DOX of 1.56 × 10⁻² mg mL⁻¹ was calculated. The DOX drug loading content (DLC) was calculated from the following relationship: DLC = mass of DOX loaded in DOX–Gd–BSA/mass of DOX–Gd–BSA × 100%, so drug loading content was characterized to be 6.0%. The cumulative DOX release of DOX in DOX–Gd–BSA is calculated according to

Cumulative DOX release [%] = (M_t/M₀) × 100%

where M_t is the total amount of DOX released from the nanoparticle at time t, and M₀ is the amount of DOX initially loaded into the nanoparticle.

In vitro and in vivo MRI characterization

In in vitro MRI assessment of magnetic nanoparticle, T₁ was obtained by using a 3.0 Tesla MR scanner (Verio, Siemens, Munich, Germany). The T₁-weighted signals were obtained via the method of spin echo acquisition. For in vivo MRI, the MRI effects of magnetic nanoparticle were evaluated in female nude mice (≈25 g). Nanoparticles with the predetermined amount were injected into the tumor bearing nude mice via the tail vein. The concentration of Gd in DOX–Gd–BSA nanoparticles was determined by ICP-AES. Images were obtained at different time intervals post injection. The relative signal intensities in the region of interest (ROI) were recorded for analysis.

Cytotoxicity test

The cytotoxicity of the nanoparticles Gd–BSA, DOX–Gd–BSA, free DOX against hepatoma cells were investigated using the conventional MTT assay by measuring the cell viabilities. Briefly, hepatoma cells were seeded into a 96-well plates at 5000 cells per well. After overnight incubation with DMEM at 37 °C exposed to 5% CO₂ atmosphere, Gd–BSA, DOX–Gd–BSA and free DOX were then added. The concentration of the Gd–BSA and DOX–Gd–BSA was diluted with culture medium to obtain a concentration range of 7.5 mg L⁻¹ to 960 mg L⁻¹. The concentration of the free DOX was diluted with culture medium to obtain a concentration range of 0.45 mg L⁻¹ to 57.6 mg L⁻¹. After overnight incubation, the cell culture medium in each well was substituted with 100 μL fresh medium and 20 μL sterile filtered MTT (5 mg mL⁻¹). After incubation for 4 hour, the culture medium was removed carefully, and then DMSO (150 μL) was added into each well to dissolve the MTT formazan crystals. After the plates were cultured at 37 °C for 10 min and kept for 10 min on a shaking machine at room temperature. The absorbance of each well was measured at 570 nm using a Multiscan MK3 plate reader. The relative cell viability was calculated as cell viability (%) (OD_sample/OD_control), where the OD_control was obtained in the absence of drugs while the OD_sample was obtained in the presence of drugs.

Cellular uptake study

Confocal laser scanning microscopy (CLSM) was applied to monitor the behavior of the cellular uptake and the intracellular release of DOX loaded in DOX–Gd–BSA–FITC by using hepatoma cells as the model cells. BSA–FITC was synthesized according to the reported work.¹⁵ The hepatoma cells were incubated with DOX–Gd–BSA–FITC for different hours (0.5, 1, 2, 4) in a humidified 5% CO₂ atmosphere at 37 °C. After the removal of the culture medium of the wells, the cells were completely washed 3 times with PBS. Then the cells were fixed by 4% paraformaldehyde for 10 min at room temperature. After rinsing the unnecessary paraformaldehyde with PBS, the cell nuclei were stained with DAPI (blue) and then were rinsed again with PBS. CLSM images were obtained by using confocal microscope (TCS SP2).

Characterizations

The obtained Gd–BSA and DOX–Gd–BSA nanoparticles were dispersed in DI water to investigate the size and distribution of the nanoparticles by Dynamic Light Scattering (Malvern Instruments Ltd, Worcestershire, UK) with the scattering angle fixed at 90°; transmission electron microscopy (TEM) images were acquired by H7100 electron microscope with an acceleration voltage of 100 kV. In the process of preparing TEM samples, a drop (10 μL) of the resultant solution was placed on a holey carbon-coated copper grid and the drop was wicked through the back side of the grid with filter paper. X-ray photoelectron spectroscopy (XPS) was carried out by means of a RBD upgraded PHI-5000C ESCA system (RBD Enterprises, USA) with A1Kα radiation (hν = 1486.6 eV). Mass spectrometric measurements were performed using a 5800 MALDI-TOF-TOF (AB SCIEX) instrument. An accelerating voltage of 20 kV was used. Mass spectra were recorded in the reflect mode. Circular dichroism spectrum test was obtained by using Circular dichroism spectrum machine (j-810, JASCON Co., Ltd, Japan). UV-vis tests were performed according to the reported work.¹⁶ They were obtained by using an UV-vis spectrophotometer (UV759S, Q/YXL270, Shanghai Precision &scientific instrument Co., Ltd). The absorbance spectra of the nanoparticles were acquired in the range of 200–800 nm wavelength. The in vivo MRI assessment was investigated by using a 3.0 Tesla MR scanner (Verio, Siemens, Munich, Germany) at 25 °C. The relaxometry was operated using a 1.4 T minispecmq60 NMR Analyzer (Bruker, Germany) with the reported protocol.¹⁷ The nanoparticles were suspended at the Gd concentrations in range of 0.0546 mM to 0.874 mM for characterization.

Treatment of in vivo hepatoma xenograft model

Severe combined immunodeficiency disease (SCID) mice were injected with 10 million hepatoma cells. Treatment was initiated when the tumors reached a reasonable size (2–3 weeks after tumor inoculation). Tumor-bearing mice were injected with DOX–Gd–BSA at DOX concentration of 5 mg kg⁻¹ every two days. The control group was injected with PBS. The tumor sizes were measured by calipers three times a week and the volume was calculated according to the formula [(tumor length) × (tumor width)²]/2. Relative tumor volumes were calculated as v/v₀ (v₀ is the tumor volume before injection). Mice were weighted with the relative body weights compared to their initial weight.

Results and discussion

Synthesis and characterization of Gd–BSA and DOX–Gd–BSA nanoparticles

The synthetic route of Gd–BSA and DOX–Gd–BSA nanoparticles is described in Fig. 1. A biomineralization process that was directed by BSA was employed to fabricate Gd–BSA nanoparticle. BSA was chosen as a scaffold to sequester and interact with Gd ions, which has been used to synthesize several nanoparticles with different composition including gold,¹⁸ gadolinium¹⁹ etc. A mild alkaline condition (pH 8.4) was used in biomineralization process to activate BSA to entrap Gd ions, as well as facilitate the hydrolysis of GdCl₃ into Gd₂O₃ and Gd(OH)₃. Then the BSA–Gd solution was dialyzed with the deionized water to remove the excess Gd³⁺. It is reported that the carboxyl groups and thiol groups of BSA contribute to form the magnetic resonance contrast agents like gadolinium–BSA and superparamagnetic iron oxide–BSA nanoparticles.^19–21 For drug loading, DOX·HCl was introduced into the Gd–BSA solution, with the addition of triethylamine to desalinate HCl in DOX·HCl. In this process, the theranostic nanoparticles DOX–Gd–BSA were formed via either coordination bonding between Gd and DOX or hydrophobic and ionic interactions between BSA and DOX. In addition to chelate capability with metal ions, BSA could also serve as transporters for organic compounds via hydrophobic and ionic interactions.^22–24 The solution was dialyzed in darkness and freeze-dried for later experiments.

Fig. 1 The schematic illustration of the process to form the nanoparticles Gd–BSA and DOX–Gd–BSA.

TEM and DLS image of the nanoparticle of Gd–BSA and DOX–Gd–BSA are shown in Fig. 2. TEM images showed clearly well dispersed, uniform nanoparticles for both Gd–BSA and DOX–Gd–BSA nanoparticles attributable to highly hydrated and negatively charged BSA outer layer to prevent the nanoparticles from aggregation. Compared with Gd–BSA, the diameter of DOX–Gd–BSA is slightly increased after drug loading from 7.5 nm (Gd–BSA nanoparticles) to 9.0 nm (DOX–Gd–BSA). The mean diameter measured by DLS was larger than those acquired by TEM, which may be related to the water layer covering on the surface of the nanoparticle.

Fig. 2 TEM images of (A) nanoparticle Gd–BSA and (B) nanoparticle DOX–Gd–BSA. Nanoparticle size distributions of (C) nanoparticle Gd–BSA and (D) nanoparticle DOX–Gd–BSA.

The interaction between BSA and Gd, as well as the interaction between Gd–BSA and the drug DOX were investigated by X-ray photoelectron spectroscopy (XPS) analysis. As shown in Fig. 3A, the Gd (4d) was divided into two parts centered at the binding energy of 143.5 eV and 141.2 eV, which are attributed to Gd₂O₃ and Gd(OH)₃, respectively. These results are in consistent with the results on the reported Gd–BSA compounds.¹⁹ For DOX–Gd–BSA nanoparticle, a new peak appeared, centered at a lower binding energy of 140.4 eV, which indicated a new chemical state of gadolinium possibly attributable to the interaction of DOX and gadolinium.²⁵

Fig. 3 The Gd (4d) XPS spectra of the Gd–BSA nanoparticle (A) and DOX–Gd–BSA nanoparticle (B). The dash dot lines are the experimentally measured data points; the red lines are the data-fitted; the blue lines are the background, and other peaks of different colours are corresponding to the Gd-components analysed by XPS Peak 4.1 software.

In order to evaluate the stoichiometry of BSA : Gd, the molecular mass of BSA and the nanoparticle Gd–BSA was determined by the macromolecular mass spectrometry (MMS). The MMS is a very powerful and important tool to characterize the nanoparticles. The comparison between the MMS of BSA and Gd–BSA was showed in Fig. 4. From the mass difference between BSA and Gd–BSA, the number of the atom Gd could be calculated by the formulation (e.g. using [M + H]⁺ ions, [m(Gd–BSA) − m(BSA)]/m(Gd). A ratio of ∼3 Gd atoms per BSA was obtained which can be described as BSA–Gd₃. As a result, we can infer that the nanoparticle Gd–BSA was composed of one BSA and several Gd atoms in one single nanoparticle.

Fig. 4 Macromolecular mass spectrometry (MMS) of BSA and nanoparticle Gd–BSA.

Circular dichroism spectrum of BSA and Gd–BSA

To investigate the structural change of BSA by the addition of Gd, the CD spectra of BSA and Gd–BSA was studied. The CD spectra of BSA exhibit two negative peaks at 209 and 220 nm (Fig. 5) from the highly α-helical secondary structure. While after the addition of Gd ion, the CD intensity of BSA decreased without any significant shift of the peaks. The above results show that the conformation of BSA has changed with the loss of α-helical stability, which is possibly adapted for more efficient coordination with metal ions.^26,27

Fig. 5 Circular dichroism spectrum in the 190–350 nm range of nanoparticle BSA and Gd–BSA.

In vitro MRI

MRI is regarded as a powerful technology for long time which can detect deeply into tissue to analyze the pathological alterations, having much more advantages for clinical benefits than other methods such as optical approaches, nuclear imaging technology, like γ- and X-ray imaging and so on.²⁸ Gadolinium-based nanoparticle is a promising contrast agent due to its large magnetic moment and higher relaxivity.¹⁹ On account of the unique structure acquired with gadolinium-based nanoparticle, the in vitro MRI effect of the SPTN was investigated. The T₁ weight MR images of the nanoparticle were obtained by a clinical 3T MRI appliance. As shown in the insert of Fig. 6, the T₁ relative signal intensity of the nanoparticle is increasing gradually as the gadolinium concentration rise from 0.0546 mM to 0.874 mM. The T₁ relative signal intensity is related to the concentration of the nanoparticles DOX–Gd–BSA, and higher concentration leads to brighter signals. The relaxation rate of the nanoparticle (Fig. 6) increases linearly in company with the concentration of nanoparticle. The r₁ value of the nanoparticle is around 12.611 mM⁻¹ s⁻¹.

Fig. 6 T₁ relaxation rate (1/T₁) as a function of Gd concentration recorded for the aqueous solution (37 °C) of nanoparticle. T₁ weighted MR images (inserted panel) of magnetic nanoparticle: (I) H₂O; (II) Gd concentration 0.0546 mM; (III) Gd concentration 0.10 mM; (IV) Gd concentration 0.218 mM; (V) Gd concentration 0.437 mM; (VI) Gd concentration 0.874 mM.

In vitro drug release

According to previous report,²⁹ nanoparticles formed by coordination bond between drug and metal ions exhibits pH-responsiveness. Thus the drug release behavior of the nanoparticle DOX–Gd–BSA was studied in different pH medium (pH = 5.7, 6.3, 6.8, 7.4) at 37 °C to test the pH-responsiveness of the nanoparticle. As shown in Fig. 7, the pH value of the medium have an obvious influence on the drug release rate of DOX–Gd–BSA. The cumulative release of DOX in PBS of pH 7.4 was less than 23% within 24 hours, while the drug release amount were 25.7%, 49.8%, 56.0% in PBS of pH 6.8, 6.3, 5.7, respectively. According to these data, we found that the amount of DOX released from the nanoparticle at pH 5.7 is almost 2.2-fold higher than that of pH 7.4. These release profiles indicated that the nanoparticle DOX–Gd–BSA will be more stable in blood during in vivo circulation, whereas the drug DOX will be released quickly after reaching the extracellular area of tumor or the intracellular acidic organelle such as lysosome. It is reported that the extracellular pH of tumor is more acidic (pH 5.7–7.8) than that of blood and normal tissues.³⁰

Fig. 7 Release behavior of DOX from the nanoparticle of DOX–Gd–BSA in PBS with pH (5.7, 6.3, 6.8, 7.4) at the temperature of 37 °C.

Cytotoxicity study

The therapeutic efficiency of the DOX-loaded Gd–BSA was carried out in vitro by quantifying the cell viability of hepatoma cancer cells using the conventional MTT assay. The nanoparticle DOX–Gd–BSA, Gd–BSA, and free DOX were incubated with the hepatoma cells at different concentration. As shown in Fig. 8, the nanoparticle Gd–BSA without DOX has little influence on the cell viability of this cell line even at a high concentration of 960 μg mL⁻¹. For some of specific concentrations, the viability of the cell even seems to be higher than that of the cell without adding any materials. This might be due to the nanoparticle Gd–BSA contains the nutrition substance of amino acids that the cells needed in the process of proliferation. The viability of the cell was reduced in the higher concentration (960 μg mL⁻¹) of Gd–BSA which may result from the toxicity of the Gd element. However, both free DOX and DOX–Gd–BSA have effectively reduced the viability of the hepatoma cells in a dose-dependent fashion. In both cases, about 80% growth inhibition of the cell is achieved at the DOX concentrations of >7.2 mg L⁻¹ after 24 h incubation.

Fig. 8 Cell proliferation of hepatoma cells incubated with free DOX, Gd–BSA and DOX–Gd–BSA for 24 h at various concentrations.

The intracellular uptake behavior

The cellular uptake of the nanoparticle DOX–Gd–BSA was investigated by confocal laser scanning microscope (CLSM). The drug DOX can be easily traced under CLSM because of its inherent red fluorescence. To study the spatial distribution of DOX–Gd–BSA, cell nucleus was stained by DAPI with the blue fluorescence. In addition, DOX–Gd–BSA was labeled by the green fluorescence dye FITC to investigate the cellular uptake and intracellular drug release behavior of DOX–Gd–BSA. From Fig. 9, we can see that the green fluorescence of FITC can be detected in the cell only after half an hour incubation which indicated the uptake of the nanoparticle DOX–Gd–BSA–FITC. Moreover, we found that as the time goes on, the intensity of the red fluorescence of DOX became stronger. After 4 h incubation, the red fluorescence of DOX is found throughout the entire cells, indicating that the drug DOX was released gradually from the nanoparticle DOX–Gd–BSA–FITC after entering into the cell.

Fig. 9 CLSM images of hepatoma cell incubated with the nanoparticle DOX–Gd–BSA–FITC for different periods. (A) 0.5 h; (B) 1 h; (C) 2 h; (D) 4 h. Cell nuclei (blue) were stained with DAPI.

In vivo MR imaging

The in vivo MR imaging data of the nanoparticle DOX–Gd–BSA was illustrated in Fig. 10. Hepatoma cells were subcutaneous injected into the right back of nude mice to prepare tumor-bearing mice. The nanoparticle solution was injected to the nude mice through the intravenous injection in the following step. Then the T₁ weighted MR imaging of the mice was acquired at determined time point. The tumor site was distinguished by the white arrow. After the injection of the nanoparticle, the signal intensity enhancement was obviously found at the tumor site at 5 min post-injection. The accumulation of the nanoparticle DOX–Gd–BSA at the tumor site was possibly through the enhanced permeability and retention (EPR) effect.³¹ Afterwards, the signal intensity gradually turned weak with the time increasing. After 4 h, the relative signal intensity in the tumor site recovered to the level of the beginning prior to injection. The recovery of the signal intensity suggests that the nanoparticle can be excreted within a certain period which is important to develop safe and effective MRI contrast agents.

Fig. 10 The T₁-weighted MR imaging in vivo of the nude mice obtained (A) as a baseline and (B–F) after injection of the nanoparticle DOX–Gd–BSA at different time period: (B) 5 min, (C) 30 min, (D) 60 min, (E) 180 min, (F) 240 min. The white arrows represent xenograft tumors.

Fig. 11 The in vivo therapeutic efficacy of nanoparticle DOX–Gd–BSA. Hepatoma tumor bearing SCID mouse were treated with DOX–Gd–BSA and PBS respectively. Tumor size and the body weight of the mouse were measured three times a week.

In vivo therapeutic efficacy

In order to further study the in vivo therapeutic efficacy of the obtained nanoparticle DOX–Gd–BSA, SCID mice bearing hepatoma tumor were treated with DOX–Gd–BSA by tail injection. For the control group treated with the medium PBS, the tumor size of the mice was rapidly increased by around 6.30 fold (Fig. 11). However, the tumor size of DOX–Gd–BSA treated mice showed to be effectively inhibited with little increase which indicated a great inhibition of the tumor growth. In accordance with the above result, the body weight of the DOX–Gd–BSA treated mice decreased to 0.97 fold comparing to the PBS injected mice whose weight increased by 1.09 fold. Thus it indicated that the theranostic nanoparticle DOX–Gd–BSA has obvious therapeutic efficacy.

Conclusions

In this work, we have successfully developed a single-albumin based theranostic nanoparticle, in which the Gd ion was introduced as a contrast agent to enhance the imaging effect and also to provide capability to load the anticancer hydrophobic drug DOX. The entire synthetic process involves two steps at ambient environment without any organic solvent and was very fast, effective and sustainable. Both in vitro and in vivo studies all suggest obvious evidence that the synthetic nanoparticle exhibits excellent T₁-weighted MR imaging effect and effective cancer cell inhibition ability. Moreover, the single molecule characteristic of this theranostic nanoparticle which could enable the rapid clearance of the metal-containing nanoparticles from the body revealed the potential of this theranostic nanoparticle to be served as a promising theranostic nanoparticle for clinical application.

Acknowledgements

This work was financially supported by 973 program (2013CB967500), National Natural Science Foundation of China (81402884, 51473124, 51173136), National Science Foundation for Post-doctoral Scientists of China (2014M561511, 2015T80453) and “Chen Guang” project founded by Shanghai Municipal Education Commission and Shanghai Education Development Foundation.

Notes and references

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