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Doxorubicin-Loaded Magnetic Silk Fibroin Nanoparticles for Targeted Therapy of Multidrug-Resistant Cancer Ye Tian, Xuejiao Jiang, Xin Chen,* Zhengzhong Shao, and Wuli Yang* Cancer is one of the leading causes of mortality worldwide.[1] As cancer proliferates, tremendous efforts in biomedical research have been devoted to clinical therapy and several sorts of anticancer drugs have been aggressively marketed.[2] Nevertheless, the drug efficacy is often altered by nonspecific cell and tissue biodistribution and drug resistance,[3] and some drugs are limited by short blood circulation half-life and rapidly metabolized in vivo.[4] To get over the problems, many advanced nanometer-scale systems have been developed, parceling up the drugs and delivering them to specific locations.[5] Nanocarrier drug delivery systems (DDSs) provide advantages over the administration of free drugs, including targeting ability to enhance accumulation at tumor sites,[6] overcoming resistance by intracellular drug delivery,[7] and realizing controlled and sustained release to enhance drug bioavailability.[8] Owing to the prodigious advances in material science, a broad range of nanocarriers with diverse sizes, structures and surface properties have been designed.[3] Biocompatibility and biodegradability are required characteristics for materials employed in preparing the nanocarriers.[9] Various polymeric materials fulfilling the previous requirements have been investigated for use as a drug delivery matrix, including synthetic biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and poly(εcaprolactone) (PCL),[10] and natural polymers such as polysaccharide (cellulose, chitosan, hyaluronic acid, dextran, etc.)[11] and proteins (collagen, gelatin, elastin, albumin, etc.).[12] Silk fibroin (SF) protein, a natural product regenerated from cocoons of Bombyx mori, has demonstrated extraordinary promise in biomedical fields with mounting attention due to their good biocompatibility and tunable biodegradability.[13,14] The past decade has witnessed an increasing number of strategies to generate SF-based nanoparticles (SFNs) with the ability of capturing and releasing model drugs.[15–20] Recently, Kaplan and coworkers successfully prepared doxorubicin (DOX)-loaded SFNs that were effective to serve as a lysosomotropic anticancer nanomedicine and overcome multidrug resistance (MDR).[21] However, lack of tumor-targeting ability compromises the tumor accumulation of therapeutic agents,[6] which is detrimental to the drug efficacy. Therefore, preparing SFNs that can
Y. Tian, X. Jiang, Prof. X. Chen, Prof. Z. Shao, Prof. W. Yang State Key Laboratory of Molecular Engineering of Polymers Department of Macromolecular Science and Laboratory of Advanced Materials Fudan University Shanghai 200433, P. R. China E-mail: [email protected]; [email protected]
DOI: 10.1002/adma.201403562
Adv. Mater. 2014, DOI: 10.1002/adma.201403562
target to the tumor sites are required in order to display their properties as a DDS in vivo. Magnetic tumor targeting, using magnetic carriers and an external magnetic field (MF) focused on the tumor, has already emerged as a promising strategy to enhance drug accumulation at the tumor sites.[22] Compared with conventional tumor targeting based on molecular ligand-receptor bindings, magnetic targeting does not require complicated chemical modification of targeting ligands on the surface of nanocarriers,[23] which makes it optimal for natural-product DDSs. The independence of magnetic targeting on the specific receptors expressed on the tumor cells also expands its applicable range to a wide variety of solid tumors.[24] Nevertheless, to the best of our knowledge, the application of magnetic targeting in SF-based DDSs has been rarely reported to date. Herein, we show that DOX-loaded magnetic SFNs (DMSs) can be easily prepared using a one-step potassium phosphate salting-out strategy by including specific amounts of hydrophilic magnetic Fe3O4 nanoparticles (MNPs) in phosphate solution. The introduction of superparamagnetic MNPs not only provides magnetism for SFNs, but also realizes the artificial regulation of SFN formation and DOX entrapment behavior. Finally, the magnetic-guided drug delivery in a humanized orthotropic breast cancer model and chemotherapy performance of drug-resistant cancer is studied in the present contribution to test the abilities of DMSs to serve as a DDS in vivo. This study could be extremely useful for the advancement of SF-based nanocarrier preparation as well as the expansion beyond its use for orthodox drug delivery. The preparation of DMSs is based on a salting-out method similar to that described previously by Kaplan and coworkers[19] and a salting-out condition is chosen to fabricate SFNs with appropriate particle size and salting-out efficiency. Briefly, DOX and MNPs are firstly dispersed in potassium phosphate solution (1.25 M, pH 8) followed by the addition of SF solution and low-temperature treatment (experiment procedures are detailed in the Supporting Information). The results, which are summarized in a phase diagram (Figure 1a), vary considerably in the formation of nanoparticles: at higher DOX concentration, larger amount of MNPs is required to form DMSs, otherwise particles aggregated into non-dispersible clusters in the saltingout process. The influence of MNP concentration on particle formation is directly suggested in the corresponding transmission electron microscope (TEM) images (shown in Figure 1a). Macroscopic gelation is formed with insufficient MNPs and as the MNP concentration increases continuously, agglomeration of SF disappears and particles with uniform sphererical shape are formed, which is also suggested by the decrease of size distribution when measured by dynamic scattering light (DLS, Figure S1a and Table S1 in the Supporting Information). Some dark spots, standing for MNPs, are observed on the background
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Figure 1. Preparation and basic characterizations of DMSs. (a) Phase diagram of the salting-out results with different concentrations of DOX and MNPs: gelation (red cross); particle formation (black dot). Half-filled dots indicate particles can be observed occasionally. The salting-out results circled in the phase diagram are visualized with TEM. White bar indicates 200 nm in all images. (b) Schematic illustration of the formation of DMSs: DOX is partly adsorbed on the surface of MNPs and then capsulated in the SFNs during the salting-out generation process. The diagram is not drawn to scale. (c) T2-MRI of DMSs in aqueous solution at different Fe concentrations. (d) Entrapment efficiency of DMSs prepared with various MNP and DOX concentrations. The mean values and standard errors are from three measurements. Inset: digital image of a DMS dispersion. (e) pH-dependent release of DOX during 3 weeks. (f) DOX release profiles in the presence of proteinase (α-chymotrypsin).
of TEM images if more MNPs are added, suggesting excess MNPs that are not capsulated in the SFNs (Figure 1a). And MNPs tend to aggregate in the salting out process of SF (Figure S1b in the Supporting Information), which leads to a broader size distribution. The phenomenon that the particle generation can be regulated simply by tuning the amount of MNPs in the system is possibly related to the electrostatic and/or hydrophobic interactions among MNPs, DOX molecules and SF protein chains. In the absence of MNPs, the slightly positively charged DOX molecules (pKa 8.3)[25] give rise to the gelation of the negatively charged SF protein, whose theoretical isoelectric point (pI) is 4.53.[26] Upon the addition of negatively charged MNPs that are stabilized by citrate groups, part of DOX will be adsorbed on the surface of the MNPs, which is proved by the zeta potential increase of the MNPs dispersed in DOX solution (data are shown in Figure S2 in the Supporting Information). The assembly of SF starts with β-sheet formation induced by the salting-out effect and the Fe3+ on the surface of MNPs,[27] and with microcrystals isolated by ice (the effect of low temperature on particle formation is detailed in Figure S3 in the Supporting Information), the SF protein grows into a well-defined
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architecture to form spherical SFNs and capsulating MNPs together with DOX (this process is schemed in Figure 1b). In case of the amount of DOX increasing, more MNPs are essential to adsorb them in the formation process of DMSs, and thus the particle generation and drug entrapment behavior are related to the MNP concentration in the system. Meanwhile, MNPs work as heterogeneous nucleation sites in the salting out process and the additon of MNPs dramatically decreases the particle size of SFNs from 1.4 µm to 130 nm, which is benefit for cell internalization (Figure S4 in the Supporting Information). The Fourier transform infrared (FTIR) spectra of the obtained DMSs (Figure S5a in the Supporting Information) show that only minor changes in secondary structure on the condition of introducing MNPs in the salting-out process, and the fractions of β-sheet calculated from the deconvolution of the FTIR spectra (shown in Figure S5b and Table S2 in the Supporting Information) are relatively low. The capsulation of MNPs in the DMSs can be characterized by the thermogravimetic analysis (TGA, Figure S6a in the Supporting Information) and superparamagnetic behavior provided by MNPs (Figure S6b and Table S2 in the Supporting Information).
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Adv. Mater. 2014, DOI: 10.1002/adma.201403562
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tissue) and pH 7.4 (blood plasma).[28] This phenomenon is due to a weak electrostatic interaction between SF and DOX at low pH values (the pH-dependent release is schemed in Figure S10 in the Supporting Information).[29] The DOX release behavior in the presence of proteinase (α-chymotrypsin) is also studied (Figure 1f and Figure S11 in the Supporting Information). In 4 days, nearly all the DOX are released, much faster than that without proteinase, indicating the facilitation effect of the nanocarrier biodegradation on the drug release. To investigate the internalization and intracellular drug release behaviors of DMSs, human breast adenocarcinoma cell line (MCF-7 cells) and its multidrug resistant counterpart (MCF-7/ADR cells) are treated with free DOX and DMSs. The cellular DOX uptake is measured quantitatively by the mean fluorescence intensity per cell via flow cytometry (Figure 2a). The uptake of free DOX and DMSs in MCF-7 cells are both high, while in the drug-resistant MCF-7/ADR cells, the accumulation of DMSs is much higher than that of free DOX. As illustrated by previous reports, free DOX enters the MCF-7/ ADR cells through a flip-flop mechanism and is extruded by the overexpressing efflux P-glycoprotein (P-gp).[30] Thus the cellular uptake data suggest different internalization pathways between free DOX and DMSs. The internalization mechanism of DMSs is studied with specific endocytic inhibitors by flow cytometry in MCF-7/ADR cells (Figure 2b). Compared with DMSs without inhibitors, the cellular uptake is significantly inhibited by sodium azide (energy inhibitor) and chlorpromazine (clathrin-mediated endocytosis inhibitor) and little or no inhibition is found when cells are pretreated with
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T2-weighted magnetic resonance imaging (T2-MRI, Figure 1c) of DMSs reveals a concentration-dependent manner of the dark intensity with the T2 relaxation cofficient (r2) calculated to be 168.42 s−1 mM−1 (Figure S7 in the Supporting Information). The entrapment efficiency of DOX is concluded in Figure 1d. Although the percentage of entrapped drug declines as the amount of DOX in the system increases, the total amount of drug loaded in DMSs increases (data shown in Figure S8 in the Supporting Information). At a constant DOX concentration, the loading efficiency of DOX grows as more MNPs are added during the salting-out process until reaching a ceiling, which is attributed to the increase of DOX adsorbed on MNPs. Afterwards, the entrapment efficiency decreases resulting from the MNPs that are not capsulated in the DMSs. The loading content of DOX peaks at a MNP concentration of 100 µg mL−1, giving the value of 5.9 wt% (60.9 ng DOX/µg SF), which is about 84.0 wt% of the total drug in the system, and this sample is used for further study. Owing to the pre-loading of MNPs, the loading content value is approximately 50% higher than that for directly binding of DOX on the SFNs[21] (40 ng DOX/µg SF, Figure S9a in the Supporting Information), which makes it more effective to kill cancer cells (Figure S9b in the Supporting Information). The DOX release profiles under various environmental pH values are demonstrated in Figure 1e, in which a pHdependent release is observed: low environment pH accelerates the DOX release. At pH 5.0 (mimicking microenvironment in endosomes and lysosomes), the release rate is significantly higher than those at pH 6.5 (extracelluar environment in tumor
Figure 2. In vitro cell assays. (a) Quantitative cellular uptake analysis of DOX in MCF-7 cells and MCF-7/ADR cells after treated with DOX and DMSs. (b) Relative uptake efficiency of DMSs in MCF-7/ADR cells in the presence of various endocytosis inhibitors. *, P < 0.05 vs control. (c) Confocal microscopic images of MCF-7 cells and MCF-7/ADR cells incubated with DOX and DMSs for 3 h. White bar indicates 25 µm in the image. (d) Schematic diagram of proposed endocytosis pathways and intracellular drug delivery mechanisms of DMSs in P-gp overexpressed MCF-7/ADR cells. (e) Growth inhibition results for MCF-7 (䊊,䊉) and MCF-7/ADR (䊐,䊏) cells treated with free DOX (䊊,䊐) and DMSs (䊉,䊏) with different DOX dosage. The concentration of DMSs is shown on the top x axis.
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Figure 3. In vivo pharmacokinetics and magnetic tumor targeting of DMSs. (a) Blood circulation time of DOX and DMSs analyzed by measuring plasma concentration of DOX. The data are expressed as percentage of the injected dose (%ID). (b) A schematic drawing to illustrate magnetic tumor targeting. (c) Fluorescence imaging after intravenous injection of DMSs or DOX-loaded SFNs with an equivalent DOX dosage (5 mg kg−1). Top: in vivo images (bottom view) of mice bearing two MCF-7 tumors on both sides at 2 h post injection. Red and green circles point out tumors with and without magnet attachment, respectively. Bottom: ex vivo images of tumors and major organs at 12 h post injection. H: heart, Li: liver, S: spleen, Lu: lung, K: kidney, T+MF and T-MF: tumor in the presence and absence of MF, respectively. (d) The biodistribution of the DMSs and DOX-loaded SFNs after DMS administration and MF application at tumor, based on HPLC analysis. (e) Tumor growth curves of tumors after various treatments. ***, P < 0.001.
indomethacin (caveolae-dependent endocytosis inhibitor), colchicine (microtubule-dependent macropinocytosis inhibitor) and quercetin (inhibitor of caveolae- and clathrin-independent endocytosis),[31] which demonstrates that the uptake pathway of DMSs is energy-dependent endocytosis process mediated by clathrin. The subcellular location study via staining the nucleus and lysosomes with Hoechst 33342 and Lysotraker Green DND26, respectively, and observation with a confocal laser scanning microscopy (CLSM, Figure 2c) also reveals the uptake difference of free DOX and DMSs in MCF-7/ADR cells. The DMSs exhibit efficient intracellular delivery in both MCF-7 and MCF-7/ADR cells with colocalization in lysosome and partly entering into the nucleus in 3 h, which further indicates the lysosomotropic delivery potential of DMSs (the endocytosis pathway and intracellular delivery process are schemed in Figure 2d). From the results above, we can infer that DMSs can be taken into the multidrug resistant MCF-7/ADR cells by clathrinmediated endocytosis, which escapes the efflux induced by P-gp and thereby may overcome MDR in cancer chemotherapy. MTT (MTT is (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is conducted (Figure 2e and Figure S12 in the Supporting Information) to confirm their ability to overcome MDR. Significant growth inhibition of MCF-7 cells is observed when the cells are incubated with either DMSs or free DOX at a higher DOX concentration, and DMSs are significantly more cytotoxic to MCF-7/ADR cells than free DOX at an equivalent DOX dosage, which proves that DMSs display enough efficacy to kill the cancer cells and overcome MDR. Before in vivo chemotherapy application of DMSs, the toxicology of DMSs in female BALB/c nude mice is investigated to ensure their safety. The blood chemistry (Figure S13a in the Supporting Information) and hematology analysis (Table S3 in the Supporting Information) reveal that no obvious hepatic toxicity is induced and all of the hematological parameters in the
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treated groups appear to be normal compared with the control group. The histopathological examinations of the organ tissues (Figure S13b in the Supporting Information) show no pathological changes in liver, spleen, lung and kidney. It should be noted that the heart slice of DMSs group exhibits myocardial fiber rupture, which may be induced by the cardiac toxicity of DOX.[32] To understand the pharmacokinetics of DMSs, the plasma concentration of DOX is measured on the basis of DOX quantification by high performance liquid chromatography (HPLC, Figure 3a). The blood circulation time of DMSs is longer than that of free DOX and the area under the curve is also larger, indicating greater blood persistence and better bioavailability of DMSs.[33] Next, the magnetic tumor targeting performance is studied. Mice bearing two subcutaneous MCF-7 tumors on opposite flanks are intravenously injected with DMSs and DOX-loaded SFNs (DOX dosage 5 mg kg−1) via tail vein, and the tumors on the left side are attached to magnets (Figure 3b). After 2 hours, in vivo fluorescence imaging is conducted (Figure 3c) and DMSs are found to accumulate at the magnet-attached tumor, while no significant tumor enrichment of DOX-loaded SFNs is observed regardless of magnet attachment. Ex vivo imaging of major viscera (heart, liver, spleen, lung and kidneys) and tumor tissues also confirms the MF-guided tumor accumulation effect of DMSs (Figure 3c). Quantification analysis of DOX concentration in the tissues (Figure 3d) states that magnetic targeting can enrich DMSs and suppress the uptake in liver, which is also proved by the quantification of Fe elements in the tissues (measured by inductively coupled plasma atomic emission spectrometer, ICP-AES, Figure S14 in the Supporting Information). The effective tumor targeting of DMSs induced by external MF makes the tumor growth different from that without magnet attachment. The growth of tumors attached to magnets is greatly inhibited, when compared to the tumor
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COMMUNICATION Figure 4. Magnetic-guided chemotherapy of multidrug resistant cancer. (a) Schematic illustration of the therapeutic process of MCF-7/ADR tumors with the assistance of a magnet. (b) In vivo T2-MRI of mice after intravenous injection of DMSs. Red and green circles point out tumors with and without magnet attachment, respectively (the same as below). (c) In vivo fluorescent imaging of mice bearing MCF-7/ADR tumors after administration. (d) Ex vivo imaging of tumor tissues and major organs at 12 h post injection. Left: from a mouse with the application of MF at the tumor site. Right: from a mouse without MF application. (e) Distribution profiles of DOX and Fe element in tissues at 12 h after DMS administration at a DOX dosage of 5 mg kg−1. (f) In vivo effects of DMSs and magnet attachment on the growth of MCF-7/ADR tumor. **, P < 0.01; ***, P < 0.001. (g) Morbidity-free survival rate of mice bearing MCF-7/ADR tumors after treatment. (h) Body weight change of the mice.
without magnet attachment (Figure 3e). However, we can hardly distinguish the difference in tumor growth in spite of the MF application when treated with the non-MF responsive DOX-loaded SFNs. Being aware of the high magnetic targeting efficiency of DMSs and their ability to overcome MDR, we finally conduct the magnetic-guided chemotherapy of multidrug resistant cancer. Mice bearing MCF-7/ADR tumors are intravenously injected with DMSs and magnets are attached to the tumor sites (schemed in Figure 4a). Tumor tissue attached to the magnet appears as a dark area on the in vivo T2-MR image, while in the absence of MF, the tumor tissue appears bright (Figure 4b and Figure S15a in the Supporting Information), confirming the successful targeting of DMSs to the tumor region and their apparent MRI contrast effect. And as demonstrated in the fluorescence images (Figure 4c and Figure S15b in the Supporting Information), the DOX fluorescent signal in the magnet-attached tumor shows a remarkable increase over time after injection. The ex vivo fluorescence images (Figure 4d) and quantification of DOX and Fe levels in the tissues (Figure 4e) also confirm the MF-induced tumor targeting
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of DMSs. To determine the antitumor effect of magnetic-guided DMSs, both tumor sizes (Figure 4f) and morbidity-free survival rate (Figure 4g) are recorded after treatment. The results show that the tumors treated with PBS or drug-free magnetic SFNs (M-SFNs) grow rapidly, suggesting the tumor growth is not affected by the SF-based nanocarriers. The growth inhibition effect of MCF-7/ADR tumors with DMS treatment is better than that treated with free DOX, possibly related to better drug availability (as shown in Figure 3a). A single injection of DMSs plus MF attachment significantly suppresses of the growth of MCF-7/ADR tumors with 100% of survival rate on day 30, confirming their effect on regression of drug-resistant tumors. The body weight of mice, whose change is an indicator of systemic toxicity,[34] is simultaneously measured after receiving DMSs treatment (Figure 4h). Those treated with free DOX display obvious body weight fluctuation, suggesting its toxic effect on animals. The body weight of mice in DMSs- and M-SFNstreated groups do not differ greatly from that of PBS, indicating that the SF-based nanocarriers do not exhibit severe systemic toxicity. It is suggested that DMSs, which possess efficient anti-tumor activity under the guidance of MF together with
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low systemic toxicity, can act as a hopeful nanocarrier for the delivery of chemotherapeutic drugs and overcoming MDR. In conclusion, we have developed an approach to prepare DMSs by means of salting out SF in the presence of DOX and MNPs, and the regulation of DMSs generation and DOX entrapment are achieved in this approach through simply tuning the concentration of MNPs. The MF-induced tumor targeting ability in vivo and effective chemotherapy of multidrug resistant cancer demonstrate that the DMSs work well as a novel DDS in cancer therapy. The nanoparticle-assisted SFNs preparation approach introduces extra functions for SFNs, providing them with magnetic targeting ability, which greatly expands the clinical applications of SF-based materials, and is inspiring in the advancement of biomacromolecular materials.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was financially supported by National Science Foundation of China (Grant Nos. 20874015 and 51273047) and the “Shu Guang” project (12SG07) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. Received: August 5, 2014 Published online:
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Doxorubicin-Loaded Magnetic Silk Fibroin Nanoparticles for Targeted Therapy of Multidrug-Resistant Cancer Ye Tian, Xuejiao Jiang, Xin Chen,* Zhengzhong Shao, and Wuli Yang* Cancer is one of the leading causes of mortality worldwide.[1] As cancer proliferates, tremendous efforts in biomedical research have been devoted to clinical therapy and several sorts of anticancer drugs have been aggressively marketed.[2] Nevertheless, the drug efficacy is often altered by nonspecific cell and tissue biodistribution and drug resistance,[3] and some drugs are limited by short blood circulation half-life and rapidly metabolized in vivo.[4] To get over the problems, many advanced nanometer-scale systems have been developed, parceling up the drugs and delivering them to specific locations.[5] Nanocarrier drug delivery systems (DDSs) provide advantages over the administration of free drugs, including targeting ability to enhance accumulation at tumor sites,[6] overcoming resistance by intracellular drug delivery,[7] and realizing controlled and sustained release to enhance drug bioavailability.[8] Owing to the prodigious advances in material science, a broad range of nanocarriers with diverse sizes, structures and surface properties have been designed.[3] Biocompatibility and biodegradability are required characteristics for materials employed in preparing the nanocarriers.[9] Various polymeric materials fulfilling the previous requirements have been investigated for use as a drug delivery matrix, including synthetic biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and poly(εcaprolactone) (PCL),[10] and natural polymers such as polysaccharide (cellulose, chitosan, hyaluronic acid, dextran, etc.)[11] and proteins (collagen, gelatin, elastin, albumin, etc.).[12] Silk fibroin (SF) protein, a natural product regenerated from cocoons of Bombyx mori, has demonstrated extraordinary promise in biomedical fields with mounting attention due to their good biocompatibility and tunable biodegradability.[13,14] The past decade has witnessed an increasing number of strategies to generate SF-based nanoparticles (SFNs) with the ability of capturing and releasing model drugs.[15–20] Recently, Kaplan and coworkers successfully prepared doxorubicin (DOX)-loaded SFNs that were effective to serve as a lysosomotropic anticancer nanomedicine and overcome multidrug resistance (MDR).[21] However, lack of tumor-targeting ability compromises the tumor accumulation of therapeutic agents,[6] which is detrimental to the drug efficacy. Therefore, preparing SFNs that can
Y. Tian, X. Jiang, Prof. X. Chen, Prof. Z. Shao, Prof. W. Yang State Key Laboratory of Molecular Engineering of Polymers Department of Macromolecular Science and Laboratory of Advanced Materials Fudan University Shanghai 200433, P. R. China E-mail: [email protected]; [email protected]
DOI: 10.1002/adma.201403562
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target to the tumor sites are required in order to display their properties as a DDS in vivo. Magnetic tumor targeting, using magnetic carriers and an external magnetic field (MF) focused on the tumor, has already emerged as a promising strategy to enhance drug accumulation at the tumor sites.[22] Compared with conventional tumor targeting based on molecular ligand-receptor bindings, magnetic targeting does not require complicated chemical modification of targeting ligands on the surface of nanocarriers,[23] which makes it optimal for natural-product DDSs. The independence of magnetic targeting on the specific receptors expressed on the tumor cells also expands its applicable range to a wide variety of solid tumors.[24] Nevertheless, to the best of our knowledge, the application of magnetic targeting in SF-based DDSs has been rarely reported to date. Herein, we show that DOX-loaded magnetic SFNs (DMSs) can be easily prepared using a one-step potassium phosphate salting-out strategy by including specific amounts of hydrophilic magnetic Fe3O4 nanoparticles (MNPs) in phosphate solution. The introduction of superparamagnetic MNPs not only provides magnetism for SFNs, but also realizes the artificial regulation of SFN formation and DOX entrapment behavior. Finally, the magnetic-guided drug delivery in a humanized orthotropic breast cancer model and chemotherapy performance of drug-resistant cancer is studied in the present contribution to test the abilities of DMSs to serve as a DDS in vivo. This study could be extremely useful for the advancement of SF-based nanocarrier preparation as well as the expansion beyond its use for orthodox drug delivery. The preparation of DMSs is based on a salting-out method similar to that described previously by Kaplan and coworkers[19] and a salting-out condition is chosen to fabricate SFNs with appropriate particle size and salting-out efficiency. Briefly, DOX and MNPs are firstly dispersed in potassium phosphate solution (1.25 M, pH 8) followed by the addition of SF solution and low-temperature treatment (experiment procedures are detailed in the Supporting Information). The results, which are summarized in a phase diagram (Figure 1a), vary considerably in the formation of nanoparticles: at higher DOX concentration, larger amount of MNPs is required to form DMSs, otherwise particles aggregated into non-dispersible clusters in the saltingout process. The influence of MNP concentration on particle formation is directly suggested in the corresponding transmission electron microscope (TEM) images (shown in Figure 1a). Macroscopic gelation is formed with insufficient MNPs and as the MNP concentration increases continuously, agglomeration of SF disappears and particles with uniform sphererical shape are formed, which is also suggested by the decrease of size distribution when measured by dynamic scattering light (DLS, Figure S1a and Table S1 in the Supporting Information). Some dark spots, standing for MNPs, are observed on the background
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Figure 1. Preparation and basic characterizations of DMSs. (a) Phase diagram of the salting-out results with different concentrations of DOX and MNPs: gelation (red cross); particle formation (black dot). Half-filled dots indicate particles can be observed occasionally. The salting-out results circled in the phase diagram are visualized with TEM. White bar indicates 200 nm in all images. (b) Schematic illustration of the formation of DMSs: DOX is partly adsorbed on the surface of MNPs and then capsulated in the SFNs during the salting-out generation process. The diagram is not drawn to scale. (c) T2-MRI of DMSs in aqueous solution at different Fe concentrations. (d) Entrapment efficiency of DMSs prepared with various MNP and DOX concentrations. The mean values and standard errors are from three measurements. Inset: digital image of a DMS dispersion. (e) pH-dependent release of DOX during 3 weeks. (f) DOX release profiles in the presence of proteinase (α-chymotrypsin).
of TEM images if more MNPs are added, suggesting excess MNPs that are not capsulated in the SFNs (Figure 1a). And MNPs tend to aggregate in the salting out process of SF (Figure S1b in the Supporting Information), which leads to a broader size distribution. The phenomenon that the particle generation can be regulated simply by tuning the amount of MNPs in the system is possibly related to the electrostatic and/or hydrophobic interactions among MNPs, DOX molecules and SF protein chains. In the absence of MNPs, the slightly positively charged DOX molecules (pKa 8.3)[25] give rise to the gelation of the negatively charged SF protein, whose theoretical isoelectric point (pI) is 4.53.[26] Upon the addition of negatively charged MNPs that are stabilized by citrate groups, part of DOX will be adsorbed on the surface of the MNPs, which is proved by the zeta potential increase of the MNPs dispersed in DOX solution (data are shown in Figure S2 in the Supporting Information). The assembly of SF starts with β-sheet formation induced by the salting-out effect and the Fe3+ on the surface of MNPs,[27] and with microcrystals isolated by ice (the effect of low temperature on particle formation is detailed in Figure S3 in the Supporting Information), the SF protein grows into a well-defined
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architecture to form spherical SFNs and capsulating MNPs together with DOX (this process is schemed in Figure 1b). In case of the amount of DOX increasing, more MNPs are essential to adsorb them in the formation process of DMSs, and thus the particle generation and drug entrapment behavior are related to the MNP concentration in the system. Meanwhile, MNPs work as heterogeneous nucleation sites in the salting out process and the additon of MNPs dramatically decreases the particle size of SFNs from 1.4 µm to 130 nm, which is benefit for cell internalization (Figure S4 in the Supporting Information). The Fourier transform infrared (FTIR) spectra of the obtained DMSs (Figure S5a in the Supporting Information) show that only minor changes in secondary structure on the condition of introducing MNPs in the salting-out process, and the fractions of β-sheet calculated from the deconvolution of the FTIR spectra (shown in Figure S5b and Table S2 in the Supporting Information) are relatively low. The capsulation of MNPs in the DMSs can be characterized by the thermogravimetic analysis (TGA, Figure S6a in the Supporting Information) and superparamagnetic behavior provided by MNPs (Figure S6b and Table S2 in the Supporting Information).
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Adv. Mater. 2014, DOI: 10.1002/adma.201403562
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tissue) and pH 7.4 (blood plasma).[28] This phenomenon is due to a weak electrostatic interaction between SF and DOX at low pH values (the pH-dependent release is schemed in Figure S10 in the Supporting Information).[29] The DOX release behavior in the presence of proteinase (α-chymotrypsin) is also studied (Figure 1f and Figure S11 in the Supporting Information). In 4 days, nearly all the DOX are released, much faster than that without proteinase, indicating the facilitation effect of the nanocarrier biodegradation on the drug release. To investigate the internalization and intracellular drug release behaviors of DMSs, human breast adenocarcinoma cell line (MCF-7 cells) and its multidrug resistant counterpart (MCF-7/ADR cells) are treated with free DOX and DMSs. The cellular DOX uptake is measured quantitatively by the mean fluorescence intensity per cell via flow cytometry (Figure 2a). The uptake of free DOX and DMSs in MCF-7 cells are both high, while in the drug-resistant MCF-7/ADR cells, the accumulation of DMSs is much higher than that of free DOX. As illustrated by previous reports, free DOX enters the MCF-7/ ADR cells through a flip-flop mechanism and is extruded by the overexpressing efflux P-glycoprotein (P-gp).[30] Thus the cellular uptake data suggest different internalization pathways between free DOX and DMSs. The internalization mechanism of DMSs is studied with specific endocytic inhibitors by flow cytometry in MCF-7/ADR cells (Figure 2b). Compared with DMSs without inhibitors, the cellular uptake is significantly inhibited by sodium azide (energy inhibitor) and chlorpromazine (clathrin-mediated endocytosis inhibitor) and little or no inhibition is found when cells are pretreated with
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T2-weighted magnetic resonance imaging (T2-MRI, Figure 1c) of DMSs reveals a concentration-dependent manner of the dark intensity with the T2 relaxation cofficient (r2) calculated to be 168.42 s−1 mM−1 (Figure S7 in the Supporting Information). The entrapment efficiency of DOX is concluded in Figure 1d. Although the percentage of entrapped drug declines as the amount of DOX in the system increases, the total amount of drug loaded in DMSs increases (data shown in Figure S8 in the Supporting Information). At a constant DOX concentration, the loading efficiency of DOX grows as more MNPs are added during the salting-out process until reaching a ceiling, which is attributed to the increase of DOX adsorbed on MNPs. Afterwards, the entrapment efficiency decreases resulting from the MNPs that are not capsulated in the DMSs. The loading content of DOX peaks at a MNP concentration of 100 µg mL−1, giving the value of 5.9 wt% (60.9 ng DOX/µg SF), which is about 84.0 wt% of the total drug in the system, and this sample is used for further study. Owing to the pre-loading of MNPs, the loading content value is approximately 50% higher than that for directly binding of DOX on the SFNs[21] (40 ng DOX/µg SF, Figure S9a in the Supporting Information), which makes it more effective to kill cancer cells (Figure S9b in the Supporting Information). The DOX release profiles under various environmental pH values are demonstrated in Figure 1e, in which a pHdependent release is observed: low environment pH accelerates the DOX release. At pH 5.0 (mimicking microenvironment in endosomes and lysosomes), the release rate is significantly higher than those at pH 6.5 (extracelluar environment in tumor
Figure 2. In vitro cell assays. (a) Quantitative cellular uptake analysis of DOX in MCF-7 cells and MCF-7/ADR cells after treated with DOX and DMSs. (b) Relative uptake efficiency of DMSs in MCF-7/ADR cells in the presence of various endocytosis inhibitors. *, P < 0.05 vs control. (c) Confocal microscopic images of MCF-7 cells and MCF-7/ADR cells incubated with DOX and DMSs for 3 h. White bar indicates 25 µm in the image. (d) Schematic diagram of proposed endocytosis pathways and intracellular drug delivery mechanisms of DMSs in P-gp overexpressed MCF-7/ADR cells. (e) Growth inhibition results for MCF-7 (䊊,䊉) and MCF-7/ADR (䊐,䊏) cells treated with free DOX (䊊,䊐) and DMSs (䊉,䊏) with different DOX dosage. The concentration of DMSs is shown on the top x axis.
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Figure 3. In vivo pharmacokinetics and magnetic tumor targeting of DMSs. (a) Blood circulation time of DOX and DMSs analyzed by measuring plasma concentration of DOX. The data are expressed as percentage of the injected dose (%ID). (b) A schematic drawing to illustrate magnetic tumor targeting. (c) Fluorescence imaging after intravenous injection of DMSs or DOX-loaded SFNs with an equivalent DOX dosage (5 mg kg−1). Top: in vivo images (bottom view) of mice bearing two MCF-7 tumors on both sides at 2 h post injection. Red and green circles point out tumors with and without magnet attachment, respectively. Bottom: ex vivo images of tumors and major organs at 12 h post injection. H: heart, Li: liver, S: spleen, Lu: lung, K: kidney, T+MF and T-MF: tumor in the presence and absence of MF, respectively. (d) The biodistribution of the DMSs and DOX-loaded SFNs after DMS administration and MF application at tumor, based on HPLC analysis. (e) Tumor growth curves of tumors after various treatments. ***, P < 0.001.
indomethacin (caveolae-dependent endocytosis inhibitor), colchicine (microtubule-dependent macropinocytosis inhibitor) and quercetin (inhibitor of caveolae- and clathrin-independent endocytosis),[31] which demonstrates that the uptake pathway of DMSs is energy-dependent endocytosis process mediated by clathrin. The subcellular location study via staining the nucleus and lysosomes with Hoechst 33342 and Lysotraker Green DND26, respectively, and observation with a confocal laser scanning microscopy (CLSM, Figure 2c) also reveals the uptake difference of free DOX and DMSs in MCF-7/ADR cells. The DMSs exhibit efficient intracellular delivery in both MCF-7 and MCF-7/ADR cells with colocalization in lysosome and partly entering into the nucleus in 3 h, which further indicates the lysosomotropic delivery potential of DMSs (the endocytosis pathway and intracellular delivery process are schemed in Figure 2d). From the results above, we can infer that DMSs can be taken into the multidrug resistant MCF-7/ADR cells by clathrinmediated endocytosis, which escapes the efflux induced by P-gp and thereby may overcome MDR in cancer chemotherapy. MTT (MTT is (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is conducted (Figure 2e and Figure S12 in the Supporting Information) to confirm their ability to overcome MDR. Significant growth inhibition of MCF-7 cells is observed when the cells are incubated with either DMSs or free DOX at a higher DOX concentration, and DMSs are significantly more cytotoxic to MCF-7/ADR cells than free DOX at an equivalent DOX dosage, which proves that DMSs display enough efficacy to kill the cancer cells and overcome MDR. Before in vivo chemotherapy application of DMSs, the toxicology of DMSs in female BALB/c nude mice is investigated to ensure their safety. The blood chemistry (Figure S13a in the Supporting Information) and hematology analysis (Table S3 in the Supporting Information) reveal that no obvious hepatic toxicity is induced and all of the hematological parameters in the
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treated groups appear to be normal compared with the control group. The histopathological examinations of the organ tissues (Figure S13b in the Supporting Information) show no pathological changes in liver, spleen, lung and kidney. It should be noted that the heart slice of DMSs group exhibits myocardial fiber rupture, which may be induced by the cardiac toxicity of DOX.[32] To understand the pharmacokinetics of DMSs, the plasma concentration of DOX is measured on the basis of DOX quantification by high performance liquid chromatography (HPLC, Figure 3a). The blood circulation time of DMSs is longer than that of free DOX and the area under the curve is also larger, indicating greater blood persistence and better bioavailability of DMSs.[33] Next, the magnetic tumor targeting performance is studied. Mice bearing two subcutaneous MCF-7 tumors on opposite flanks are intravenously injected with DMSs and DOX-loaded SFNs (DOX dosage 5 mg kg−1) via tail vein, and the tumors on the left side are attached to magnets (Figure 3b). After 2 hours, in vivo fluorescence imaging is conducted (Figure 3c) and DMSs are found to accumulate at the magnet-attached tumor, while no significant tumor enrichment of DOX-loaded SFNs is observed regardless of magnet attachment. Ex vivo imaging of major viscera (heart, liver, spleen, lung and kidneys) and tumor tissues also confirms the MF-guided tumor accumulation effect of DMSs (Figure 3c). Quantification analysis of DOX concentration in the tissues (Figure 3d) states that magnetic targeting can enrich DMSs and suppress the uptake in liver, which is also proved by the quantification of Fe elements in the tissues (measured by inductively coupled plasma atomic emission spectrometer, ICP-AES, Figure S14 in the Supporting Information). The effective tumor targeting of DMSs induced by external MF makes the tumor growth different from that without magnet attachment. The growth of tumors attached to magnets is greatly inhibited, when compared to the tumor
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COMMUNICATION Figure 4. Magnetic-guided chemotherapy of multidrug resistant cancer. (a) Schematic illustration of the therapeutic process of MCF-7/ADR tumors with the assistance of a magnet. (b) In vivo T2-MRI of mice after intravenous injection of DMSs. Red and green circles point out tumors with and without magnet attachment, respectively (the same as below). (c) In vivo fluorescent imaging of mice bearing MCF-7/ADR tumors after administration. (d) Ex vivo imaging of tumor tissues and major organs at 12 h post injection. Left: from a mouse with the application of MF at the tumor site. Right: from a mouse without MF application. (e) Distribution profiles of DOX and Fe element in tissues at 12 h after DMS administration at a DOX dosage of 5 mg kg−1. (f) In vivo effects of DMSs and magnet attachment on the growth of MCF-7/ADR tumor. **, P < 0.01; ***, P < 0.001. (g) Morbidity-free survival rate of mice bearing MCF-7/ADR tumors after treatment. (h) Body weight change of the mice.
without magnet attachment (Figure 3e). However, we can hardly distinguish the difference in tumor growth in spite of the MF application when treated with the non-MF responsive DOX-loaded SFNs. Being aware of the high magnetic targeting efficiency of DMSs and their ability to overcome MDR, we finally conduct the magnetic-guided chemotherapy of multidrug resistant cancer. Mice bearing MCF-7/ADR tumors are intravenously injected with DMSs and magnets are attached to the tumor sites (schemed in Figure 4a). Tumor tissue attached to the magnet appears as a dark area on the in vivo T2-MR image, while in the absence of MF, the tumor tissue appears bright (Figure 4b and Figure S15a in the Supporting Information), confirming the successful targeting of DMSs to the tumor region and their apparent MRI contrast effect. And as demonstrated in the fluorescence images (Figure 4c and Figure S15b in the Supporting Information), the DOX fluorescent signal in the magnet-attached tumor shows a remarkable increase over time after injection. The ex vivo fluorescence images (Figure 4d) and quantification of DOX and Fe levels in the tissues (Figure 4e) also confirm the MF-induced tumor targeting
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of DMSs. To determine the antitumor effect of magnetic-guided DMSs, both tumor sizes (Figure 4f) and morbidity-free survival rate (Figure 4g) are recorded after treatment. The results show that the tumors treated with PBS or drug-free magnetic SFNs (M-SFNs) grow rapidly, suggesting the tumor growth is not affected by the SF-based nanocarriers. The growth inhibition effect of MCF-7/ADR tumors with DMS treatment is better than that treated with free DOX, possibly related to better drug availability (as shown in Figure 3a). A single injection of DMSs plus MF attachment significantly suppresses of the growth of MCF-7/ADR tumors with 100% of survival rate on day 30, confirming their effect on regression of drug-resistant tumors. The body weight of mice, whose change is an indicator of systemic toxicity,[34] is simultaneously measured after receiving DMSs treatment (Figure 4h). Those treated with free DOX display obvious body weight fluctuation, suggesting its toxic effect on animals. The body weight of mice in DMSs- and M-SFNstreated groups do not differ greatly from that of PBS, indicating that the SF-based nanocarriers do not exhibit severe systemic toxicity. It is suggested that DMSs, which possess efficient anti-tumor activity under the guidance of MF together with
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low systemic toxicity, can act as a hopeful nanocarrier for the delivery of chemotherapeutic drugs and overcoming MDR. In conclusion, we have developed an approach to prepare DMSs by means of salting out SF in the presence of DOX and MNPs, and the regulation of DMSs generation and DOX entrapment are achieved in this approach through simply tuning the concentration of MNPs. The MF-induced tumor targeting ability in vivo and effective chemotherapy of multidrug resistant cancer demonstrate that the DMSs work well as a novel DDS in cancer therapy. The nanoparticle-assisted SFNs preparation approach introduces extra functions for SFNs, providing them with magnetic targeting ability, which greatly expands the clinical applications of SF-based materials, and is inspiring in the advancement of biomacromolecular materials.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was financially supported by National Science Foundation of China (Grant Nos. 20874015 and 51273047) and the “Shu Guang” project (12SG07) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. Received: August 5, 2014 Published online:
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