Letter www.acsami.org
H2S Activated Drug Release from Protein Cages Weijian Chen,†,‡ Yajie Zhang,†,‡ Xiao Li,‡ Hong Chen,‡,§ Jian Sun,‡ and Fude Feng*,‡ ‡
Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China § Lab of Advanced Functional Materials, School of Environmental Science, Nanjing Xiaozhuang University, Nanjing 210013, P. R. China S Supporting Information *
ABSTRACT: We took advantage of gasotransmitter H2S as a chemical reaction-based trigger for controlled release of doxorubicin which is precoordinated by copper ions and enclosed in horse spleen apoferritin. The nanocomposite is stable at physiological pH and temperature before H2S activation. The drug release process avoids disassembly of protein shells and is controllable by the strong affinity of sulfide with copper ions. The in vitro cytotoxicity assay indicates the antitumor effect of doxorubicin toward tumor cells could be achievable by H2S activation.
KEYWORDS: apoferritin, hydrogen sulfide, doxorubicin, controlled drug delivery, cell imaging
H
molecules and binding affinity toward tumor cells.18,19 A general drug loading strategy relies on pH-induced assembly and disassembly process at a cost of excess drug molecules applied.20 Alternatively, drug loading is achievable by ureainduced protein denaturation and refolding, and the resulted human H-chain apoferritin caged doxorubicin (DOX, also called adriamycin) nanoparticles indicated excellent antitumor activity.21 Efficient loading of DOX in a form of copper complex into recombinant human apoferritin was reported with loading efficiency up to 73.49 wt % by Xie’s group without pH alteration step, which emphasized essential role of metal ions in enclosing DOX by unclear mechanisms.22 Recent DOX caging approaches23,24 are outlined in Table S1, which indicates that the key factors, such as loading number per protein and particle stability, to evaluate an encapsulation method vary remarkably. The strategy these approaches undertake for DOX release is limited to passive diffusion of DOX or disassembly of protein shell in acidic condition. Currently, the method for activatable release of therapeutic drugs, except gaseous molecules, from intact apoferritin cages remains unavailable without destruct of cage integrity. We previously revealed that hydrophobic ruthenium(II) polypyridyl complexes could be enclosed into horse spleen apoferritin (apo-HSF) at 4 °C, and escaped in a first-order kinetics at 37 °C, likely due to the enlarged channels at elevated temperature.25 Without anchor sites for metal ions, the cage
ydrogen sulfide (H2S) has been validated as a thirdgeneration endogenous gasotransmitter because of its critical roles in numerous physiological and pathological processes such as Down’s syndrome and Alzheimer’s disease.1−3 More or less associated with its biological functions, H2S participates as a nucleophile, reductant, or metal chelator in chemical reactions.4−6 These properties of H2S have inspired increasing research interest in drug design, biosensing and bioimaging, etc.7,8 Recently, Yan’s and Lin’s groups separately reported drug loaded polymeric vesicles bearing azide moieties that are H2S-reducible to result in H2S-responsive vesicle disassembly or charge reversal event.9−11 As compared to wellestablished triggers like GSH, enzymes, pH and light, the use of H2S as an environmental stimuli for controlled drug release from delivery vehicles is still in its infancy.12,13 H2S is membrane permeable, widely found in various tissues, and reactive toward heavy metals with high specificity and efficiency, which allow for potential application in liberating drugs from metal bonding. Proteins are natural drug delivery platform featuring good degradability and high biocompatibility.14 Degraded proteins are bioavailable without byproducts. Remarkably, serum albumin carrying anticancer drug paclitaxel has been approved by FDA for treating metastatic breast cancers,15 and more clinical trials are under way.16,17 One of comparable multifunctional proteins is nanocage-structured ferritin assembled by 24 subunits. Deionized ferritin, termed apoferritin, has found applications in nanomedicine such as nanoreactors, mimic enzymes, contrast imaging materials, and drug carriers because of its spacious cavity available for hosting wide ranges of guest © XXXX American Chemical Society
Received: August 20, 2017 Accepted: September 15, 2017 Published: September 15, 2017 A
DOI: 10.1021/acsami.7b12524 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
is replaced by H2S which penetrates freely through the protein shell and binds copper ion firmly. The loss of coordination interaction with copper ion leads to depletion of DOX from attached sites in the inner surface and subsequent DOX diffusion through shell channels into outer solution. The size of free DOX molecule is approximately half that of Cu2+/DOX preformed in 2:1 stoichiometry of DOX to copper ion,26 in favor of DOX escape upon H2S activation. We employed H2S fast releasing agent Na2S in buffer as H2S substituent, which is more acceptable than direct use of gaseous H2S.27,28 The method could overcome current circumstances toward controlled uncaging of antitumor drugs. CuDOX NP was carefully purified to remove nonspecifically absorbed drug molecules. According to the Cu2+/DOX absorption intensity at 510 nm and protein concentration examined by BCA assay, DOX was loaded up to ∼128 moieties per protein with an efficiency of 49.1%. The copper was coloaded with an efficiency up to 94.4% on a basis of ICP-OES analysis. The size and morphology of CuDOX NP were characterized by high-resolution transmission electron microscopy (HR-TEM). As shown in Figure 1a, uranyl acetate-stained CuDOX NP were visualized in uniformed core−shell shapes, which confirm intact protein cage structures. The accumulation of CuDOX in the inner cavity was revealed by nonstained HRTEM imaging (Figure S2) which indicates CuDOX clusters as punctas in the size comparable to protein cavity. The hydrodynamic diameter of CuDOX NPs was estimated by dynamic light scattering (DLS) technique as 15.8 ± 1.9 nm, slightly larger than that of apo-HSF (13.5 ± 1.6 nm) (Figure 1b). There were negligible changes of particle size after a 2week storage in PBS at 4 °C. Meanwhile, the zeta potential of protein cages was nearly constant before and after the incorporation of Cu2+/DOX (−24.1 mV versus −23.5 mV). The composition of Cu2+/DOX complex in PBS solution and in the protein cages was evidenced by the absorption spectra. It is known that Cu2+ coordinates DOX in 1:2 or 1:1 ratio on the carbonyl and phenolate oxygen of DOX,26,29 and
interior has limited binding affinity with the enclosed compounds. The stability of composites depends on the metals used. Leakage is blocked if the compound of interest is coordinated with copper ion which is attached to the inner cage of apo-HSF. This assumption raises a possibility to establish a copper ion-dependent platform that is H2S responsive by abolishing the above coordination interaction. Herein, we employed deironized commercial horse spleen ferritin as a container for copper-complexed doxorubicin. However, different from reported recombinant human apoferritin which encapsulated Cu2+/DOX at a constant neutral pH,22 Apo-HSF formed aggregates with Cu2+/DOX (Figure S1). Alternatively, as elucidated in Scheme 1, Cu2+/DOX is Scheme 1. Schematic Representation of (a) Reaction between H2S and CuDOX, Which Is Shown in a Front-View Geometry, and (b) Method for Drug Incorporation into Apo-HSF Cavity and H2S Activated Drug Releasea
a
The grey, blue, orange, white, and red spheres in DOX and CuDOX structures represent C, N, Cu, H, and O atoms, respectively.
enclosed by the widely accepted pH-induced unfolding and refolding process. The resulted water-soluble nanocomposite, termed CuDOX NP, is stable at physiological pH, unless DOX
Figure 1. (a) TEM image of CuDOX NP. (b) DLS analysis of apo-HSF and CuDOX NP. (c) UV−vis absorption spectra of free DOX, CuDOX, apo-HSF, and CuDOX NP in PBS buffer (pH 7.4) at DOX concentration of 33 μg/mL. (d) Changes in UV−vis absorption spectra of CuDOX NP in PBS buffer (pH 7.4) before and after a 4-week storage at 4 °C. B
DOI: 10.1021/acsami.7b12524 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 2. (a) Plot of fluorescence intensity at 558 nm of CuDOX (5.1 μM) in PBS solution (pH 7.4) as a function of the Na2S/Cu2+ molar ratio (λEx = 478 nm). The cumulative time-dependent DOX release from CuDOX NP solution with or without Na2S at (b) pH 7.4 and (c) pH 6.4 at 37 °C following the same procedure. Inset: photographs of CuDOX NP solution after Na2S-treatment with increasing Na2S/Cu2+ ratios from left to right.
In contrast, DOX release was accelerated by the presence of Na2S which was initially added in the outside solution, and approached a plateau at 6 h post reaction (Figure 2b). The release rate was boosted as the initial ratio Na2S/Cu2+ increased from 0.2:1 to 4:1. The total release percentage reached 35.7% and 46.4% for Na 2 S/Cu 2+ 1:1 and 4:1, respectively, accompanied by a color change of reaction mixture from purple to dark orange. Even for Na2S/Cu2+ as low as 0.2:1, DOX release was achieved at 5.3%, which was twice that of control. This result demonstrates the sensitive response of the delivery system activated by H2S. Similar results were obtained with sulfide-activated DOX releasing at weakly acidic condition (pH 6.4, Figure 2c). It seems that DOX retardation took place. As a result of Na2S treatment on CuDOX NP, in the circular dichroism (CD) spectra (Figure S8), the positive peak at 193 nm remained constant to show that the β sheet structures were not affected. However, the absorption at 207 nm was slightly attenuated, accompanied by the red-shifted band from 222 to 226 nm. The changes in CD spectra reveals conversion of α helix to other structures happened, which may impact transportation of DOX in the channels composed of α helices. To investigate intracellular drug release that is controllable by chemical reaction, we employed fluorescent microscopy to image intracellular DOX that emits red fluorescence from copper-free form. Relatively high DOX concentrations (0.5, 1, 1.5, and 2 μM) were applied to acquire bright fluorescent images (Figure 3a). DOX or CuDOX NP was incubated in serum-free medium with HeLa cells for 2 h before imaging. As control for DOX treatment, cell membrane permeable DOX was accumulated in cells, especially in the nucleus. Red fluorescence was seen in the cytosol when CuDOX NP was loaded at a concentration of 1 μM or higher, indicating efficient cellular uptake of protein particles likely via receptor-mediated endocytosis.32 However, low DOX concentration (0.5 μM) for CuDOX NP led to dim red fluorescence in cells. For H2S group, HeLa cells were pretreated with slow H2S releasing agents AP39 and ADT−OH (0.2 μM, structurally shown in Figure S9), respectively, before loading CuDOX NP. ADT− OH/CuDOX NP cotreatment afforded similar results to CuDOX NP with DOX visible in the cytosol at high DOX concentrations (1−2 μM) and weakly detectable at 0.5 μM DOX. Interestingly, AP39/CuDOX NP treated cells were visualized with enhanced bright red fluorescence in nuclei even at 0.5 μM DOX, suggesting accumulation of uncaged DOX in nuclei (see Figure S10 for high-resolution images). This result agrees with the previous report that apoferritin, H-apoferritin in
which form is dominant highly relies on the pH applied. However, formation of stable complex is rather slow,30 and there is debate with respect to the composition of Cu2+/DOX complex in neutral condition.26,31 In general, the 1:1 Cu2+/ DOX complex has red-shifted absorption as compared to the 1:2 counterpart, due to the increased number of deprotonation on phenolate oxygen. As shown in Figure 1c, free DOX had maximum absorption at 478 nm in PBS (pH 7.4), which was red-shifted to 510 nm when DOX was bound by copper ions primarily in 1:1 ratio. More profound redshift occurred with incorporated Cu2+/DOX. The CuDOX NP solution was stable without detectable spectral changes over a period of 4-week storage at 4 °C and pH 7.4, implying reverse conversion of incorporated Cu2+/DOX to another form did not happen (Figure 1d). DOX was emissive by excitation at 478 nm in PBS, and entirely quenched when bound to Cu2+ (Figure S3). Recovery of DOX emission was expected if H2S was added as Cu2+ scavenger into CuDOX solution. The fluorescence intensity was monitored at 558 nm during titration of Na2S. As shown in Figure 2a, DOX emission enhanced gradually and reached a plateau when Na2S concentration raised to approximately the same level of Cu2+, in good agreement with the requirement for immobilizing all of copper in a form of CuS. At the same time, the color of reaction mixture was restored from purple to orange (Figure S4), suggesting that the presence of Na2S can promptly induce dissociation of DOX from Cu2+. Investigation of DOX leakage from apo-HSF was carried out by incubation of CuDOX in a dialysis bag against PBS buffer at 37 °C and pH 7.4. According to the linear work curve (Figure S5), DOX release percentage was achieved as a ratio of the estimated DOX amount in the outside solution to the total amount of DOX. The detected absorption spectrum of the outside solution with a maximum at 478 nm showed characteristic band shape (Figure S6) of free DOX (Figure 1c), which suggests that copper ions were retained in the protein inside the dialysis bag and allows sensitive fluorescencebased quantification of released DOX without interference from copper ions. Spontaneous DOX leakage was detected as only 2.6% over 10 h (Figure 2b) and 4.1% over 24 h (Figure S7), respectively. This finding confirms excellent stability of CuDOX NP in neutral condition at 37 °C, unlike the previously reported system which indicated rapid spontaneous DOX leakage (Table S1).22 Diverse factors, such as ferritin source, DOX loading method, purification procedure and location of DOX, may contribute to the remarkable difference. C
DOI: 10.1021/acsami.7b12524 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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In summary, we provided a proof-of-concept approach employing H2S as a trigger to control drug release from protein cages without protein disassembly process. H2Sactivated DOX release was achieved by disconnecting the CuDOX coordination interaction at physiological pH condition, and the release rate was dependent on the H2S/Cu2+ molar ratio. In vitro cell imaging studies showed the stable CuDOX NP could be internalized by tumor cells and induced by AP39 to liberate DOX in nuclei. The CuDOX NP was found to benefit minimizing DOX premature release, attenuating DOX cytotoxicity and restoring the antitumor effect upon H2S activation. The combination of H2S controlled release with antitumor drug has potential application in disease therapeutics. The cytoprotective effects by H2S against cardiomyopathy38 potentially attenuate DOX induced dose-dependent cardiotoxicity which often limits its clinical use.39,40 Thus, the mutual role of H2S in therapeutics and dose-dependent activation of caged drugs would motivate more approaches in combination therapy in future.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b12524. Experimental details and figures (PDF)
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Figure 3. (a) Fluorescent microscope images for cellular uptake and distribution of Dox species in HeLa cells after 2 h-treatment by DOX species at DOX concentration of 0.5−2 μM. λEx = 527.5−552.5 nm, λEm = 605−655 nm. (b) Cell viabilities of HeLa cells after 24 h of treatment with various DOX species.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Fude Feng: 0000-0002-5348-5959 Author Contributions
particular, could activate nuclear translocation rather than release DOX by rapid degradation in the lysosome.33 It is known that AP39 is highly productive in donating H2S and contributes to elevated level of intracellular H2S as compared to ADT−OH,34 well consistent with the in vitro DOX imaging data. H2S releasing capability of AP39 is considered to rely on the intracellular esterase-mediated metabolic pathway,35,36 which supports that DOX release was correlated to the increase of intracellular H2S level. The in vitro antitumor effect was studied by MTT assay on HeLa cells that were treated with different DOX species at the same DOX loading (0.001, 0.01, 0.1, and 1.0 μM) in DMEM medium containing 10% FBS for 24 h. As shown in Figure 3b, free DOX exhibited high cytotoxicity as its concentration was larger than 0.01 μM. However, no antitumor effect was detected for CuDOX NP group at low DOX concentration (0.001−0.1 μM), and cotreatment with ADT−OH did not affect the behavior of CuDOX NP. The cell viabilities were maintained at ∼60% even when maximum DOX concentration (1 μM) was applied. Interestingly, in comparison to CuDOX NP (or ADT−OH/CuDOX NP), it seems AP39/CuDOX NP cotreatment could induce lowered cell viability, particularly at high DOX concentration (0.1−1 μM) with statistical significance (P < 0.05; Student’s t test, shown in Figure S11), correlated to the improved level of H2S generation. As compared with free DOX, the reduced toxicity of AP39/ CuDOX NP cotreatment could be primarily due to the following factors: prevention of DOX release by protein shell, cytoprotection by H2S,37 and limited availability of H2S to CuDOX NP.
†
W.C. and Y.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We’re grateful to Prof. Wei Wang (Nanjing University) for help with DLS measurements. We thank the National Basic Research Program of China (2015CB856300), the National Natural Science Foundation of China (21474046), 1000 Young Talent Program, Interdisciplinary Training for Graduate Students of Nanjing University (2016CL11), Collaborative Innovation Center of Chemistry for Life Sciences, and the Program for Changjiang Scholars and Innovative Research Team in University for financial support.
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