Hexahistidine-metal assemblies: A promising drug delivery system

Acta Biomaterialia 90 (2019) 441–452

Contents lists available at ScienceDirect

Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

Full length article

Hexahistidine-metal assemblies: A promising drug delivery system Wenjuan Huang a, Pengyan Hao a, Jianghui Qin a, Shan Luo a, Tinghong Zhang b,c, Bo Peng b,c, Hao Chen a,⇑, Xingjie Zan a,b,c,⇑ a

School of Ophthalmology and Optometry, Eye Hospital, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang Province 325035, PR China Wenzhou Institute of Biomaterials and Engineering, CNITECH, CAS, Wenzhou, Zhejiang Province 325001, PR China c Engineering Research Center of Clinical Functional Materials and Diagnosis & Treatment Devices of Zhejiang Province, Wenzhou Institute of Biomaterials and Engineering, CAS, Wenzhou, Zhejiang Province 325001, PR China b

a r t i c l e

i n f o

Article history: Received 28 December 2018 Received in revised form 23 March 2019 Accepted 29 March 2019 Available online 3 April 2019 Keywords: Hexahistidine Coordination polymer Nanomedicine Responsive release Endocytosis

a b s t r a c t It is of considerable interest to construct an ideal drug delivery system (i.e., high drug payload, minimal cytotoxicity, rapid endocytosis, and lysosomal escape) under mild conditions for disease treatment, tissue engineering, bioimaging, etc. Inspired by the coordinative interactions between histidine and metal ions, we present the facile synthesis of hexahistidine (His6)-metal assembly (HmA) particles under mild conditions for the first time. The HmA particles presented a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release. In addition, HmA encapsulation largely improved the antitumor ability of camptothecin (CPT) relative to free CPT. By capitalizing on these promising features in drug delivery, HmA will have great potential in various biomedical fields. Statement of Significance It is of considerable interest to construct an ideal drug delivery system (i.e., high drug payload, minimal cytotoxicity, rapid endocytosis, and lysosomal escape) under mild conditions. Inspired by the coordinative interactions between histidine and metal ions, we present for the first time the facile synthesis of Hexahistidine (His6)-metal assembly (HmA) particles under mild conditions. The HmA particles exhibited a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release. By capitalizing on these promising features in drug delivery, HmA will have great potential in various biomedical fields. Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Nanomedicine is revolutionizing traditional drug administration because such therapeutics can change drug biodistribution and pharmacokinetics, permeate the cell membrane, deliver drugs to specific cell populations, and release drugs at a designed site in a controllable manner [1]. Inspired by these advantages, various drug delivery systems (DDSs) such as liposomes, capsules, dendrimers, inorganic nanoparticles, and micelles have been developed in recent decades, and methods have shed light on the treatment of several refractory diseases [2]. Although a variety of ⇑ Corresponding authors at: School of Ophthalmology and Optometry, Eye Hospital, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang Province 325035, PR China (X. Zan). E-mail addresses: [email protected] (H. Chen), [email protected] (X. Zan). https://doi.org/10.1016/j.actbio.2019.03.058 1742-7061/Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

strategies have been envisioned, exploring new DDSs remains highly desired to drive their further applications. In recent years, assemblies constructed by coordinative interactions between metal ions and organic ligands, such as metalorganic frameworks (MOFs) and metal coordination polymers (MCPs) [3,4], have drawn increasing attention in the field of drug delivery [5] because of their high payload, multiresponsive properties and fast lysosomal escape [6–8]. Drugs can be encapsulated in these assemblies through postloading or in situ encapsulation. It has been demonstrated that the in situ method has a higher loading capacity and is time-saving than the postloading method [9]. These assemblies have achieved great success in loading various kinds of drugs, especially small-molecule drugs. Compared to small-molecule drugs, bioactive molecules (protein, peptide, and DNA) exhibit high biological activity and specific function, along with various applications in biomedicine, tissue repair, and life science [10–12]. Because of the high sensitivity of these molecules

442

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

to environmental changes, a mild encapsulation process is required to store their bioactivity. For most current metal-organic assemblies, issues such as harsh conditions during the assembly process, limited pore size, and cytotoxicity of organic ligands greatly restricted their further application to loading various drugs. Thus far, only a few systems have been successfully demonstrated to load various kinds of drugs [9,13–15]. Bioassemblies, inspired by interactions between biomolecules (i.e., a -helix, b -sheet, and base pairs in DNA), have been applied for decades to construct drug delivery vehicles [16–18]. Such bioassemblies can be obtained under mild conditions and can avoid cytotoxicity, but these structures still suffer from low loading capacity, inefficient endosomal escape, and limited loadable materials. Taking advantage of those bioassemblies, metal-organic coordinative assemblies were developed as drug delivery vehicles, which have drawn increasing attention because of their high loading capacity, multiresponsive properties and tunable structures [6–8]. Polyhistidine tags have high affinities for many metal ions and are often used for the purification of recombinant proteins by immobilized metal ion chromatography [19]. Zinc, the second most abundant trace metal in human bodies after iron, is the only metal that appears in all enzyme classes [20]. Interactions between zinc ions and proteins have been studied for decades. In recent years, hierarchical assemblies of zinc and poly-his6-boligostyrene were generated [21], a multicomponent coordination self-assembly strategy was used to generate smart nanodrugs showing tumor-specific delivery and therapeutic effects [22], and protein particles assembled from histidine-rich protein and divalent metal ions were explored [23]. Here, we present a facile synthesis of His6-metal assemblies (HmAs) under mild conditions; these assemblies feature a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release, showing their potential in numerous potential applications in various biomedical fields. Considering that His6 can coordinate with many metal ions and can be integrated into peptides or proteins by recombinant or conjugation techniques [24], this strategy will facilitate the construction of protein/peptide-metal assemblies as a DDS.

2. Experimental procedures 2.1. General materials 99%), Zinc nitrate hexahydrate (Zn(NO3)2
6H2O, polyvinylpyrrolidone (PVP, Mw 58 k), 4-(2-hydroxyethyl)-1-pip erazineethanesulfonic acid (HEPES), bis-(2-hydroxyethyl)amino-tr is(hydroxymethyl)methane (Bis-tris), sodium hydroxide (NaOH), hydrochloric acid (HCl), fluorescein sodium (Fl.), rhodamine B (Rho), calcein, dexamethasone (DXM), and camptothecin (CPT) were purchased from Aladdin (China). All peptides with purity  99%, His3, His6, His9, His12, FITC-His3, FITC-His6, FITCHis9, FITC-His12, C-terminal end-capped His6 (AcHis6), N-terminal end-capped His6 (His6NH2), both-terminal end-capped His6 (AcHis6NH2), glycine6, glutamine6, and lysine6 were purchased from KareBay Biochem, Inc. and used without further purification. HeLa cell lines and dendritic cell lines were provided by ATCC. All materials for cell culture were purchased from GIBCO. A ProLongÒ Gold anti-fade kit, LysoTracker Red DND-99, and DAPI were purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo. Anhydrous dimethyl sulfoxide (DMSO), lysozyme (Lys), bovine serum albumin (BSA), FITC-dextran-4 k, FITC-dextran-40 k, FITC-dextran-500 k, FITC-dextran-2000 k, fluorescein isothiocyanate (FITC), genistein,

chlorpromazine, amiloride, nocodazole, and Triton were purchased from Sigma-Aldrich. Dialysis tubes were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. 2.2. Characterization X-ray diffraction patterns were acquired on a Scintag XDS-2000 diffractometer. The size and zeta potential of the HmA and HmA@Drug particles were measured with a Zetasizer Nano ZS instrument (Malvern, UK). The morphology of HmA nanoparticles was detected by a SEM unit (FESEM, SU8010 HITACHI, Japan) and a TEM unit (FEI Talos F200S microscope, USA). Fourier transform infrared spectroscopy (FTIR) spectra and Raman spectra of His6 and HmA were acquired with an FTIR spectrometer (Tensor II Bruker, Germany) and a Raman spectrometer (inVia Qontor; Renishaw, UK), respectively. Fluorescence spectra and UV–Vis spectra of free Fl. and HmA@Fl. were detected with a fluorescence spectrometer (FLUOROMAX-4 Horiba, France) and a UV–Vis spectrometer (Lambda 25 Perkin Elmer, USA), respectively. A microplate reader (Varioskan LUX Multimode; Thermo, USA) was used to quantitatively analyze the absorbance of CCK-8-containing medium and the fluorescence intensities of F-HmA, Rho, and calcein-containing medium. Cell imaging was performed b yconfocal laser scanning microscopy (CLSM) (A1 Nikon, Japan) or fluorescence microscopy (DMi8 Leica, Germany). The cellular uptake efficiency of F-HmA was detected by flow cytometry (CytoFLEX Beckman Coulter, USA). 2.3. Preparation of FITC-labeled proteins The proteins labeled with FITC were employed as previously reported [25] Typically, 1 mL of DMSO with 11 mg of FITC was added dropwise to the protein solution (100 mg in 20 mL of buffer solution with sodium bicarbonate at pH = 9.0) under stirring. After overnight reaction, the solution was dialyzed against sodium bicarbonate buffer for 3 days with dialysis tubing (molecular weight cut-off (MWCO) of 20 kDa). The whole procedure was performed in a 4 °C dark room. The dialyzed FITC-protein solution was freeze-dried to obtain the protein powder, which was stored at 80 °C before use. 2.4. Synthesis of HmA particles Under sonication (ultrasonic power of 300 W and frequency of 80 KHz), 100 lL of Zn(NO3)2
6H2O (30 mg mL1) was added dropwise to a 2 mL solution (8.4 mg of His6 and 10 mg of PVP) buffered with 50 mM HEPES at a pH of 8.0. Subsequently, the color of the solution changed to light blue and was further sonicated for another 30 min to complete the reaction. The HmA particles were collected by centrifugation (12000 rpm, 10 min), washed three times with deionized H2O, stored at 4 °C, and sonicated 1 min before characterization and the cellular test. To identify the study factors (pH, ratio of zinc ions to His6, chain length of polyhistidine, and C- and/or N-terminal capping) after generating HmA, a similar procedure was performed. Only the investigated factor was varied without changing the other conditions. The pH values were exactly controlled using NaOH (0.1 M) and HCl (0.1 M). FITC-labeled polyhistidine (F-Hisn) was used to test the yield percentage of HmA from polyhistidine, according to the following equation:

Yield ð%Þ ¼

VolO  AO

 VolS  AS VolO  AO at 489 nm

at 489 nm

at 489 nm

 100%

where AOat489nm and ASat489nm represent the UV–Vis absorbance of FITC at 489 nm in the original and supernatant solutions,

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

443

respectively, and VolO and VolS represent the volume of the original and supernatant solutions, respectively.

1 mL at each tested point) is the volume of discarded medium; and W total is the weight of the total drug in HmA particles.

2.5. Drug encapsulation

2.7. Cytotoxicity assays

To encapsulate various types of drugs into HmA particles, only slight modifications were needed during the HmA synthesis procedure. Hydrophilic model drugs (Fl., Rho, FITC-dextran-4 k, FITC-dextran-40 k, FITC-dextran-500 k, FITC-dextran-2000 k, FITC-lysozyme, and FITC-BSA) were codissolved with the His6 solution at the desired concentration. For encapsulation of the hydrophobic model drug (DXM and CPT), methanol was used as the reaction medium instead of H2O without changing the above standard procedure. In brief, 2 mL drug-containing solutions (8.4 mg of His6 and 10 mg of PVP) buffered with 50 mM HEPES at a pH of 8.0 were prepared. Subsequently, 100 lL of Zn(NO3)2
6H2O (30 mg mL1) was added dropwise to the drug-containing solutions under sonication. After the generation of HmA@Drug particles, the particle collection and washing procedures were the same as those used for the synthesis of HmA. The encapsulation concentrations mentioned below were the concentrations of drug-containing reaction solutions (volume of 2 mL). The EE % and LC wt% of drugs were calculated according to the following equations:

HeLa cells were seeded in 96-well plates at a density of 1  104 cells/well. After 24 h of incubation at 37 °C in a 5% CO2 atmosphere, the culture medium was removed and the cells were washed with PBS buffer. Then, HmA suspended in DMEM at various concentrations (20 mg mL1, 40 mg mL1, 60 mg mL1, 80 mg mL1, and 100 mg mL1) was added. Cells that were not exposed to treatment were used as the control. The cells were further cultured for 24 h, 48 h, or 72 h before testing. A standard CCK8 assay was used to determine the cell viability, and five repeats were conducted for each point. In brief, the test cells in plates were washed three times with PBS to remove the treatments, the media were replaced with 100 lL of fresh DMEM containing 10 lL of CCK-8 solution, and the plates were incubated for 2 h at 37 °C in 5% CO2 atmosphere. The plates were read at 490 nm using a microplate reader. HmA at a concentration of 50 mg mL1 was used for the following cellular tests.

ðbÞ EEð%Þ ¼

VolO  AO

ðcÞ LC ðwt%Þ ¼

 VolO  AS VolO  AOatQ nm

at Q nm

at Q nm

 100%

W added drug  EEð%Þ  100% W added polyhistidine  Yieldð%Þ

where AO at Q nm and AS at Q nm represent the UV–Vis absorbance for drugs at the quantitative position of original and supernatant solutions, respectively; VolO and VolS are the volume of the original and supernatant solutions, respectively; and W added drug and W added polyhistidine are the weight of the added drugs and added polyhistidine, respectively. The absorbance peaks at 490 nm for Fl.; 553 nm for Rho B; 496 nm for FITC-dextran-4 k, FITC-dextran40 k, FITC-dextran-500 k, and FITC-dextran-2000 k; 499 nm for FITC-lysozyme; 492 nm for FITC-BSA; and 243 nm for DXM were used for quantitative analysis by UV–Vis spectroscopy. Three repeats were conducted for each sample. 2.6. In vitro release The in vitro release process was tested according to a method previously reported by other research groups [26,27]. In brief, 5 mL of HmA@Drug (containing 5 mg of Fl. or FITC-dextran-40 k) was added to a 10 mL dialysis tube (MWCO of 3.5 kDa for Fl. and 300 kDa for FITC-dextran-40 k). Drug release was carried out by incubating dialysis tubing containing HmA@Drug in 45 mL of various mother media (Bis-Tris buffer solution at pH = 4.5, 5.8, 6.5, and 7.2 and 0.9% NaCl at pH = 7.2) at room temperature under 200 rpm stirring in a dark room. Finally, 1 mL of the test solution was withdrawn at regular time intervals, followed by the addition of the same volume of fresh medium, and quantitative analysis by UV–Vis spectroscopy was performed. The medium was discarded after testing. The accumulative release (%) at each time point was obtained from the following equation:

ðdÞ Accumulative release ð%Þ ¼

P C t V t þ i C i  V i 100 % W total

where C t and C i are the concentration of the drug in the mother medium at testing time point (t) and the concentration of the drug in the discarded medium at testing time points (i) before t, respectively; V t is the volume of mother medium at time t; V i (actually

2.8. Flow cytometry assays HeLa cells and DC cells were seeded in 24-well plates at a density of 1  105 cells/well. After incubation for 24 h, F-HmA (50 mg mL1)-containing DMEM was added to the cells and incubated for different time intervals. The cellular uptake efficiency was quantitatively measured by flow cytometry. Additionally, FHmA@Rho (50 mg mL–1F-HmA, 10 mg mL1 Rho) was tested on the HeLa cells under the same conditions by flow cytometry. Five repeats were conducted for each sample. 2.9. Lysosome formation analysis HeLa cells were seeded on 24-well-sized glass at a density of 4  104 cells/well. After 24 h of incubation, F-HmA (50 mg mL1)containing DMEM was added and incubated for another 2 h, 4 h, 8 h, or 24 h. At different time points, the acidic compartments of the cells were stained with LysoTracker Red DND-99. The nucleus was stained with DAPI for CLSM. To determine the effect of F-HmA concentrations (0, 0.1, 0.5, and 1 mg mL1) on lysosome formation, only the acidic compartments of the cell were stained with LysoTracker Red DND-99 at 2 h of incubation and were observed by fluorescence microscopy. Three repeats were conducted for each sample. 2.10. Treatment with endocytosis inhibitors Cells were seeded on 24-well-sized glass at a density of 4  104 cells/well. After 24 h incubation, the cells were pretreated with endocytosis inhibitors (chlorpromazine (5 mg mL1), genistein (100 mg mL1), nocodazole (5 mg mL1), and amiloride 1 (25 mg mL )) for 2 h. Subsequently, F-HmA (50 mg mL1)containing DMEM was added to the pretreated cells and incubated for another 2 h. Then, the cells were observed by CLSM. For quantitative analysis, triton (10 mg ml1) was added and shaken for 30 min until the cells were permeabilized. The fluorescent intensity was tested using a microplate reader. The excitation/emission wavelengths were 500/550 nm for F-HmA. Five replicates were conducted for each sample. 2.11. Intracellular fate of HmA and the drugs Rho and calcein were chosen as the model drugs. HeLa cells were seeded on 24-well-sized glass at a density of 4  104 cells/

444

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

well. After 24 h of incubation, F-HmA@Rho (Rho, 10 mg mL1)- or HmA@Calcein (calcein, 10 mg mL1)-containing DMEM was added to the cells and incubated for 2 h. Then, the culture media were removed, the cells were washed with PBS to remove the treatments, and the time was reset to 0 h. The media were replaced with fresh DMEM and incubated for the desired time (4 h, 8 h, or 24 h). At each time point, the media were removed and injected into a 96-well plate for quantitative analysis using a microplate reader, and the cells were observed by CLSM after staining the nucleus with DAPI. The excitation/emission wavelengths were 425/475 nm for DAPI (nucleus), 500/550 nm for F-HmA and calcein, and 570/620 nm for Rho. Five repeats were conducted for each sample. 2.12. Cytotoxicity of HmA@CPT HeLa cells were seeded in 96-well plates at a density of 1  104 cells/well. After 24 h of incubation, HmA@CPT-containing DMEM (1 lM, 5 lM, and 9 lM) was added to the cells. After another 24 h of incubation, the cytotoxicity of HmA@CPT was tested by the standard CCK-8 assay. Five repeats were conducted for each sample. 2.13. Statistical analysis Student’s t-test was used to assess the significance of the difference in mean values between two groups. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001; ‘‘NS” stands for not significant. 3. Results and discussion 3.1. Mechanism of and factors in generating HmA In a typical experiment, HmA particles were generated by the dropwise addition of zinc ions into a His6 solution under sonication

at pH 8. The His6 solution was colorless before the addition (Fig. S1a) and changed to light blue (Fig. S1b) after the addition and sonication, indicating the generation of HmA particles. Dynamic lighting scattering (DLS) (Fig. 1a) analysis further confirmed this result and suggested an average size of 60 nm and a small polymer dispersity index (PDI) of 0.19. The zeta potential of the HmA nanoparticles was approximately +10 mV (Fig. S1c). The morphologies of the HmA nanoparticles were observed by SEM (Fig. 1b) and TEM (Fig. 1c). As indicated in Fig. 1b and c, the HmA nanoparticles showed a narrow size distribution, which was consistent with the DLS results, but a smaller particle size of 25 nm was observed by SEM and TEM, suggesting that the larger size of the HmA nanoparticles observed in the DLS test is due to hydration. When following the same procedure but without adding His6, no color change or precipitation was observed, indicating that the presence of His6 was critical to particle generation. Considering the universal coordination ability of zinc [5], we hypothesized that the generation of HmA particles is induced by coordination between the imidazole groups in the side chains of His6 and the zinc ions, as illustrated in Fig. 1c. To confirm our hypothesis, a series of experiments were designed. End-capped His6 peptides were used to avoid coordinative or complex interactions between carboxyl and amino groups and zinc ions. Similar phenomena were observed when using the C-terminal ((His)6-NH2), N-terminal ((Ac-(His)6) or both-terminal end-capped (Ac-(His)6-NH2) derivatives (Fig. S2a, S2b) instead of -(His)6. Use of other poly(amino acid)6 derivatives instead of histidine, such as glycine, glutamine, and lysine, led to no color change or precipitation. All of these control experiments suggest that HmA is generated through the coordinative interactions between the side chain imidazole groups of His6 and zinc ions. According to previous reports [13,28], at pH values lower than the pKa of the imidazole groups in the side chains, the imidazole groups are protonated and lose their ability to coordinate. To further prove the coordinative effect, the pH of

Fig. 1. Size distribution (a), SEM image (b), and TEM image (c) of HmA nanoparticles. Schematic of the coordination process of His6 and zinc ions at pH = 8 to generate the HmA particles (d). The scale bars are 200 nm.

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

the reaction mixture was adjusted to be lower than 6.0. As expected, in all of the above experiments, no particles or aggregates could be produced. To further confirm the coordinative interactions and explore the mechanisms underlying the generation of HmA, we conducted FTIR and Raman spectroscopy. FTIR and Raman spectroscopy are two common complementary techniques for investigating the secondary structure of proteins or peptides, which could undergo changes during coordination. As shown in Fig. 2a, the spectra of His6 and HmA displayed substantial differences in several major regions, namely, the NAH stretching vibrations in the amide I (1750–1600 cm1) and amide II (1600–1500 cm1) regions. Specifically, in the amide I region, the bands at 1675 cm1 and 1655 cm1 can be attributed to b sheet and random coil conformations, respectively, and the bands at 1628 cm1 and 1602 cm1 are related to the b-turn conformation and the degree of crystallinity, respectively [17,29]. The appearance of a shoulder peak at 1655 cm1 suggested the presence of random coil configurations in both His6 and HmA. The shift of the band at 1628 cm1 in His6 to lower wavenumbers (1602 cm1) in HmA suggested that the degree of crystallinity of HmA was higher than that of His6 or that HmA had an increased proportion of b-turns, which most likely originated from the shorter hydrogen bonds between b sheets [30]. These findings indicated that zinc ions induced an ordered structure of the His6 moieties in HmA, a finding that was consistent with those given in other reports [24]. In the NAH stretching vibration region, the broad peak centered at 3039 cm1 for His6 was attributed to the hydrogen bonding between the NAH groups. This band shifted to a higher wavenumber (centered at approximately 3351 cm1) in the spectrum of HmA, indicating that the hydrogen bonding between the N–H groups was strengthened by the added zinc ions [30]. In the amide II region, the broad peak centered at 1539 cm1 for His6 was attrib-

445

uted to the overlapping bands from in-plane NAH bending and C@N bending vibrations (1500 cm1) and imidazole ring stretching (1523 cm1) [29,31]. After adding zinc ions, the peak became narrower and shifted to lower wavenumbers (centered at 1517 cm1) in the spectrum of HmA, indicating the existence of interactions between the imidazole rings and the zinc ions. Compared with FTIR spectroscopy, which mainly provides functional group and structural information, Raman spectroscopy is a more useful tool for studying the protonation, coordination, and tautomers of histidine, especially the interactions between metal ions and histidine [32] The most sensitive region in the Raman spectra (1750–1350 cm1) of His6 and HmA is displayed in Fig. 2b, exhibiting distinct intensities and wavenumbers in the amide I and amide II region. The broad peak centered at 1683 cm1 in the amide I region can be attributed to the presence of b sheet structures. Compared with the spectrum of His6, the spectrum of HmA exhibited a slightly narrower peak centered at 1683 cm1, indicating a higher proportion of b-sheet structures in HmA than in His6, a result that was consistent with the FTIR results. The peaks related to vibration modes were labeled, as illustrated in Fig. 2c. The characteristic peaks of protonated (① in Fig. 2c) and deprotonated (② Ns-H and ③ Np-H in Fig. 2c) imidazole rings can be found in the His6 spectrum [33,34], indicating that these two states are both present at Ph 6. The characteristic peaks of the metal-imidazole binding, including zinc ions bridging imidazoles (④ in Fig. 2c) and metalimidazole tautomers (⑤ Ns-Zn and ⑥ Np-Zn in Fig. 2c), were found in the spectrum of HmA [21,35], confirming the coordinative interactions between the zinc ions and imidazole groups in the His6 side chains. In addition, at pH < 6, no peaks corresponding to the deprotonated species or metal-imidazole tautomers were observed even in the presence of zinc ions. Moreover, the XRD pattern of the generated HmA particles did not show sharp diffraction peaks (Fig. S2c), indicating the HmA particles were amorphous [21].

Fig. 2. FTIR (a) and Raman (b) spectra of HmA (red) and His6 (black); schematic of the His6 and zinc ion coordination process under different pH conditions and the corresponding status of the imidazole ring: protonated ① and deprotonated ② ③ imidazole ring before coordination, and different binding modes of the metal and imidazole ring ④ (metal bridging imidazole) ⑤ ⑥ (metal-imidazole tautomer) after coordination. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

446

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

Considering that a coordinative interaction induced the generation of the HmA particles, any factors influencing this interaction could change the physical or chemical properties of the HmA particles. We roughly evaluated the effect of factors that influence the generation of the HmA particles by measuring the size, PDI, zeta potential, and yield of polyhistidine in the generated HmA particles. The results demonstrated that, in addition to the abovementioned effects of pH, both the molar ratio of zinc ions to the monomer unit of polyhistidine and the chain length of the polyhistidine monomers were critical factors. As listed in Table S1, as the molar ratio of zinc ions to histidine increased, although the yield reached approximately 100% for all tested ratios, the size varied from 58 nm to 3751 nm, and the PDI could not be controlled. The chain length also played a critical role in HmA generation. Among the tested polyhistidine derivatives, HmA showed increased size and size distribution with increasing chain length, except for His3. HmA generated from His3 showed the largest size (472.4 nm) with a very wide size distribution and the lowest yield (42.32%), even though it was the shortest polyhistidine monomer. The reason for this phenomenon is not yet clear, but we are working to elucidate the principle. Overall, among the HmA derivatives prepared, HmA generated from His6 had the lowest particle size (60 nm) with an acceptable size distribution (PDI 0.21) and a high yield (98.76%). Therefore, we established the optimal HmA generation conditions (pH = 8, 1:1 M ratio of zinc ions to His6, and optimal chain length of His6) to construct HmA for the following experiments. 3.2. Drug encapsulation and release The model drug-encapsulating HmA (HmA@Drug) particles were produced by mixing zinc nitrate and His6 with the model drugs in water (or methanol for hydrophobic drugs). Taking Fl. as the first model drug, two of the most important parameters for evaluating the drug loading ability, namely, the encapsulation

efficiency (EE %) and loading capacity (LC wt%), were thoroughly investigated. On addition of zinc ions, the transparent solution (Fig. S3a) became milky (Fig. S3b), indicating the generation of HmA@Fl. particles. The size distribution as evaluated by DLS (Fig. S3c) was similar to that of the HmA particles. However, the average size was 169 nm, which was larger than that of the empty HmA particles. This difference may be due to the carboxyl group of Fl., as the electronic interaction between the carboxyl group and His6 could affect the coordination interaction between the zinc ions and His6. As shown in Fig. 3a, both the EE % and LC wt% of HmA@Fl. were strongly dependent on the concentration of Fl. The EE % could reach as high as 98.5% at an Fl. concentration of <1 mg mL1, although it steadily decreased as the Fl. concentration increased. However, the LC wt% increased with increasing concentrations of Fl., and the maximum loading capacity (as high as 532 mg/g, LCm = 53 wt%), indicating Fl. encapsulation saturation, was reached at an Fl. concentration above 9 mg/mL. Considering that the molecular weight of Fl. is too low, the polymer dextran, with a molecular weight of 40 kDa, was used to further verify the loading ability of the HmA particles. Dextran-40 k was selected because its monodisperse molecular weight is close to that of most proteins (on the order of tens of thousands of daltons) and because it is a representative neutral molecule under physiological conditions. Fig. 3b shows the dependence of EE % and LC wt% on the concentration of dextran-40 k. Although the loading behavior of dextran-40 k is different from that of Fl. (as observed by comparing Fig. 3b and a), similar trends in the concentration dependence of Fl can be seen, and the LCm values reached as high as 438 mg/g. In addition, the size (Fig. S4a and b) and size distribution (Fig. S4c and d) were independent of the initial concentration of the drug for encapsulation (Fl. and dextran-40 k). Drugs possess a wide variety of chemical and physical properties, such as large or small molecular sizes, negative or positive charges, hydrophobicity or hydrophilicity (and concomitant variations in water solubility), and biological and pharmacological activities. To

Fig. 3. EE % and LC wt% of Fl. (a) and dextran-40 k (b) for HmA particles at different drug concentrations. Release profile of HmA@drug nanoparticles as determined by UV/Vis spectrophotometry. Release of Fl. from HmA@Fl. nanoparticles in solutions with pH = 4.5 and pH = 7.2, 0.9% NaCl solution (c). Release of Fl. (5 mg) and dextran-40 k (5 mg) from HmA@Fl. (dashed line) and HmA@dextran-40 k (solid line) at various pH values in Bis-Tris solutions (d).

447

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452 Table 1 Characteristics of different HmA@drug particles. Drugs

Fl.

Rho

Mw Water solubilitya Activitya Chargesb EE LC Size PDI Zeta potential

376 Y N  94.55 30.31 167 0.19 10

479 Y N + 36.50 33.60 129.1 0.117 12.9

Dextran4k

40 k

500 k

2000 k

4k Y N n 91.32 29.27 62.11 0.22 10.4

40 k Y N n 87.91 28.18 72.71 0.20 10.7

500 k Y N n 86.33 27.67 71.49 0.21 11.1

2000 k Y N n 84.39 27.05 90.68 0.21 11.4

Lys

BSA

DXM

14 k Y Y + 76.59 24.55 134 0.23 9.4

66 k Y Y  99.47 31.88 139.7 0.12 0.42

392 N N n 96.2 30.83 118.23 0.23 29

The encapsulation concentrations of the drugs were all 1 mg mL1. a (Y) stands for ‘‘yes”, (N) stands for ‘‘no”. b (+) stands for ‘‘positively charged”, () stands for ‘‘negatively charged”, (n) stands for ‘‘neutral”. The unit of EE is ‘‘%”, LC is ‘‘wt%”, size is ‘‘nm”, zeta potential is ‘‘mV”.

prove the universality of the encapsulation, we encapsulated a variety of common drugs with diverse chemical and physical properties in the HmA particles. With the initial drug concentration fixed at 1 mg/mL, the EE %, LC wt%, size, PDI, and zeta potential of the generated HmA@Drug particles were tested, and the results are listed in Table 1. Overall, for all tested drugs, except Rho, the HmA showed EE % and LC wt% values analogous to those for Fl. The zeta potentials of the generated HmA@Drug particles were centered at +10 mV, with the exception of the HmA@BSA particles at  0.49 mV. Although the size of the generated HmA@Drug particles varied with the encapsulated drugs from 62 nm (for dextran-4 K) to 167 nm (for Fl.), they displayed small PDI values (PDI < 0.2 for most tested drugs), suggesting that HmA@Drug particles are small and have a narrow size distribution, which is suitable for applications in nanomedicine. Proteins play important roles in biological systems and disease treatments, as they have high biological activity and specificity, and the number of the proteins used as therapeutic agents has increased in recent decades [36–38]. Two representative proteins (a basic protein (lysosome) with isoelectric point (IEP) at 11.2 and an acidic protein (BSA) with IEP at 4.7) were applied as examples. As expected, HmA could encapsulate these proteins in a similar manner as that of small molecules and was much more effective than other protein loading systems, such as porous silica and CaCO3 [39,40]. In addition, HmA could encapsulate hydrophobic drugs (DXM) because the coordinative interaction between His6 and zinc ions was not hindered by organic solvents. Although the HmA showed high efficiency encapsulating most of the tested model drugs, different EE and LC values were observed for different drugs. These divergent values were attributed to the varied interaction mode of drugs with His6 or zinc ions during the production of HmA. There are multiple possible interaction modes, including charge interaction between negatively charged drugs and the positive charge of His6, hydrogen bonding interactions, and coordinative interaction between zinc ions and carboxyl or amino groups from the drugs. As shown in Table 1, the negatively charged drugs (Fl. and BSA) showed a better EE than the positively charged drugs (Rho and Lys). This difference is mainly attributed to the fact that positively charged His6 has better electrostatic interactions with the negatively charged drugs than the positively charged drugs. The strategy was effective for encapsulating neutral model drugs mainly because of hydrogen bonding interaction. Thus, the multiple interaction modes of His6 or the zinc ions ensured the ability of HmA to encapsulate various drugs with drastically different chemical and physical properties. It is reasonable to speculate that this system can be used for the co-encapsulation of multiple drugs simultaneously, which is critical for treating many diseases that require combinational drugs, especially cancers [41]. Compared with traditional drug delivery vehicles (liposomes, micelles, dendrimers, and inorganic porous particles such as SiO2

and CaCO3), HmA exhibits a much higher loading capacity [39,40] and is comparable to metal–organic frameworks (MOFs) in a recent review [5]. As reported, more than 50 types of MOFs have been developed for drug delivery, and many of them have shown considerable drug loading capacities ranging from 1.5 wt% to 65.5 wt%. The LCm reported here, i.e., 53 wt% for Fl. by HmA, surpasses that of most reported MOFs. As summarized in that review, only 4 MOFs showed LCm performances superior to those of HmA. Only specific drugs or model drug molecules were reported, namely, 5-fluorouracil for Zn-CDDB (LCm 53.5 wt%), ibuprofen for MIL-101 (LCm, 58 wt%), diclofenac sodium for ZJU-800 (LCm, 58 wt %), and doxorubicin (DOX) for PAA@ZIF-8 (LCm, 65.5 wt%). However, harsh conditions such as organic solvents, heat, or extreme pH were sometimes required to produce these carriers, thereby limiting the loading of unstable drugs. Moreover, the small pore size of MOFs limits the kinds of drugs that they can carry [5]. Obviously, compared to MOFs, HmA displayed much greater flexibility in loading various drugs under gentler conditions, without losing LCm values. To reveal the incorporation mode, fluorescence and UV–Vis spectra were recorded before and after the generation of HmA@Fl. particles. As shown in Fig. S5a (fluorescence spectra), the emission peak at 512 nm for free Fl. shifted to 527 nm after encapsulation. In Figure S5b (UV–Vis spectra), the absorbance peak at 489 nm for free Fl. shifted to 502 nm after encapsulation. At different initial Fl. concentrations, a redshift is reflected by the color change after encapsulation (before: Fig. S5c, and after: Fig. S5d). Consistent with previous reports [9], a redshift indicates that Fl. is encapsulated inside the HmA and not adsorbed onto the surface, which is schematically illustrated in Fig. S5e. In addition, the images of HmA@BSA (Fig. S6a) and HmA@Dextran-40 K (Fig. S6b) illustrate their similar color changes to those of HmA@Fl. after encapsulation, suggesting that the incorporation mode of these model drugs is consistent with that of Fl. In other control experiments, drug encapsulation in HmA was successful only when the drugs were present during HmA generation. Once the HmA is generated, it is difficult to load drugs into the HmA by incubating the HmA with the drugs, further indicating the encapsulation mode. Furthermore, the zeta potentials for most HmA@Drug particles were very close to that of HmA. However, in some cases, such as BSA, the zeta potential decreased slightly, suggesting that BSA binds to HmA. As a control, after incubating the HmA particles in BSA solution, the zeta potential of HmA did not change, and no adsorbed BSA was detected by UV–Vis spectroscopy, indicating BSA was encapsulated into the HmA even in the case of HmA@BSA. The stability of HmA, HmA@Fl., and HmA@Dextran-40 k particles was investigated by creating 100-fold (v/v) dilutions of their suspensions with deionized water and incubating the suspensions for 24 h. The DLS results are shown in Fig. S7; the average sizes and

448

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

PDI of the HmA, HmA@Fl., and HmA@Dextran-40 k particles are stable under extreme dilution conditions for 24 h. However, a slight aggregation of the particles was observed in phosphate buffer or in solutions containing phosphate ions. A possible reason for this aggregation is the competition for the zinc ions between His6 and the phosphate anions in the buffer, which can capture the zinc ions to aggregate the HmA. However, slight ultrasonication can suitably redisperse the HmA particles. The release profiles of the HmA@Drug particles are demonstrated in Fig. 3. To avoid the issue of aggregation caused by anions in the buffer, Bis-Tris buffer was chosen. As a drug delivery vehicle, HmA will experience a decrease in pH during the maturation of the lysozyme after being endocytosed by the cell. Therefore, the release profiles of the HmA@Drug particles at different pH values are an important characteristic that affects drug efficacy. First, the low critical pH value to disassemble the HmA was carefully determined. As shown in Fig. 3a, a burst release of Fl. (100% released in 5 min) was observed at pH 4.5 (Fig. 3c). Above pH 4.5, three pH values (5.8, 6.5, and 7.2) were selected to describe the release profile of the HmA@Drug particles in detail. A good ability to retain the loaded Fl. (<5% released in 7 days) was observed at pH 7.2. These results indicated that HmA particles decomposed at low pH (4.5) and were stable at neutral pH. Fl. and dextran-40 k were separately used as model small-molecule and macromolecular drugs, respectively, and tested in Bis-Tris buffer solutions with various pH values. As shown in Fig. 3d, all samples of HmA@Fl. (dashed line) exhibited rapid release at the beginning of the incubation period (<30 h), which could be attributed to the dissolution of the free Fl. attached onto the shell of the HmA particles, and the release finally reached a plateau after 7 days of incubation. As expected, the lower pH values led to faster drug release and higher cumulative release (8% released at pH = 7.2; 53% at pH = 6.5; 97% at pH = 5.8 in 7 days). The HmA@Dextran-40 k particles (solid line) showed similar release trends at all tested pH values,

but a slower release and a slightly lower cumulative release plateau, which might be attributed to the higher molecular weight and the difference in interaction mode with His6 of dextran. After the rapid release over the first several hours, the subsequent release was dependent on the pH; for example, at pH = 5.8, the particles rapidly released 100% of the drug, but at pH = 7.2, <8% of the dextran-40 k was released. This release profile is similar to that of the ZIF-8@DOX system reported by Haoquan Zheng et al, [13] which showed promising results for cancer treatment. 3.3. Endocytosis process and mechanism Before further cellular experiments, the cytotoxicity of the HmA particles was evaluated by CCK-8 assays. HeLa cells were incubated with increasing concentrations of HmA (20–100 lg mL1) for 24 h, 48 h, and 72 h to test the cytotoxicity, and the results are shown in Fig. 4a, Fig. S8a, and S8b, separately, respectively. Cell viabilities ranged from 99.9% to 129.4% at 24 h and remained at 89.7% when incubated with HmA at a concentration of 100 lg mL1 for 72 h. These results indicate that HmA is not or minimally cytotoxic toward HeLa cells and is suitable for use as a drug delivery vehicle. FITC-labeled His6 (F-His6) was used to generate labeled HmA particles, which are referred to as F-HmA, and flow cytometry was employed to monitor the cellular uptake efficiency of F-HmA. After incubating HeLa cells with F-HmA (50 lg mL1) for different time intervals (15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h), the cells were analyzed by flow cytometry (Fig. S9). As shown in Fig. 4b, the proportion of fluorescent HeLa cells initially increased with increasing incubation time and then reached a plateau of 96.53% within 2 h of incubation, thus indicating a fast endocytosis process, and the mean fluorescence intensity increased with increasing incubation time over the whole test period. To confirm this result, cells of another phenotype (dendritic cells) were subjected to the same experiment. As shown in Fig. S10, similar trends

Fig. 4. Cell viability of HeLa cells when incubated with different concentrations of HmA for 24 h (a). Fluorescence-positive rate and mean fluorescence intensity of HeLa cells when incubated with F-HmA for different times as detected by flow cytometry (b). Confocal images of HeLa cells when incubated with F-HmA (green) for 2 h and then with LysoTracker for 30 min (c). The nuclei were stained by DAPI (blue). The scale bar is 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

(increase in fluorescence vs. time and increase in mean fluorescence intensity vs. time) were observed. To further elucidate the cellular uptake profile, after 2 h of incubation with F-HmA particles, the cells were stained with LysoTracker Red DND-99 (used to label lysosomes) and imaged by confocal laser scanning microscopy (CLSM). As shown in Fig. 4c, the F-HmA (green) and lysosomes (red) were fully delocalized. Considering the time necessary for the lysosomes to mature [42], samples subjected to longer incubation times were also imaged (Fig. S11). For all samples, the delocalization phenomena remained similar for up to 2 h of incubation, and very few colocalization spots were observed even after 24 h of incubation. According to previous reports, the increased particle concentration could increase the number of intracellular lysosomes [43]. However, the number of LysoTracker labels was independent of the concentration of F-HmA particles used during the incubation (Fig. S12), even at concentrations as high as 1 mg/mL. According to the above LysoTracker data, most of the HmA particles did not pass through the lysosome pathway after entering the cell. It is essential to clearly delineate the endocytic mechanism of the HmA particles. We examined the effects of known pharmacological inhibitors on the cellular uptake of these particles. Chlorpromazine, a known inhibitor of clathrin-mediated endocytosis, is involved in the disassembly of clathrin and the recycling of LDL receptors to the membrane during clathrin-mediated endocytosis [44]. Nocodazole, an antitumor agent, was applied to interfere with the polymerization of microtubules [45]. Genistein can block caveolae-mediated endocytosis by inhibiting the tyrosine kinases involved in this process [46]. Amiloride, an inhibitor of Na+/H+ exchange, was employed to inhibit micropinocytosis [47]. After treatment with each drug separately for 2 h, the cells were treated with F-HmA for another 2 h. As shown in Fig. 5a, notable decreases in fluorescence intensity were observed for all samples, indicating that the internalization of F-HmA was dependent on all four pathways (clathrin-mediated endocytosis, microtubule-mediated endocytosis, caveolae-mediated endocytosis, and micropinocytosis). By dissolving the cell membrane using 0.1% Triton, the effects of these inhibitors could be quantified, and the results are displayed in Fig. 5b. Compared to the other inhibitors, genistein had the greatest inhibitory effect (as high as 81%), followed by amiloride (43%), nocodazole (28%), and chlorpromazine (27%). The potent inhibitory effect shown by genistein strongly suggested that the endocytosis of HmA particles was mainly caveola-mediated, while micropinocytosis, clathrin-mediated endocytosis, and microtubules played minor roles. As reported by others [46,48], caveola-mediated endocytosis did not go through the lysosome maturation process or the endosome escape process; rather, the particles directly released their cargoes, a result that was consistent with the above LysoTracker results. This intracellular process preserved the bioactivities of the loaded biomacromolecules to the greatest extent possible. In addition, the materials transported by caveola-mediated endocytosis could easily penetrate into solid tumors, indicating that our HmA particles have great potential as antitumor DDSs [49]. The size and zeta potential of nanoparticles are known to have considerable effects on the endocytosis process [50]. Accordingly, the drugs loaded into the HmA could change the endocytosis process, as the size and zeta potential varied after drug loading. However, compared with the fluorescence images of F-HmA (Fig. 4), the images of F-HmA@Rho at 2 h incubation (Fig. 6a) did not exhibit much difference in endocytosis, even though they had different sizes (60 nm and 129 nm for HmA and HmA@Rho, respectively). Once the experimental time was extended, the fluorescent intensity per cell of HmA@Rho increased (Fig. S13), similar to that of F-HmA (Fig. 4b). There are multiple potential reasons for these phenomena. In addition to size and zeta potential, the shape, sur-

449

Fig. 5. Confocal images of HeLa cells following incubation with F-HmA nanoparticles for 2 h after pretreatment with endocytosis inhibitors (chlorpromazine, nocodazole, amiloride and genistein). Control, untreated (a). Percent of F-HmA uptake in HeLa cells exposed to different inhibitors compared to the control (untreated) group (b). The scale bar is 100 lm. *P < 0.05, **P < 0.01, and ***P < 0.001, ‘‘NS” stands for not significant.

face composition, surface architecture, and the tested cell lines are also critical factors to the endocytosis process of nanoparticles [51,52]. Even for particles having the same size and surface chemistry, they might display different endocytosis processes, as reported by various research groups [53]. After drug loading, HmA@Drug particles are different from HmA in various aspects including size, surface composition, and zeta potential. Some might be positive for endocytosis, but some are negative, which makes the endocytosis process complex and makes it difficult to compare HmA with the HmA@Drug particles.

3.4. Intracellular fate of HmA and drugs To explore the intracellular fate of the HmA particles and the loaded drugs, HeLa cells were incubated with F-HmA@Rho for 2 h and then imaged by CLSM. The 3D images rebuilt by combining the cross-sectional images (Fig. S14) vividly illustrate the internalization of the F-HmA@Rho particles. After 2 h of incubation, the extracellular F-HmA@Rho particles were removed and the time was reset to 0 h. Then, the cells were further cultured and imaged at different time intervals after removing the extracellular particles. As shown in Fig. 6a, both F-HmA and the loaded model drug Rho were clearly present within the cells at 0 h (top panel), although their signals were faint at 4 h (middle panel). The signal of F-HmA had almost disappeared compared to that of Rho. The images at other time intervals (Fig. S15) exhibited the same phenomenon: over time; the signal of F-HmA almost disappeared, while that of Rho became faint. To further determine the fate of

450

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

Fig. 6. Confocal images of HeLa cells when incubated with F-HmA@Rho for 2 h (the time was reset to 0 h) (top panel); then the free F-HmA@Rho in the medium was washed away, and the cells were incubated for another 4 h (second panel) (a). Confocal images of HeLa cells when incubated with HmA@calcein for 2 h (the time was reset to 0 h) (third panel); then the free HmA@calcein in the medium was washed away, and the cells were incubated for another 4 h (bottom panel) (b). The scale bars are 20 lm in (a) and 100 lm in (b). The nuclei were stained by DAPI (blue). Fluorescence intensities of F-HmA, rhodamine B, and calcein outside the HeLa cells were quantified at different time intervals after washing away the free nanoparticles in the culture medium (c). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Image of HmA@CPT under a UV lamp (a). Viabilities of HeLa cells following treatment with HmA@CPT and free CPT for 24 h (b).

the loaded drug, calcein was chosen as the model drug, as it cannot pass through the cell membrane [54], and the same procedure as that used for F-HmA@Rho was followed. As shown in Fig. 6b, the fluorescence intensity of intracellular calcein 4 h after removing the extracellular HmA@calcein particles (bottom panel) was comparable to that observed at 0 h. The independence of fluorescence

intensity from incubation time was further confirmed after prolonged incubation times (Fig. S16). To analyze the fate of the F-HmA particles and the loaded drugs, F-HmA, F-HmA@Rho, and F-HmA@calcein were separately incubated with the HeLa cells again. However, in this case, the culture media were collected at different incubation times after removing the extracellular HmA@Drug particles. Their corresponding fluorescence intensities were measured and are shown in Fig. 6c. With increasing incubation time, the fluorescence intensity of extracellular FITC and Rho initially increased and then reached a plateau after 3 h, whereas calcein remained at a very low intensity from the beginning to the end of the experiment. The extracellular fluorescence intensity results were consistent with the CLSM observations: both suggested that the F-HmA particles undergo disassembly and release of their loaded drugs intracellularly. Furthermore, the model drugs diffusing outside the cytomembrane or staying inside cytomembrane was strongly dependent on their own intrinsic physicochemical properties. Here, Rho is able to diffuse out of the cytomembrane, while the calcein is not. Notably, the entire process of the endocytosis, disassembly and escape of F-HmA was very rapid (4 h), much more rapid than that of most reported delivery systems (from hours to days) [55].

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

451

3.5. Cytotoxicity of HmA@CPT

References

Furthermore, we evaluated the utility of HmA as a DDS to load camptothecin (CPT). As a chemotherapeutic drug, CPT induces cell apoptosis by inhibiting DNA topoisomerase I in the cytoplasm [56]. We demonstrated the successful encapsulation of CPT in HmA nanoparticles (EE: 37.8%) (Fig. 7a) and evaluated the antitumor activity of HmA@CPT. The lower EE of CPT than the other model drugs might be due to the limited interaction of CPT with His6 and zinc ions, which is determined by its chemical structure. We know that the release profile of HmA@CPT is critical to studying the cytotoxicity of CPT. However, CPT has very low solubility in the water, which makes testing the release profile difficult. Considering that the encapsulation mode of CPT in HmA is similar to that of other drugs and that drug release is caused by the disassembly of HmA at different pH values, it is reasonable to speculate that the release profile of CPT is similar to that of the other drugs that were already tested. HmA@CPT-treated HeLa cells showed much higher cytotoxicity than that treated with free CPT (Fig. 7b). Because of the low solubility of CPT in water, CPT barely penetrates through the cytomembrane, which can be overcome with the assistance of HmA. After endocytosis, CPT was quickly released into the cytoplasm and played a killing role in the cells.

[1] S. Ravindran, J.K. Suthar, R. Rokade, P. Deshpande, P. Singh, A. Pratinidhi, R. Khambadkhar, S. Utekar, Pharmacokinetics, metabolism, distribution and permeability of nanomedicine, Curr. Drug Metab. 19 (2018) 327–334. [2] S. Hossen, M.K. Hossain, M.K. Basher, M.N.H. Mia, M.T. Rahman, M.J. Uddin, Smart nanocarrier-based drug delivery systems for cancer therapy and toxicity studies: a review, J. Adv. Res. 15 (2019) 1–18. [3] M. Oh, C.A. Mirkin, Chemically tailorable colloidal particles from infinite coordination polymers, Nature 438 (2005) 651–654. [4] H. Maeda, M. Hasegawa, T. Hashimoto, T. Kakimoto, S. Nishio, T. Nakanishi, Nanoscale spherical architectures fabricated by metal coordination of multiple dipyrrin moieties, J. Am. Chem. Soc. 128 (2006) 10024–10025. [5] M.X. Wu, Y.W. Yang, Metal-organic framework (MOF)-based drug/cargo delivery and cancer therapy, Adv. Mater. 29 (2017) 1606143. [6] H.J. Lee, W. Cho, S. Jung, M. Oh, Morphology-selective formation and morphology-dependent gas-adsorption properties of coordination polymer particles, Adv. Mater. 21 (2009) 674–677. [7] K. Liang, R. Ricco, C.M. Doherty, M.J. Styles, S. Bell, N. Kirby, S. Mudie, D. Haylock, A.J. Hill, C.J. Doonan, P. Falcaro, Biomimetic mineralization of metalorganic frameworks as protective coatings for biomacromolecules, Nat. Commun. 6 (2015) 7240. [8] C.H. Kuo, Y. Tang, L.Y. Chou, B.T. Sneed, C.N. Brodsky, Z. Zhao, C.K. Tsung, YolkShell nanocrystal@ZIF-8 nanostructures for gas-phase heterogeneous catalysis with selectivity control, J. Am. Chem. Soc. 134 (2012) 14345–14348. [9] J. Zhuang, C.H. Kuo, L.Y. Chou, D.Y. Liu, E. Weerapana, C.K. Tsung, Optimized metal-organic-framework nanospheres for drug delivery: evaluation of smallmolecule encapsulation, ACS Nano 8 (2014) 2812–2819. [10] C.M. Calabrese, T.J. Merkel, W.E. Briley, P.S. Randeria, S.P. Narayan, J.L. Rouge, D.A. Walker, A.W. Scott, C.A. Mirkin, Biocompatible infinite-coordinationpolymer nanoparticle-nucleic-acid conjugates for antisense gene regulation, Angew. Chem. Int. Ed. Engl. 54 (2015) 476–480. [11] R. Roder, T. Preiss, P. Hirschle, B. Steinborn, A. Zimpel, M. Hohn, J.O. Radler, T. Bein, E. Wagner, S. Wuttke, U. Lachelt, Multifunctional nanoparticles by coordinative self-assembly of his-tagged units with metal-organic frameworks, J. Am. Chem. Soc. 139 (2017) 2359–2368. [12] L. Tan, H. Li, Y. Qiu, D. Chen, X. Wang, R. Pan, Y. Wang, S. Zhang, B. Wang, Y. Yang, Stimuli-responsive metal-organic frameworks gated by pillar[5]arene supramolecular switches, Chem. Sci. 6 (2015) 1640–1644. [13] H. Zheng, Y. Zhang, L. Liu, W. Wan, P. Guo, A.M. Nystrom, X. Zou, One-pot synthesis of metal-organic frameworks with encapsulated target molecules and their applications for controlled drug delivery, J. Am. Chem. Soc. 138 (2016) 962–968. [14] C. He, C. Poon, C. Chan, S.D. Yamada, W. Lin, Nanoscale coordination polymers codeliver chemotherapeutics and siRNAs to eradicate tumors of cisplatinresistant ovarian cancer, J. Am. Chem. Soc. 138 (2016) 6010–6019. [15] D. Liu, C. Poon, K. Lu, C. He, W. Lin, Self-assembled nanoscale coordination polymers with trigger release properties for effective anticancer, Therapy, Nat. Commun. (2014) 4182. [16] L. Ding, Y. Jiang, J. Zhang, H.A. Klok, Z. Zhong, pH-sensitive coiled-coil peptidecross-linked hyaluronic acid nanogels: synthesis and targeted intracellular protein delivery to CD44 positive cancer cells, Biomacromolecules 19 (2018) 555–562. [17] B. Shivu, S. Seshadri, J. Li, K.A. Oberg, V.N. Uversky, A.L. Fink, Distinct betasheet structure in protein aggregates determined by ATR-FTIR spectroscopy, Biochemistry 52 (2013) 5176–5183. [18] B. Breit, W. Seiche, Self-assembly of bidentate ligands for combinatorial homogeneous catalysis based on an A-T base-pair model, Angew. Chem. Int. Ed. Engl. 44 (2005) 1640–1643. [19] H. Block, B. Maertens, A. Spriestersbach, N. Brinker, J. Kubicek, R. Fabis, J. Labahn, F. Schafer, Reprint of: immobilized-metal affinity chromatography (IMAC): a review, Protein Expr. Purif. 463 (2011) 439–473. [20] W. Maret, Zinc biochemistry: from a single zinc enzyme to a key element of life, Adv. Nutr. (2013) 82–91. [21] F. Jehle, P. Fratzl, M.J. Harrington, Metal-tunable self-assembly of hierarchical structure in mussel-inspired peptide films, ACS Nano 12 (2018) 2160–2168. [22] S. Li, Q. Zou, Y. Li, C. Yuan, R. Xing, X. Yan, Smart peptide-based supramolecular photodynamic metallo-nanodrugs designed by multicomponent coordination self-assembly, J. Am. Chem. Soc. 140 (2018) 10794–10802. [23] H. López-Laguna, U. Unzueta, O. Conchillo-Solé, A. Sánchez-Chardi, M. Pesarrodona, O. Cano-Garrido, E. Voltà, L. Sánchez-García, N. Serna, P. Saccardo, R. Mangues, A. Villaverde, E. Vázquez, Assembly of histidine-rich protein materials controlled through divalent cations, Acta Biomater. 83 (2019) 257–264. [24] A.S. Knight, J. Larsson, J.M. Ren, R. Bou Zerdan, S. Seguin, R. Vrahas, J. Liu, G. Ren, C.J. Hawker, Control of amphiphile self-assembly via bioinspired metal ion coordination, J. Am. Chem. Soc. 140 (2018) 1409–1414. [25] P. Shi, S. Luo, B. Voit, D. Appelhans, X. Zan, A facile and efficient strategy to encapsulate the model basic protein lysozyme into porous CaCO3, J. Mater. Chem. B 6 (2018) 4205–4215. [26] S. Sun, J. Li, X. Li, B. Lan, S. Zhou, Y. Meng, L. Cheng, Episcleral drug film for better-targeted ocular drug delivery and controlled release using multilayered poly-epsilon-caprolactone (PCL), Acta Biomater. 37 (2016) 143–154. [27] Y. Meng, S. Sun, J. Li, K. Nan, B. Lan, Y. Jin, H. Chen, L. Cheng, Sustained release of triamcinolone acetonide from an episcleral plaque of multilayered polyepsilon-caprolactone matrix, Acta Biomater. 10 (2014) 126–133.

4. Conclusions In summary, we present the first reported production of HmA under mild conditions based on the coordinative interaction between metal ions and peptides, His6. The HmA particles displayed many features of an ideal DDS: average size 60 nm with a narrow size distribution (PDI < 0.2); wide encapsulating scope of drugs, from small molecules (Mw of several 100 Da) to polymers (Mw as high as 2000 kDa) and with diverse physical characteristics (various wettabilities, bioactivities, and charges); high loading capacity (53%) and loading efficiency (>90% for mostly tested); and pH-responsive drug release (15% of the load released at pH = 7.4, burst release (100%) at pH = 4.5 in minutes, or slow but almost 100% release around pH = 5.5 over weeks). In vitro experiments demonstrated that HmA had minimal cytotoxicity, rapid internalization, the ability to bypass the lysosomes, and fast intracellular drug release. With the assistance of the caveola-mediated endocytosis mechanism, the drugs could be rapidly released into the cytoplasm. In addition, the intracellular retention of the released drugs was found to be strongly dependent on the physical and chemical properties of the drugs. HmA encapsulation greatly improved the antitumor activity of CPT relative to free CPT. By capitalizing on these promising features, the universal coordination of His6 with metal ions, and the development of recombinant or bioconjugation techniques to integrate His6 into peptides or proteins, HmA will have great potential in various biomedical fields.

Acknowledgments This work was supported by the National Natural Science Foundation of China (81601079), startup funding from the Wenzhou government (WIBEZD2014002-02), and Wenzhou Science and Technology Bureau (G20180034).

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.actbio.2019.03.058.

452

W. Huang et al. / Acta Biomaterialia 90 (2019) 441–452

[28] T.J. Moyer, J.A. Finbloom, F. Chen, D.J. Toft, V.L. Cryns, S.I. Stupp, pH and amphiphilic structure direct supramolecular behavior in biofunctional assemblies, J. Am. Chem. Soc. 136 (2014) 14746–14752. [29] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 1767 (2007) 1073–1101. [30] P.O. Andersson, M. Lundquist, L. Tegler, S. Borjegren, L. Baltzer, L. Osterlund, A novel ATR-FTIR approach for characterisation and identification of ex situ immobilised species, ChemPhysChem 8 (2007) 712–722. [31] F. Wang, C.L. Feng, Metal-ion-mediated supramolecular chirality of lphenylalanine based hydrogels, Angew. Chem., Int. Ed. Engl. 57 (2018) 5655–5659. [32] H. Takeuchi, Raman structural markers of tryptophan and histidine side chains in proteins, Biopolymers 72 (2003) 305–317. [33] L.E. Valenti, M.B. Paci, C.P. De Pauli, C.E. Giacomelli, Infrared study of trifluoroacetic acid unpurified synthetic peptides in aqueous solution: trifluoroacetic acid removal and band assignment, Anal. Biochem. 410 (2011) 118–123. [34] M.E. Rousseau, T. Lefevre, L. Beaulieu, T. Asakura, M. Pezolet, Study of protein conformation and orientation in silkworm and spider silk fibers using raman microspectroscopy, Biomacromolecules 5 (2004) 2247–2257. [35] A. Reinecke, G. Brezesinski, M.J. Harrington, pH-responsive self-organization of metal-binding protein motifs from biomolecular junctions in mussel byssus, Adv. Mater. Interfaces 4 (2017) 1600416. [36] S. Mitragotri, P.A. Burke, R. Langer, Overcoming the challenges in administerin biopharmaceuticals: formulation and delivery strategies, Nat. Rev. Drug Discovery 13 (2014) 655–672. [37] K. Lee, M. Rafi, X. Wang, K. Aran, X. Feng, C. Lo Sterzo, R. Tang, N. Lingampalli, H.J. Kim, N. Murthy, In vivo delivery of transcription factors with multifunctional oligonucleotides, Nat. Mater. 14 (2015) 701–706. [38] V. Postupalenko, D. Desplancq, I. Orlov, Y. Arntz, D. Spehner, Y. Mely, B.P. Klaholz, P. Schultz, E. Weiss, G. Zuber, Protein delivery system containing a nickel-immobilized polymer for multimerization of affinity-purified histagged proteins enhances cytosolic transfer, Angew. Chem., Int. Ed. Engl. 54 (2015) 10583–10586. [39] H. Zhang, J. Peng, X. Li, S. Liu, Z. Hu, G. Xu, R. Wu, A nano-bio interfacial protein corona on silica nanoparticle, Colloids Surf B 167 (2018) 220–228. [40] D.V. Volodkin, N.I. Larionova, G.B. Sukhorukov, Protein encapsulation via porous CaCO3 microparticles templating, Biomacromolecules 5 (2004) 1962– 1972. [41] C. He, K. Lu, D. Liu, W. Lin, Nanoscale metal-organic frameworks for the codelivery of cisplatin and pooled siRNAs to enhance therapeutic efficacy in drug-resistant ovarian cancer cells, J. Am. Chem. Soc. 136 (2014) 5181–5184. [42] R. Logan, A.C. Kong, J.P. Krise, Time-dependent effects of hydrophobic aminecontaining drugs on lysosome structure and biogenesis in cultured human fibroblasts, J. Pharm. Sci. 103 (2014) 3287–3296.

[43] C. Orellana-Tavra, S.A. Mercado, D. Fairen-Jimenez, Endocytosis mechanism of nano metal-organic frameworks for drug delivery, Adv. Healthcare Mater. 5 (2016) 2261–2270. [44] A.I. Ivanov, Exocytosis and endocytosis, Preface, Methods Mol. Biol. 440 (2008) v–vi. [45] Y.C. Chang, P. Nalbant, J. Birkenfeld, Z.F. Chang, G.M. Bokoch, GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA, Mol. Biol. Cell 19 (2008) 2147–2153. [46] J. Rejman, A. Bragonzi, M. Conese, Role of Clathrin- and Caveolae-Mediated Endocytosis in Gene Transfer Mediated by Lipo- and Polyplexes, Mol. Ther. 12 (2005) 468–474. [47] L. Li, T. Wan, M. Wan, B. Liu, R. Cheng, R. Zhang, The effect of the size of fluorescent dextran on its endocytic pathway, Cell Biol. Int. 39 (2015) 531– 539. [48] G. Sahay, D.Y. Alakhova, A.V. Kabanov, Endocytosis of nanomedicines, J. Controlled Release 145 (2010) 182–195. [49] M. Sung, X. Tan, B. Lu, J. Golas, C. Hosselet, F. Wang, L. Tylaska, L. King, D. Zhou, R. Dushin, J.S. Myers, E. Rosfjord, J. Lucas, H.P. Gerber, F. Loganzo, Caveolaemediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1), Mol. Cancer Ther. 17 (2018) 243–253. [50] M. Ekkapongpisit, A. Giovia, C. Follo, G. Caputo, C. Isidoro, Biocompatibility, endocytosis, and intracellular trafficking of mesoporous silica and polystyrene nanoparticles in ovarian cancer cells: effects of size and surface charge groups, Int J Nanomedicine 7 (2012) 4147–4158. [51] V. Forest, M. Cottier, J. Pourchez, Electrostatic interactions favor the binding of positive nanoparticles on cells: a reductive theory, Nano Today 10 (2015) 677– 680. [52] N. Oh, J.-H. Park, Endocytosis and exocytosis of nanoparticles in mammalian cells, Int. J. Nanomed. 9 (Suppl 1) (2014) 51–63. [53] L. Zhang, P. Hao, D. Yang, S. Feng, B. Peng, D. Appelhans, T. Zhang, X. Zan, Designing nanoparticles with improved tumor penetration: surface properties from the molecular architecture viewpoint, J. Mater. Chem. B 7 (2019) 953– 964. [54] M. Javadi, W.G. Pitt, C.M. Tracy, J.R. Barrow, B.M. Willardson, J.M. Hartley, N.H. Tsosie, Ultrasonic gene and drug delivery using eLiposomes, J. Controlled Release 167 (2013) 92–100. [55] J. Gilleron, W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U. Schubert, K. Manygoats, S. Seifert, C. Andree, M. Stoter, H. Epstein-Barash, L. Zhang, V. Koteliansky, K. Fitzgerald, E. Fava, M. Bickle, Y. Kalaidzidis, A. Akinc, M. Maier, M. Zerial, Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape, Nat. Biotechnol. 31 (2013) 638–646. [56] Y.H. Hsiang, R. Hertzberg, S. Hecht, L.F. Liu, Camptothecin induces proteinlinked DNA breaks via mammalian DNA topoisomerase I, J. Biol. Chem. 260 (1985) 14873–14878.