Article Cite This: Biomacromolecules 2017, 18, 3498-3505
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Bioreducible Peptide-Dendrimeric Nanogels with Abundant Expanded Voids for Efficient Drug Entrapment and Delivery Dan Zhong,† Zhaoxu Tu,† Xiao Zhang,† Yachao Li,† Xianghui Xu,*,†,‡ and Zhongwei Gu*,†,‡ †
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, P.R. China College of Materials Science and Engineering, Nanjing Tech University, Nanjing, Jiangsu 210009, P.R. China
‡
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S Supporting Information *
ABSTRACT: Dendrimer-based nanoplatforms have exhibited wide prospects in the field of nanomedicine for drug delivery, without great success due to many predicaments of cytotoxicity, high cost, and low yield. In this work, we report a feasible strategy on dynamic cross-linkings of low-generation peptide dendrimers into bioreducible nanogels for efficient drug controlled release. With a facile fabrication, the disulfide cross-linking of biocompatible peptide dendrimers successfully possess well-defined and stable nanostructures with abundant expanded voids for efficient molecular encapsulation. More importantly, high reducing condition is capable of triggering the cleavage of disulfide bonds, the disintegration of peptidedendrimeric nanogels, and stimuli-responsive release of guest molecules. The bioreducible nanogels improve antitumor drug internalization, contribute to endosomal escape, and realize intracellular drug controlled release. The doxorubicin-loaded nanogels afford high antitumor efficiency and reduce the side effects to BALB/c mice bearing 4T1 tumor. Therefore, dynamic cross-linkings of low-generation dendrimers into smart nanogels will be an alternative and promising strategy to resolve the dilemmas of current dendrimer-based nanocarriers as well as develop innovative nanoplatforms.
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INTRODUCTION Macromolecular nanoparticle-based therapeutics have profoundly impacted the landscape of clinical anticancer chemotherapy, attributed to prolonged drug circulation time, enhanced tumor accumulation, and reduced adverse effects to normal organs (such as clinical formulations of PEGylated Doxil and protein-based Abraxane).1,2 Dendrimers, as the fourth new class of synthetic macromolecules, are of particular interest in the development of advanced delivery nanoplatforms owing to their unique structures (including precise, monodisperse, highly branched, and nanoscale properties),3 which inherently provide abundant peripheral groups and interior cavities for drug conjugation and encapsulation.4 Notably, supramolecular drug encapsulation with dendrimeric pockets is capable of avoiding additional chemical modification of dendrimers and bioactive drugs, maintaining intact pharmacological activity, and improving water solubility of hydrophobic drugs (e.g., camptothecin, doxorubicin, and paclitaxel).5 The drug loading capacity of dendrimer-based nanocontainers largely depends on the growth generations, and higher generation of dendrimers often affords larger internal void spaces and more densely packed surfaces for efficient drug entrapment and storage.6 For this reason, higher generations (≥5) of dendrimeric nanocarriers, such as poly(amido amine) (PAMAM) dendrimers and peptide dendrimers,7−9 are widely used to encapsulate various drugs for powerful tumor therapy. Nevertheless, the superior difficulty to the manufacture of © 2017 American Chemical Society
precise high-generation dendrimers results in low production rate and expensive cost.10 Moreover, high-generation dendrimers usually fail regarding conspicuous toxicity in vitro and in vivo.11 These dilemmas severely impede the clinical translation of dendrimer-based nanocontainers for drug delivery. As a result, endowing a new generation of dendrimer-based nanocarriers with robust load capacity and excellent biocompatibility is urgently needed in the promotion of clinical perspectives. In recent years, assembling low-generation dendrimers has become a promising trend to create biocompatible nanovehicles with plentiful supramolecular voids for highly efficient drug control release.12 Previously, we have successfully developed capsid-like nanoparticles hierarchically self-assembled from Generation 2 peptide dendrimers and linear polypeptides as pH-responsive drug nanovehicles.13−15 Furthermore, selfassembly of tailor-made low-generation dendrimers is able to gain multifunctional nanoplatforms for sequential tumorspecific targeted delivery and multidrug resistance reversal.16−19 However, analogous to linear polymeric assemblies, a major Special Issue: Organized Peptidic Nanostructures as Functional Materials Received: May 7, 2017 Revised: July 17, 2017 Published: July 20, 2017 3498
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
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Biomacromolecules
Scheme 1. Schematic Illustration for (i) Chemical Structures of Peptide Dendrimer and Disulfide Cross-Linker, (ii) Dynamic Cross-Linkings of the Low-Generation Peptide Dendrimers for Drug Encapsulation, and (iii) Intracellular Redox-Responsive Disintegration of Nanogels for Site-Specific Drug Delivery
Herein, we report a novel type of bioreducible nanovehicles based on disulfide cross-linked low-generation peptide dendrimers for antitumor drug entrapment and site-specific delivery (Scheme 1). With a facile divergent approach, we accurately synthesized low-generation peptide dendrimers as biodegradable and biocompatible building blocks for nanostructural fabrication. In addition, a custom-built disulfide derivate was designed as bioreducible linker for dynamic crosslinking of low-generation peptide dendrimers into well-defined nanogels. These nanogels are expected to hold satisfactory colloidal stability and drug loading capacity during blood circulation and tumor extracellular environment, which have a relatively moderate reducing condition corresponding to 2−20 μM glutathione (GSH).27,28 Conversely, tumor intracellular high GSH concentration (∼10 mM) is capable of triggering the redox cleavage of disulfide linkages and disintegration of nanogels, inducing the rapid release of antitumor drugs at the tumor site. This work puts forward a feasible approach to expand dendrimeric voids based on bioreducible peptide−
drawback of supramolecular dendritic nanoassemblies is instability with the environmental changes in the drug delivery (such as dilution and blood circulation), leading to premature disintegration and drug leakage.20 Covalent cross-linking must be a straightforward and practical strategy to stabilize dendritic systems,21,22 and recent research suggests that covalent crosslinking of low-generation dendrimers generates efficient, lowcytotoxicity, and readily available nanocarriers for gene delivery and magnetic resonance imaging.23,24 On the other hand, intermolecular cross-linking generates abundant voids among low-generation dendrimers, allowing for increasing drug loading capacity. More importantly, tailoring stimuli-responsive cross-linkers (e.g., acid-cleavable acetal linkages and reducible disulfide bonds) for stabilizing the supramolecular dendritic systems is expected to realize stimuli-triggered disintegration for site-specific drug delivery.25,26 Based on the considerations mentioned above, it is reasonable to believe that dynamic crosslinkings of low-generation dendrimers will open a new avenue to develop dendrimer-based nanocontainers. 3499
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
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Biomacromolecules
The mixture solution was dispersed in 10.0 mL of PBS and persistently stirred for 12 h. Free drug was removed by extensive dialysis against deionized water at 4 °C using dialysis membrane tube (Spectra/Por, MWCO 1000). The DOX-loaded BPDNs (D-BPDNs) were obtained after freeze-drying in the dark. The size and zeta potential of DBPDNs were determined by DLS, and their nanostructures were determined by scanning electron microscopy (SEM, S-4800 Hitachi, Japan) and transmission electron microscopy (TEM, Tecnai G2 F20 S-TWIN, USA). The drug loading content (DLC) of D-BPDNs was determined using a fluorescence spectrophotometer with excitation wavelength at 480 nm according to the following formula:
dendrimeric nanogels (BPDNs) for robust and stable drug encapsulation, as well as attain the goal of site-specific delivery of antitumor drug for efficient tumor inhibition in vitro and in vivo.
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EXPERIMENTAL SECTION
Synthesis of Low-Generation Peptide Dendrimers (PDs). The Generation 3 peptide dendrimers (G3 PDs) were synthesized with lysine (K) as branching units using a divergent method.29 First, pentaerythritol (0.30 g, 1 equiv), Boc-Lys(Boc)−OH (4.58 g, 6 equiv), HOBT (2.41 g, 8 equiv), HBTU (6.71 g, 8 equiv), and DIEA (3.6 mL, 10 equiv) were dissolved in dimethylformamide (DMF) with 48 h reaction under nitrogen atmosphere at room temperature. After removal of DMF, the residue was dissolved in dichloromethane (DCM) to wash with NaHCO3 solution (∼1 M) and HCl solution (∼1 M) 3 times. Following drying (MgSO4) and concentration, the crude product was purified by silica-gel column chromatography using DCM:methanol (20:1; v/v) as the eluent. The protected G1 PDs (2.50 g, 1 equiv) was treated with TFA (10.6 mL, 80 equiv) for 6 h to put off tert-butyloxycarbonyl groups. Then, we carefully repeated these processes to obtain G3 PDs, and detailed procedures can be found in the Supporting Information (Schemes S1−S3). The synthetic products were characterized by 1H NMR spectra (400 MHz, Bruker Avance II NMR spectrometer, Germany) and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectra (Bruker Autoflex III, Germany) (Figures S1−S6). Synthesis of Dimethyl L-Cystinate Bis(acrylamide)s (DCBs). Dimethyl L-cystinate (H-Cys-OMe)2·2HCl, 2.20 g, 1 equiv) and (CH3CH2)3N (2.7 mL, 3 equiv) were dissolved in 20 mL DCM in an ice bath. Then, methacryloyl chloride (1.9 mL, 3 equiv) was dropwise added into the mixture solution under nitrogen atmosphere with a magnetic stirrer for 24 h at room temperature. After removal of DCM, the crude product was purified by silica-gel column chromatography (DCM:methanol; 20:1). The detailed synthetic procedures and characterizations were presented in Supporting Information (Scheme S4 and Figures S7−S8). Fabrications and Characterizations of BPDNs. We prepared BPDNs through disulfide cross-linking of low-generation dendrimers via Michael addition with different molar ratios of G3 PDs and DCB linkers (Table S1). In brief, a certain amount of DCBs was slowly added into the G3 PD-included methanol solution. After bubbling with nitrogen for 10 min, the cross-linking reaction proceeded at 50 °C for 48 h. Then, the mixture solution was cooled down to room temperature and precipitated with excess anhydrous diethyl ether 3 times. The obtained solid was dissolved and dialyzed against distilled water in a membrane tube (Spectra/Por, MWCO 8000). After freezedrying, the BPDNs could be used for characterizations and applications. Viscosity measurement was performed with an Ubbelohde viscometer (inner diameter 0.5 mm) at 25 °C in water. Size and zeta potential were measured by dynamic light scattering (DLS, Malvern NANO ZS90, UK) at 25 °C in water. The nanostructure of BPDNs was performed by atomic force microscope (AFM, MFP-3D-BIO, USA). The reduction responsive properties of BPDNs were studied by monitoring the size and turbidity changes in the presence of GSH (10 μM or 10 mM) with phosphate buffered solution (PBS) at 37 °C. For turbidity measurement, the transmittance at 550 nm was detected by UV−vis spectrometer (Specord 200 Plus, Germany). Molecular Encapsulation and Controlled Release. BPDNs were dissolved in the pyrene solution (6 × 10−7 M) with a certain concentration of BPDNs (20 μg mL−1), and then two samples were treated with 10 μM or 10 mM GSH, respectively. The emission spectra of pyrene were obtained using a fluorescence spectrophotometer (Hitachi F-7000, Japan) with excitation wavelength of 330 nm, and the emission fluorescence intensity at 372 and 383 nm was recorded to calculate the ratios of I372/I383. The experiments were conducted in triplicate. To prepare drug-loaded BPDNs, antitumor doxorubicin (DOX, 2.5 mg) and BPDNs (10.0 mg) were first dissolved in 1.0 mL of DMSO.
DLC(%) = [mass of drug in D‐BPDNs/total mass of D‐BPDNs] × 100% The in vitro release profiles of DOX from D-BPDNs were studied using dialysis membrane tubes (Spectra/Por, MWCO 1000) in various media at 37 °C. In short, D-BPDNs (1.0 mg mL−1) were dissolved in PBS (pH 7.4) with different concentrations of GSH (0, 10 μM, or 10 mM). Then, 1.0 mL of D-BPDNs solution was transferred to a dialysis membrane tube and dialyzed against 25.0 mL of the same media. At preset time intervals, 1.0 mL of release media was sampled and 1.0 mL of fresh media was replenished. The amount of DOX released from DBPDNs was detected by fluorescence spectrometer. The release experiments were conducted in triplicate. In order to visualize BPDNs under confocal microscopy, fluorescein isothiocyanate (FITC, Fanbo Biochemicals, China) was used to label BPDNs. With a typical reaction, BPDNs (50.0 mg) was dissolved in 5.0 mL of DMSO. Meanwhile, FITC (0.5 mg, 1 wt % of BPNDs) was solubilized in 1.0 mL DMSO and dropwise added into BPDN solution. The mixture was stirred in the dark for 12 h, and free FITC was removed by dialysis against water. In Vitro Cytotoxicity Assay. Mouse breast 4T1 tumor cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA) and 1% (v/v) penicillin−streptomycin (Hyclone, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. To assess the in vitro cytotoxicity of DBPDNs, 4T1 cells were seeded in 96-well plates at a density of 8 × 103 cells per well for 24 h. Then, the cells were treated with 100 μL of fresh media containing D-BPDNs, DOX, DOX·HCl, or BPDNs for another 24 h. After rinsing with PBS 3 times, the tumor cells were treated with 100 μL of FBS-free medium containing 10% (v/v) CCK-8 (Dojindo Laboratories, Japan) for an additional 2 h. Cell viability (%) was measured by a Varioskan Flash microplate reader (Thermo Fisher Scientific, USA) to read the absorbance data at 450 nm. The cell viability was calculated according to the following formula: Cell viability(%) = (ODsample − ODbackground ) /(ODcontrol − ODbackground ) × 100% Cellular Uptake and Intracellular Delivery. To quantify the cellular internalization, 4T1 cells were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured for 24 h. Then, the cells were treated with BPDNs (100 μg mL−1), DOX (10 μg mL−1), DOX· HCl (10 μg mL−1), or D-BPDNs (100 μg mL−1, 10% DOX loading) for 2 h. After removal of the culture medium, the 4T1 cells were washed with PBS 3 times. Then, 4T1 cells were harvested and resuspended in 500 μL of PBS for analysis. Quantitative drug internalization was determined by fluorescence activated cell sorter (FACS, BD Biosciences, USA) with 2 × 104 gated events. To track the intracellular delivery of D-BPDNs, 4T1 cells were seeded on glass bottom dishes at a density of 1 × 104 cells/well and allowed to adhere for 24 h. After exposure to FITC-labeled D-BPDNs (10 μg mL−1 DOX) for 1, 3, or 6 h, the 4T1 cells were stained with LysoTracker (Molecular Probes, USA) and observed with confocal laser scanning microscopy (CLSM, Leica TCP SP5, Germany). In Vivo Antitumor Treatment. The animal experiments were approved by the ethics committee of Sichuan University. BALB/c mice (20−22 g, 6−8 weeks) were purchased from Chengdu Dashuo Laboratory Animal Center. Solid tumor models were built by 3500
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
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Figure 1. Macromolecular and nanostructural characterizations of bioreducible peptide-dendrimeric nanogels. (A) 1H NMR spectra of peptide dendrimers (green line, in D2O/DMSO-d6), dimethyl L-cystinate bis(acrylamide)s (blue line, in DMSO-d6), and BPDNs (red line, in D2O). (B) Viscosity measurements of BPDNs and PDs in aqueous solutions with the different concentration at 25 °C, and the inset photos showing the Tyndall effects of BPDNs and PDs. (C) Size distribution in aqueous solution and (D) AFM image (including size and height profile along the red line) of the BPDNs. subcutaneously inoculating with 4T1 cells (1 × 106/mouse) at the right flank. When the tumor volumes reached about 150 mm3, the mice were randomly divided into 4 groups (n = 5) for administration with normal saline, BPDNs, DOX·HCl, or D-BPDNs via the tail vein every 3 days for 4 times (5.0 mg DOX/kg body weight). The tumor volume was measured by electronic digital vernier caliper and calculated by the formula: V [mm3] = LW2/2, where L and W were the length and width of the tumor. At the end of the antitumor treatment (18 days), solid tumors and hearts were excised and fixed in 4% (v/v) formalin saline. We stained the tumor and heart sections with hematoxylin and eosin (H&E) for histopathological analysis. The tissue slices were observed using an inverted fluorescence microscope (Leica DMI 4000B, Germany).
ppm (l) of terminal −NH2 in G3 PDs significantly decreased (Figure 1A). In the meantime, the signals of methyl protons in DCBs changed from h (δ = 1.85 ppm) to h′ (δ = 1.22 ppm), due to reduction of CC double bonds.30,31 These results manifested the successful cross-linkings of peptide dendrimeric aggregations into nanogels. To further confirm the cross-linked networks in BPDNs, viscosity variation was determined using an Ubbelohde viscometer at 25 °C in an aqueous solution. In the low concentration range (100−500 μg mL−1), the viscosity of G3 PDs increased with a slope of 0.04, implying a linear relationship between concentration and viscosity (Figure 1B).13 As expected, BPDNs showed higher viscosity values (slope = 0.31) than those of single PDs, attributed to the increase in molecular weight of cross-linked peptide dendrimers. Additionally, an obvious Tyndall effect was observed in the BPDN solution (100 μg mL−1) due to the presence of abundant colloidal nanoparticles,32 while the Tyndall effect could not be found in the PD solution. Next, we turned to study the BPDN nanostructures. Fully considering the average size distribution and polydispersity index of BPDNs (Table S1 and Figure S11), the nanogels with an average diameter of 91.1 ± 5.6 nm were selected as representative nanoplatforms for the following studies (Figure 1C). AFM image demonstrated the three-dimensional structures of BPDNs, which had well-defined spherical nanostructures around 100−200 nm (Figure 1D). The difference between AFM and DLS results might be attributed to the different detection conditions, because AFM images reflected the waterless dimension of the nanogels adhering on mica substrates, but DLS distribution showed the hydrodynamic diameter at the swollen state in water.33 Taken
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RESULTS AND DISCUSSION Fabrications and Characterizations of BPDNs. The lowgeneration peptide dendrimers were successfully synthesized with accurate molecular structures according to the divergent method. The apparent peaks at m/z 649.72, 1675.15, and 3726.20 in MALDI-TOF mass spectra all agreed with the theoretical molecular weights of protonated G1, G2, and G3 PDs (649.85, 1675.25, and 3726.05), respectively (Figures S4− S6). As for disulfide cross-linkers of DCBs, MALDI-TOF mass spectrum and 1H NMR results provided direct evidence of successful introduction of CC double bonds into dual terminals of dimethyl L-cystinate (Figure S7 and Figure S8). After Michael reactions among peripheral amino groups (−NH2) of G3 PDs and CC double bonds of disulfide linkers in methanol, the aggregations of G3 PDs should be cross-linked into the bioreducible nanogels with the dendrimeric network. As shown in the 1H NMR results, the signals of vinyl protons in disulfide linkers shifted from g (δ = 5.72 and 5.42 ppm) to g′ (δ = 3.20 ppm), and the signals at δ = 1.95 3501
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
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For another, we monitored the turbidity changes with different GSH concentrations to disclose redox degradation of BPDNs, since the cleavage of cross-linked networks in nanogels would result in the decrease of the turbidity.36,37 Observably, the transmittance of BPDN solution (500 μg mL−1) increased in the presence of 10 mM GSH, owing to redox-dependent degradation of nanogels (Figure 2B). As control groups, the transmittance of BPDN solution had no obvious alternation within 12 h incubation with the existence of 10 μM GSH and in the absence of GSH, confirming the stability of BPDNs at the normal conditions and tumor microenvironments. These observations suggested that BPDNs would provide stable nanostructures with interior voids for molecular entrapment, while intracellular reducing conditions would cleave crosslinked networks of nanogels. Molecular Entrapment and Controlled Release. We first used pyrene as a hydrophobic probe that can sense the polarity of local environment, to explore the molecular loading capacity and stimuli-sensitivity of BPDNs. When BPDNs were added into the pyrene solution (pH 7.4), the fluorescence intensity of pyrene increased and the I372/I383 ratio decreased (Figure 3A), declaring that pyrene was encapsulated into the network cavities of BPDNs.14 As expected, high reduction conditions (GSH 10 mM) induced the decrease of fluorescence intensity and the increase of the I372/I383 ratio, suggesting redox-dependent destruction of nanogels and release of guest molecules. BPDNs could resist the degradation of 10 μM GSH for stable pyrene loading without fluorescence alteration. The pyrene loading capacity of BPDNs testified that disulfide crosslinked low-generation dendrimers were capable of serving as smart nanoplatforms for molecular encapsulation and stimuliresponsive delivery. Next, a broad-spectrum anticancer drug of hydrophobic DOX was trapped into nanogels to acquire DOX-loaded BPDNs (D-BPDNs) at PBS (pH 7.4). After dialysis and freezedrying, DLS analysis showed that the average diameter of DBPDNs was about 100 nm and the zeta potential was −15.9 mV (Figure 3B). Based on fluorescence of FITC-labeled BPDNs and DOX, the CLSM images revealed that DOX (red fluorescence) was encapsulated into nanogels (green fluorescence) with completely overlapping fluorescence. It is worth mentioning that the maximum DOX loading capacity of BPDNs reached up to 16.8% (w/w), much higher than that of single low-generation PDs (7.3%, w/w). Such a high DOX loading capacity of BPDNs indicated that disulfide crosslinkings of low-generation PDs efficiently expanded dendrimeric voids for robust drug entrapment. SEM and TEM images showed that D-BPDNs also exhibited as well-defined nanoparticles (∼100 nm, Figure 3C), which was consistent with DLS results. The in vitro release profiles showed that 10 mM GSH contained in PBS triggered the rapid release of encapsulated DOX, and the half-life of drug release was only 5 h (Figure 3D). However, the drug release amount was just around 20% after 48 h with 10 μM GSH and without GSH. The redoxresponsive DOX release should be ascribed to the disintegration of cross-linked networks and a drastic decrease in payload. These results proved that BPDNs indeed could encapsulate the drug for transportation effectually, but BPDNs would be destroyed at high reductive conditions (10 mM GSH) for drug delivery. Therefore, BPDNs were alternative candidates for intracellular site-specific delivery of anticancer drugs.
together, these results suggested that our strategy succeeded in cross-linking of the low-generation PDs into nanogels with dendrimeric networks. Redox Sensitivity of BPDNs. To investigate the stimuliresponsiveness of BPDNs, we monitored the size of our nanogels under different conditions. In PBS (pH 7.4) without reducing agents, BPDNs still maintained approximately 100 nm in diameter after 12 h incubation (Figure 2A), suggesting the
Figure 2. Redox sensitivity of BPDNs. (A) Size variations of BPDNs in response to the different GSH concentrations at the preset time points as determined by DLS. (B) Turbidity changes of BPDN solutions (500 μg mL−1) as a function of different GSH concentrations as monitored by measuring the transmittance at 550 nm (means ± standard deviation (SD), n = 3). Inset: photographs of BPDN dispersions in 10 mM GSH at the time points of 0 and 12 h.
excellent stability of BPDNs under normal physiological condition. In the meantime, the nanogels could conserve size stability in an aqueous solution for 1 week (Figure S12). The stable and well-defined nanostructure could hopefully contribute to tumor passive targeting via enhanced permeability and retention (EPR) effects. In the presence of 10 mM GSH mimicking the tumor intracellular reductive conditions,34 the size of BPDNs quickly increased into about 250 nm within 0.5 h due to disruption of cross-linked network in BPDNs. Along with the extension of time, the nanogels swelled into large aggregations with size of about 1 μm within 10 mM GSH for 12 h, which upon dilution to a low concentration dispersed into a small size (Figure S13 and Figure S14).35 In contrast, little change in the size of the nanogels could be observed with the existence of 10 μM GSH analogous to the tumor extracellular environment. In addition, no obvious size variation on disulfide-omitted nanogels (the synthesis and characterization of cross-linkers without disulfides can be found in the Supporting Information) could be observed in the presence of 10 mM GSH, supporting the disulfides playing an important role in the fabrication of stimuli-responsive nanogels (Figure S15). 3502
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
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Biomacromolecules
Figure 3. Molecular encapsulation and stimuli-responsive release. (A) Pyrene fluorescence emission spectra and I372/I383 ratios in water with or without BPDNs (20 μg mL−1) after 12 h incubation with different concentration of GSH (0, 10 μM, or 10 mM). Excitation wavelength: 330 nm (n = 3). (B) Size distribution in an aqueous solution of D-BPDNs. Inset: fluorescence microscopy images for FITC-labeled D-BPDNs, including FITCBPDN (green) channel, DOX (red) channel, and the overlay (yellow). (C) SEM and TEM images of D-BPDNs. (D) In vitro release profiles of DBPDNs in PBS (pH 7.4) containing different GSH concentration at 37 °C (means ± SD, n = 3).
Figure 4. In vitro antitumor treatment of D-BPDNs. (A) Cell viability of 4T1 tumor cells after incubation with BPDNs, DOX, DOX·HCl, or DBPDNs for 24 h, respectively (means ± SD, n = 5). (B) Flow cytometric profiles of 4T1 tumor cells after exposure to BPDNs, DOX, DOX·HCl, or D-BPDNs for 3 h. (C) CLSM images for 4T1 tumor cells after treatment with FITC-labeled D-BPDNs for 1, 3, or 6 h, including FITC-labeled BPDN channel (green), DOX channel (red), LysoTracker-stained lysosome channel (blue), and overlay of previous images. (D) Cell viability of 4T1 tumor cells pretreated with 10 mM or without GSH-OEt after incubation with the different concentrations of D-BPDNs (means ± SD, n = 5, *p < 0.05, **p < 0.01). 3503
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
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Figure 5. In vivo antitumor efficacy of D-BPDNs. (A) Tumor volume variation and (C) relative body weight of BALB/c mice bearing 4T1 tumor treated with saline, BPDNs, DOX·HCl, or D-BPDNs by intravenous injection (5 mg DOX kg−1 body weight, means ± SD, n = 5, *p < 0.01). (B) Tumor and (D) heart weights of BALB/c mice bearing 4T1 tumor after 18 d treatment (means ± SD, n = 5, *p < 0.01). (E) Histological images of H&E stained tumor and heart tissues after 18 d treatment.
In Vitro Cytotoxicity and Cellular Uptake. Following the confirmation of the redox-triggered release of D-BPDNs, we turned to determine their in vitro antitumor activity against 4T1 tumor cell line by CCK-8 assay. As shown in Figure 4A, bioreducible nanogels markedly improved antitumor effects of DOX to 4T1 tumor cells, and BPDNs down-regulated the IC50 value of DOX (the concentration causing 50% growth inhibition, 5.08 μg mL−1) into 2.04 μg mL−1. BPDNs showed no obvious cytotoxicity to 4T1 tumor cells and mouse fibroblast 3T3 cells (Figure S16) even at a high concentration, supporting favorable biocompatibility of bioreducible crosslinkings of low-generation peptide dendrimers. The high antitumor efficiency of D-BPDNs was probably due to enhanced bioavailability of poorly soluble drugs. Next, we utilized fluorescence sorting to quantify the drug internalization of D-BPDNs. After 3 h incubation with different formulations, the fluorescence intensity of the D-BPDN group was stronger than that of the DOX group as determined by flow cytometry measurement (Figure 4B and Figure S18), supporting that BPDNs could facilitate the cellular uptake of antitumor drugs. Afterward, the intracellular delivery of D-BPDNs in 4T1 tumor cells was monitored by CLSM imaging. After exposure to the FITC-labeled D-BPDNs for 1 h, DOX fluorescence (red) were colocated with the green fluorescence of FITClabeled BPDNs (Figure 4C), highly overlapping with LysoTracker-stained lysosomes (blue). When the incubation time was increased to 3 h, a small portion of red DOX fluorescence disassociated with the FITC-labeled BPDNs and dispersed in the cytoplasm. After 6 h incubation, major red and green fluorescence no longer overlapped with blue lysosome, and red fluorescence widely distributed in the cytoplasm. These phenomena manifested that D-BPDNs smoothly transported the drug into tumor cells, escaped from the lysosome, and accomplished redox-responsive drug delivery. To clarify the importance of bioreducible features in drug delivery, the 4T1 tumor cells were pretreated with glutathione monoethyl ester (GSH-OEt, 10 mM) for 2 h to elevate intracellular reducing
condition as well as accelerate DOX release.38 As shown in Figure 4D, our D-BPDNs provided much more efficient inhibition activity to GSH-OEt pretreated 4T1 tumor cells, since accelerating cleavage of cross-linked networks would rapidly export more antitumor drugs to suppress tumor growth. In Vivo Antitumor Efficacy of D-BPDNs. To evaluate in vivo antitumor efficacy of D-BPDNs, BALB/c mice bearing 4T1 tumor were used as animal models following intravenous injection for 4 times (5 mg DOX kg−1). During the course of the treatment, the tumor volumes were recorded in Figure 5A. After a treatment course, the tumor volume in the salineadministered group (∼800 mm3) sharply increased to over 2fold that of the D-BPDN-administered group (∼350 mm3, *p < 0.01). Tumor inhibition efficiency of D-BPDNs was comparable to positive control of DOX·HCl. At the end of the treatment, the average tumor weight of the control group was about 4.5 times that of D-BPDN-treated group (Figure 5B, *p < 0.01), verifying successful tumor suppression of D-BPDNs. One of the major purposes in developing chemotherapy nanoplatforms is to reduce severe systematic toxicity and the damage on normal tissue. As shown in Figure 5C, about 10% decrease of body weight was observed in the DOX·HCl group (*p < 0.01), whereas BPDNs mitigated the DOX impact on the mouse body weight. Furthermore, cardiac weight loss notably occurred in the DOX·HCl group (Figure 5D), resulting from known DOX cardiotoxicity.39 It was concluded that bioreducible nanogels not only assisted the DOX to exert high antitumor activity in vivo, but also overcame the side effects of DOX chemotherapy. Finally, histological studies of tumor and heart slices were carried out to determine antitumor effects and heart injury. The D-BPDN administration caused remarkable damage in tumor tissue with loose structure and distinct karyopyknosis, while dense tumor cells were observed in the saline and BPDN treated groups (Figure 5E). The histological studies of heart slices showed that there were disordered and impaired cells in the DOX·HCl group, whereas no apparent lesions were 3504
DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
Article
Biomacromolecules
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observed in the D-BPDN group. Altogether, BPDNs successfully improved therapeutic effects and reduced side effects for tumor chemotherapy.
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CONCLUSIONS In summary, we have developed bioreducible dendrimer-based nanogels based on disulfide cross-linkings of low-generation peptide dendrimers for antitumor drug delivery. The peptide− dendrimeric nanogels offered well-defined nanostructures and abundant interior voids for drug encapsulation. The high GSH concentration corresponding to tumor intracellular condition was able to strongly destroy the networks of nanogels and rapidly release guest molecules. As expected, BPDNs were biocompatible and robust nanoplatforms to facilitate drug cellular uptake, endosomal escape, and stimuli-responsive delivery. In vivo antitumor evaluation suggested that our DBPDNs efficiently inhibited the tumor growth of BALB/c mice bearing 4T1 tumor, and reduced unwanted side effects in chemotherapy. We believe that this work will afford an alternative approaches to develop biocompatible, easily available, and stimuli-responsive dendrimer-based nanoplatforms for nanomedical applications.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00649. Materials and methods, experimental details, additional data, schemes, figures, and table (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Xianghui Xu: 0000-0002-8885-0848 Zhongwei Gu: 0000-0003-1547-6880 Author Contributions
D. Zhong and Z. Tu contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC, 81361140343, 51503128, 21674067, and 81621003), and Scientific Research Foundation for Outstanding Young Scholars in Sichuan University (2016SCU04A19).
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DOI: 10.1021/acs.biomac.7b00649 Biomacromolecules 2017, 18, 3498−3505
