Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular

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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 278−289

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Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in a Tumor Microenvironment Guihua Ye,† Yajun Jiang,† Xiaoying Yang,† Hongxiang Hu,‡ Beibei Wang,† Lu Sun,† Victor C. Yang,†,‡ Duxin Sun,‡ and Wei Gao*,†,‡

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†

Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China ‡ College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48108, United States S Supporting Information *

ABSTRACT: Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid−polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-L-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pHe: 7.0−6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pHendo: 6.5− 4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use. KEYWORDS: sequential drug delivery systems, pH sensitive, tumor target, poly-L-histidine, iNGR

1. INTRODUCTION

impediments limiting the antitumor efficacy of liposomal DOX as well as other kinds of nanoparticles.8 Especially, when poly(ethylene glycol) (PEG) was introduced to coat nanoparticles, a commonly used method to realize long circulation, the highly hydrophilic PEG corona with a large steric badly hinders the nanoparticle interaction with the negatively charged cell membrane and suppresses their cellular uptake.9,10 Moreover, despite some of the particles being successfully endocytosed by tumor cells, most of them are trapped in acid

Delivering chemotherapeutic agents by nanoparticles is considered to be a promising strategy for enhancing their therapeutic response and reducing the side effects. The first Food and Drug Administration-approved liposomal drug, PEGylated liposomal doxorubicin known as Doxil, was released in the market in 1995 and is being extensively used in clinic for the past 20 years.1 However, clinical data from different trials during these years reveal conflicting results on their therapeutic benefits over free doxorubicin (DOX); several clinical studies have demonstrated that PEGylated liposomal DOX does not exhibit advanced antitumor efficacy than free DOX in long-term treatments.2−7 Inefficient cellular uptake and intracellular drug release at the tumor site are two major © 2017 American Chemical Society

Received: October 20, 2017 Accepted: December 20, 2017 Published: December 20, 2017 278

DOI: 10.1021/acsami.7b15978 ACS Appl. Mater. Interfaces 2018, 10, 278−289

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of pH-triggered two-step phase transition of iNGR-LPNs (A) and the delivery of antitumor drug DOX (B). The DOX-loaded iNGR-LPNs were first accumulated in the tumor site driven by the electron paramagnetic resonance (EPR) effect and iNGR-specific binding to CD13 on tumor vessels. At the TME (pHe: 7.0−6.5), the particle undergoes first phase transition to facilitate nanoparticle uptake, simultaneously releasing a portion of encapsulated DOX; after internalization, at the acid endolysosome (pHendo: 6.5−4.5), the particle dissociates and induces endolysosome escape to release DOX into the cytoplasm; then free DOX enters into the nucleus, exerting cytotoxicity.

has an ability of endolysosome escape and shows a great enhancement of intracellular drug release.15 To solve the cellular uptake and intracellular delivery problems at the same time, several sequential or step-by-step drug-delivery systems have recently been developed.16 For instance, multistage pHresponsive liposomes containing a synthesized smart lipid (1,5dioctadecyl- L-glutamyl 2-histidyl-hexahydrobenzoic acid, HHG2C18) are prepared, which is capable of switching the surface charge from highly negative to positive at the TME to enhance the cellular uptake and sequentially causing endolysosomal bursting after endocytosis.17 The sequential drug-delivery systems exhibit promising efficacy in animal experiments; however, they often contain complex structures or require multiple steps or chemical synthesis in preparation, which are not appropriate for large-scale manufacture. In this study, we aim to solve both the cellular uptake and intracellular drug release problems using a simple-structured pH-sensitive nanoparticle. PHIS is employed in the nanoparticle as a pH-sensitive switch. PHIS is a widely used biomaterial with good biocompatibility and biodegradability.18 The imidazole groups in its side chain undergo protonation at weak acidic pH, allowing PHIS chain transits from the hydrophobic neutral to hydrophilic cationic form. When PHIS delivers drugs or biomacromolecules into the acid endolysosome, it can trigger endolysosome escape and release the cargoes into the cytoplasm.19 Recently, Iwasaki et al. have demonstrated that PHIS is also a highly efficient cellpenetrating peptide that can be applied as a vector to facilitate the cellular uptake of the cargoes.20

endolysosomes and unable to release the drugs into the cytoplasm.11 One of the most promising strategies to promote tumorspecific cellular uptake and intracellular drug delivery is to design “smart” bioresponsive nanoparticles, which undergo particle transition or drug release in response to the tumor microenvironment (TME) signals (redox potential, acid pH, or unique enzymatic activity).12,13 Acid- or enzyme-triggered deshielding of the PEG chain or layer at the TME has been proved to be better for cellular uptake.10 Charge conversional nanoparticles, which carry a negative charge for enhanced circulation and then transform to positive at the TME, are also considered to be an effective method to increase cellular uptake because surface charge crucially affects tumor-specific cellular uptake and intracellular delivery.10 For instance, the acidification of cancerous sites (pH of TME, pHe: 7.0−6.5) and the intracellular endolysosomes (pH of endolysosome, pHendo: 6.5−4.5) create an ideal pH gradient for controlling the delivery of drugs. Du et al. have prepared a surface chargeswitchable nanoparticle, which is negatively charged at physiological pH (∼7.4), but undergoes charge reversion from negative to positive at pHe to enhance cellular internalization.14 To facilitate intracellular drug delivery, Bae et al. have developed a micellar system using poly-L-histidine (PHIS), a special pH-sensitive material that can trigger endolysosome escape via the so-called “proton sponge” effect. The micellar system was fabricated by PHIS-PEG2000 and poly(L-lactide)-PEG2000, and the triggering pH was tailored to 6.0 by optimizing the ratio of the two polymers. The micelle 279

DOI: 10.1021/acsami.7b15978 ACS Appl. Mater. Interfaces 2018, 10, 278−289

Research Article

ACS Applied Materials & Interfaces

penicillin−streptomycin were purchased from Basal Media Ltd. (Shanghai, China). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Healthy female BALB/c nude mice (18−20 g, approximately 6 weeks old) were supplied by the Peking University Health Science Center. Before the test, the mice were kept at 25 °C and 55% of humidity for 1 week, with free access to standard water and food. All animal experiments conducted in this study were performed under the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2. Methods. 2.2.1. Preparation of LPNs. The LPNs were synthesized by a single-step nanoprecipitation method.26 In brief, PHIS (4.1 mg) was dissolved in 1 mL of DMSO as the organic phase. EPC and DSPE-PEG (7:3, molar ratio, totally 5.4 mg) were dissolved in Na2B4O7 solution (2 mM, pH 9.0) containing 4% ethanol and heated to 65 °C. The PHIS DMSO solution was then added into the preheated EPC/DSPE-PEG solution dropwise (1 mL/min) under violent stirring to obtain a mixture (DMSO/Na2B4O7 solution; 1:10, v/v), followed by continuous stirring for 2 h at room temperature to allow spontaneous self-assembly. Then, the solution was dialyzed against Na2B4O7 solution (2 mM) with cellulose ester membranes (molecular weight cutoff 3500 Da, Spectrum Medical Industries, Rancho Dominguez, CA) for 24 h. The final LPN solution was obtained and stored at 4 °C. The iNGR-modified LPNs were prepared with the same procedure, except replacing a portion of DSPE-PEG with iNGR-DSPE-PEG (DSPE-PEG/iNGR-DSPE-PEG = 7:3, molar ratio). To prepare the DOX-loaded LPNs, DOX·HCl (1 mg) was first reacted with TEA in DMSO for 2 h to eliminate HCl and then added dropwise to the PHIS solution under continuous stirring for another 2 h. The rest of the preparation process was the same as described above. For the in vivo imaging test, the near-infrared fluorescent probe DiR was loaded into LPNs or iNGR-LPNs. The preparation process was the same as that of LPNs, except for the addition of DiR into the PHIS solution. 2.2.2. Synthesis of iNGR-PEG-DSPE. To synthesize iNGR-PEGDSPE, iNGR was conjugated to the terminal NHS group of DSPEPEG2000-NHS, according to the previous reports.27 Briefly, iNGR and DSPE-PEG2000-NHS (1:1.5, molar ratio) were dissolved in anhydrous DMSO at pH 8.0−8.5 adjusted by trimethylamine (TEA). The reaction solution was continuously stirred under nitrogen gas for 5 days at 30 °C. After the reaction, the solution was dialyzed against deionized water for 48 h with cellulose ester membranes (molecular weight cutoff 3500 Da, Spectrum Medical Industries, Rancho Dominguez, CA) to remove the unreacted iNGR and DSPEPEG-NHS. The solution was then lyophilized and stored at −20 °C. The final product was confirmed by an Axima-CFR plus (Kratos Analytical Ltd, Manchester, UK) mass spectrometer. 2.2.3. Characterization of DOX-Loaded LPNs and iNGR-LPNs. 2.2.3.1. Particle Size Distribution and Zeta Potential. The particle size distribution and surface charge of LPNs and iNGR-LPNs were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK) at 25 °C. The morphology of LPNs and iNGR-LPNs was observed by transmission electron microscopy (TEM) (Hitachi HT7700, Japan). The TEM samples were prepared by adding the iNGR-LPN solution onto a carbon-coated copper grid, which was stained for 10 min with phosphotungstic acid solution (2%) at room temperature and dried in the air. 2.2.3.2. Encapsulation and Loading Efficiencies (EE % and LE %). The dialysis method was used in the preparation of LPNs to remove free DOX, as described in section 2.2.1. The amount of DOX after dialysis was measured to calculate the EE % and LE %. The concentration of DOX was quantified by an ultraviolet−visible spectrophotometer (Hitachi, Japan) at 488 nm. The EE % and LE % of the LPNs and iNGR-LPNs were calculated according to the following formulas

The modification of tumor-specific antibody or ligand on the surface of nanoparticles, known as the active-targeting strategy, improves the tumor accumulation of nanoparticles.21 Tumorpenetrating peptide iNRG (CRNGRGPDC) is a recently developed target moiety. iNRG contains two sequence motifs: a tumor-homing motif NRG that binds to CD13 on tumor vessels and tumor cells; a cryptic CendR motif that induces greater tumor penetration of coupled nanoparticles.22 Previous research from several individual groups demonstrated that iNRG enhances tumor accumulation and penetration of its anchored nanoparticle.23−25 Herein, we designed a smart lipid−polypeptide hybrid nanoparticle (LPN) with a simple structure, which was successfully prepared via a single-step nanoprecipitation method, potentially suitable for scale-up.26 As illustrated in Figure 1, the LPN consists of a PEGylated lipid monolayer shell (for long circulation and good biocompability) and a pHsensitive PHIS core (for pH responsive) and loaded with antitumor drug DOX. A tumor-specific ligand iNGR was modified on the surface of the particles for tumor targeting. Particularly, we observed that the LPN undergoes a two-step phase transition at the tumor acid pH: (i) at the TME (pHe: 7.0−6.5), the protonated segments of the polyhistine chain moves to the surface of the particle, the particle swells, and its surface potential turns from negative to neutral or positive, facilitating the cellular uptake; simultaneously, a portion of encapsulated DOX releases from the particle, further increasing the cellular uptake. (ii) after internalization, at acid endolysosome (pHendo: 6.5−4.5), the particle dissociates and induces endolysosome escape to release drugs into the cytoplasm. To evaluate the therapeutic efficacy of iNRGmodified LPN (iNRG-LPN), we established the 4T-1 murine breast tumor model that represents the late-stage aggressive breast carcinoma in clinic, poorly responding to traditional chemotherapy such as free DOX treatment. By using the tumor model, we compared the cellular uptake, cytotoxicity, in vivo antitumor efficacy, and systemic toxicity of DOX-loaded iNRG-LPNs with free DOX to evaluate the improvements of therapeutic response.

2. MATERIALS AND METHODS 2.1. Materials. PHIS [Mw: 3035, >95% by high-performance liquid chromatography (HPLC)] was synthesized by GL Biochem Co., Ltd (Shanghai, China). CRNGRGPDC (iNGR, 97.62% by HPLC) was synthesized by ChinaPeptides Co., Ltd. (Shanghai, China). DSPE-PEG (PEG, Mw: 2000), DSPE-PEG-NHS (PEG, Mw: 2000), egg yolk phosphatidylcholine (EPC), and cholesterol were purchased from Advanced Vehicle Technology Pharmaceutical Co., Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). 1,1-Dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Triethylamine (TEA) was purchased from Shanghai Titanchem Co., Ltd. (Shanghai, China). Na2B4O7·10H2O and anhydrous dimethylsulfoxide (DMSO) were purchased from Aladdin Industrial Corporation (Qingdao, China). All other agents used in this study are of analytical grade. The mouse mammary tumor cell line (4T1) was obtained from the Institute of Basic Medical Science at the Chinese Academy of Medical Science (Beijing, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% penicillin−streptomycin at 37 °C with 5% CO2 in a humidified environment. DMEM and FBS were provided by Biological Industries USA, Inc. (Connecticut, USA). Trypsin−ethylenediaminetetraacetic acid (EDTA) solution and 280

DOI: 10.1021/acsami.7b15978 ACS Appl. Mater. Interfaces 2018, 10, 278−289

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ACS Applied Materials & Interfaces

2.2.8. In Vivo Imaging Test. The biodistributions of LPNs and iNGR-LPNs were determined using an in vivo image method. First, 4T1 cells were subcutaneously inoculated into the right flanks of female BALB/c nude mice. When the tumor volume reached circa 100 mm3, each mouse was injected via the tail vein with near infrared fluorescence probe DiR-loaded LPNs or iNGR-LPNs (0.4 μg/100 μL). At each time point (1, 3, 6, 8, 20, 24, 36, and 48 h), the mice were anesthetized with isoflurane, and the fluorescent and X-ray images were captured by an in vivo imaging system (FX Pro; Kodak, Rochester, NY, USA). After 48 h, the mice were killed. The tumors and major organs were excised and imaged. 2.2.9. In Vivo Antitumor Efficacy and Systemic Toxicity Tests. The in vivo test was conducted on BALB/c mice bearing 4T1 murine mammary. First, 4T1 cells with the number of approximately 1 × 106 for each mouse were subcutaneously inoculated in the right flanks of female BALB/c mice. When the tumors reached about circa 100 mm3, the mice were randomly divided into six groups (n = 8) and administrated via the tail vein with one of the following regimens on day 1, 4, 7, 11, and 14: group 1 = PBS (pH 7.4); group 2 = free DOX (5 mg/kg in DOX); group 4 = LPNs (10 mg/kg in DOX); and group 5 = iNGR-LPNs (10 mg/kg in DOX). Group 1 served as the control group. The survival rate of the animals (n = 6, minus the two mice scarified for the histological analysis) was calculated for each group until all animals were dead. The survival curve was drawn, and the median survival time of each group was calculated using the Log rank test with GraphPad V5.0. The mice weight was measured every other day after the treatments. For histological analysis, we randomly chose two mice from each group and sacrificed them on day 25 to collect the tumor and heart for histological section. The heart sections were stained with hematoxylin and eosin (H&E) to evaluate the DOXrelated cardiotoxicity. For the tumor sections, CD31 stain was performed to measure the tumor microvessel density, and Ki 67 stain was used to evaluate the tumor cell proliferation. All histopathologic specimens were observed via light microscopy (Wuhan Goodbio Technology Co., Ltd., Wuhan, China). 2.2.10. Statistical Analysis. All tests were performed at least three times, unless otherwise specified. Data were reported as means ± standard deviation, unless otherwise specified. The differences among groups were evaluated by a two-tailed Student’s t test analysis. A p value less than 0.05 or 0.01 was considered to be statistically significant or highly significant.

EE % = (the amount of DOX in LPNs afte dialysis /the amount of DOX input) × 100%

LE % = (thethe amount of DOX in LPNs after dialysis /the weight of DOX loaded LPNs) × 100% 2.2.4. Observation of pH-Induced Phase Transition of the LPNs. The variation of pyrene fluorescence that was loaded into the LPNs can reflect the dissociation process of the LPNs.28 Test tubes were first filled with pyrene acetone solution and dried under N2, avoiding light. LPN solution was added to the tubes and sonicated for 30 min and placed at room temperature for 1 h. HCl solution was added dropwise to adjust the pH from 7.5 to 4.0. At different pH values, the fluorescence emission spectrum of pyrene was measured with excitation wavelength set at 339 nm. The intensity ratio of the first and third peaks (I373/I383) in the emission spectrum was recorded. The phase-transition process of the LPNs with pH can also be tracked by measuring the variation of zeta potential, size distribution, and morphology. HCl solution was added dropwise to the LPN solution to adjust the pH. At each preset pH, the zeta potential and size of the particles were measured by the DLS analysis using a Malvern Zetasizer Nano ZS (Malvern, UK) at 25 °C, and the morphology of the particles were observed by TEM (Hitachi HT7700, Japan). 2.2.5. In Vitro Drug Release Test at Different pH Values. The DOX release profile from the LPNs was tested by a dialysis method. Briefly, LPNs and iNGR-LPNs were added into a dialysis bag (3500 Da, Shanghai Jinsui Bio-Technology Co., Ltd) and placed into 30 mL of phosphate-buffered saline (PBS) solution (10 mM, pH 7.4, 6.5 or 5.0) as the dialysis medium under continuous shaking at 37 °C for 24 h. At each time point (0.5, 1, 2, 4, 8, 12, and 24 h), 1 mL of the outer phase was withdrawn and replaced with an equal volume of the dialysis medium. The concentration of DOX was determined by an HPLC system (Agilent 1260 Infinity, USA) with an octadecylsilica column (Agilent ZORBAX SB-C18, 4.6 × 250 mm, 5 μm) and an ultraviolet detector at 488 nm. The flow rate was 1 mL/min. The mobile phase consisted of water [1.44 g of sodium dodecyl sulfate and 0.68 mL of H3PO4 in 500 mL of deionized water], methanol, and acetonitrile (40:5:50, v/v). 2.2.6. Flow Cytometry Analysis. The cellular internalization of various DOX formulations was analyzed by flow cytometry. 4T1 cells (approximately 1 × 105) were seeded into 12-well plates and incubated for 24 h. Then, the medium was replaced with the culture medium (pH 6.8) containing free DOX, DOX-loaded LPNs, or iNGR-LPNs at the concentration of 5 μg/mL DOX. After 2 h of incubation, the cells were harvested by trypsin−EDTA, washed three times with cold PBS solution, and examined by a FACScan flow cytometer (BD FACSVerse, USA) with excitation and detection wavelengths set at 488 and 575 nm, respectively. The number of collected cells was over 10 000 for each sample. 2.2.7. In Vitro Cytotoxicity Assays. The in vitro cytotoxicity of various DOX formulations against 4T1 cells was evaluated by the MTT assay. Briefly, a volume of 100 μL of cell suspension (approximately 8000 cells) was added into 96-well plates and incubated for 24 h. The cells were treated with different concentrations of free DOX, DOX-loaded LPNs, or iNGR-LPNs (DOX concentrations from 0.01 to 10 μg/mL diluted by DMEM at pH 6.5). After 24 h of incubation, the cells were washed three times with PBS. A volume of 100 μL of DMEM containing 20 μL of MTT (5 mg/mL in PBS) was added into each well and incubated for another 4 h, avoiding light. Finally, the medium was replaced by 200 μL of DMSO, and the plate was shaken via an IS-RDD3 Thermostat oscillator (Texas, USA) at 100 rpm for 10 min. The absorbance of each well was measured at 570 nm by a Thermo Multiskan GO microplate reader (Thermo, UK). The cell inhibition rate was calculated as the percentage of cell viability compared to the control group; and the concentration of DOX causing 50% cell inhibition (IC50) was calculated for each group by the linear regression analysis.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of DOXLoaded iNGR-LPNs. The LPNs were successfully prepared via a single-step nanoprecipitation method, also being called as the modified nanoprecipitation method, which was previously used to prepare the similar-structured polymer core−lipid shell nanoparticles.26,29 This method avoids time-consuming preparation steps, having advantages for large-scale manufacture.26 Briefly, lipids (EPC and DSPE-PEG) were dispersed in Na2B4O7 solution at 65 °C to ensure proper dispersion. The PHIS and DOX solutions were then added into the preheated EPC/DSPE-PEG solution dropwise under violent stirring. As the solubilities of PHIS and DOX are low in Na2B4O7 solution, they precipitate together to form the hydrophobic core. Simultaneously, lipids (EPC and DSPE-PEG) predispersed in Na2B4O7 solution self-assemble around the hydrophobic core to form a lipid monolayer covered by a PEG shell; the process is driven by the hydrophobic interaction between the hydrophobic chain of the lipids and the core (Figure 2A).29,30 The mixture was then dialyzed in Na2B4O7 buffer (pH 9.0) to remove DMSO and free DOX that were not encapsulated into the particles. The Na2B4O7 buffer was used as the dialysis medium, according to the previous reports on preparation of polyhistine-based nanoparticles.30,31 We compared the Na2B4O7 buffer with the PBS buffer; the drug281

DOI: 10.1021/acsami.7b15978 ACS Appl. Mater. Interfaces 2018, 10, 278−289

Research Article

ACS Applied Materials & Interfaces

Figure 2. Preparation and characterization of DOX-loaded LPNs. (A) Schematic illustration. The particle size distributions (B) and the typical TEM image (C) of the optimized DOX-loaded LPNs formulation. Influence of PEGylation and lipid-to-PHIS ratio (D1) on the size (D2) and pH sensitivity (D3) of the LPNs. F1, F2, F3, F4, F5, and F6 represent six formulations of LPNs. For each formulation, the ratio of DOX to the total weight of materials is 1:9.5.

loading efficiency and stability was higher by using Na2B4O7 buffer than by using the PBS buffer. When used in the body, we either diluted the nanoparticle solution or changed the medium to PBS or saline by ultrafiltration. The formulation of DOX-loaded LPNs was optimized as DSPE-PEG 3.2 mg/mL, EPC 2.2 mg/mL, PHIS 4.1 mg/mL, and DOX 1 mg/mL. The obtained LPNs were around 100 nm with a narrow size distribution and were negatively charged at pH 7.4 (Figure 2B and Table 1). The representative TEM photograph shows a typical spherical shape (Figure 2C). DOX was encapsulated into LPNs with high EE (84.59% ± 2.19%) and LE (9.08% ± 0.33%) (Table 1). PEGylation and lipid-to-PHIS mass ratio are two important factors for the formation of ideal LPNs. As shown in Figure 2D2, the formulations (F1, F2, and F3) without the addition of DSPE-PEG show an obvious aggregation, as indicated by the large average diameter (620−740 nm) and a wide size distribution (PDI: 0.56−0.95). An increase in the total lipid (EPC and DSPE-PEG) to PHIS mass ratio from 25 to 75% does not prevent the aggregation, which demonstrates that even a thick lipid layer to cover the hydrophobic core is insufficient to stabilize the LPNs. On the contrary, when

DSPE-PEG (30% of total lipid) was added into the formulations, the size of LPNs (F4, F5, and F6) turned into 100−200 nm with a narrow size distribution. This indicates that surface PEGylation plays a crucial role in stabilizing LPNs from aggregation, thus resulting in smaller and uniform nanoparticles. Next, we tested the pH sensitivity of F4, F5, and F6 by measuring the change of zeta potential. As shown in Figure 2D3, all three formulations show a negative charge at pH 7.4, suggesting a good coverage of the PHIS core by the PEGylated lipid membrane. However, only F6 (total lipid− PHIS = 1:3, mass ratio) shows an obvious charge reversal at pH ∼6.5, whereas F4 and F5 with a thicker lipid layer (total lipid−PHIS mass ratio = 1:1 or 3:1, mass ratio) merely undergo a slight potential increase with pH and remain negative at pH 4.5. The results suggest that the pH-sensitive property of LPNs requires a proper thickness of the lipid layer to make a good cover of the hydrophobic core at physiological pH, while at the same time allowing the remolding by protonated PHIS at acidic pH. 3.2. pH-Stimulated Phase Transition of LPNs. Most of the reported PHIS formed pH-sensitive micelles or nanoparticles that undergo pH-stimulated transition at pH 6.5−4.5 (the pH of endolysosome, pHendo) and exert the proton sponge effect to facilitate the endolysosome escape.15,32 Herein, the LPNs hold a special two-step phase transition property at acidic pH; thus, not only it can induce endolysosome escape after endocytosis but also facilitate the cellular uptake of nanoparticles at the TME, as illustrated in Figure 1A. To prove the hypothesis, we first monitored the pyrene fluorescence intensity variation when pH drops. The pyrene probe was encapsulated into the LPNs. Pyrene can sense the micropolarity change inside of the LPNs and reflect the dissociation of the particles via the intensity ratio of the first and third peaks (I373/I383) in the emission spectra profile.28 As shown in Figure

Table 1. Characterization of DOX-Loaded LPNs and iNGRLPNs (n = 3)

size (nm, Z-average) polydispersity index (PDI) zeta potential (mV) EE (%) LE (%)

empty LPNs

LPNs

iNGR-LPNs

114.59 ± 6.06 0.26 ± 0.07

124.14 ± 8.44 0.28 ± 0.05

116.73 ± 4.18 0.25 ± 0.02

−7.99 ± 0.43

−7.96 ± 1.34 84.59 ± 2.19 9.08 ± 0.33

−8.12 ± 1.58 87.98 ± 3.62 7.64 ± 0.70 282

DOI: 10.1021/acsami.7b15978 ACS Appl. Mater. Interfaces 2018, 10, 278−289

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ACS Applied Materials & Interfaces

Figure 3. pH-stimulated two-step phase transition of LPNs. (A) Change of the pyrene fluorescence intensity (I373/I383) with pH. Pyrene was preloaded into LPNs. (B) Zeta potential of LPNs at different time points at pH 5.5, 6.5, and 7.4. (C) Size distributions and TEM images (D) of LPNs at pH 5.5, 6.5, and 7.4. The scale bar is 200 nm.

3A, the I373/I383 value is consistent at pH 7.4−6.5 but undergoes a sharp decline at pH 6.5−4.5. The result demonstrated that the LPNs remained integrated at pH 7.4− 6.5, but gradually dissociated at a pH lower than 6.5. To further prove the nanoparticle’s phase change, we tested the size distribution and morphology of LPNs at pH 7.4, 6.5, or 5.5. As shown in Figure 3C,D, at pH 7.4, the LPNs remained stable as uniform spherical particles with approximately 100 nm in diameter. When the medium pH dropped to 6.5, the pH of the tumor environment (pHe), the average diameter of the particles increased to 141 ± 32 nm. From the TEM image, we can see that the particles remain in spherical shape but with a relatively larger size and a less uniform distribution at pH 6.5 than that at pH 7.4. When the medium pH dropped further to 5.5 (pHendo), a nonuniform size distribution was observed; large particles as well as irregular aggregations are seen in TEM images (Figure 3C). The results indicate that LPNs dissociate after swelling at pH 5.5. Interestingly, as shown in Figure 2D3 (line F3), while slowly adding hydrogen ions into the medium, the potential of the LPNs showed a sharp increase at pH 7.4−

6.5 and converted to positive at around pH 6.5 (pHe) and then continuously increased until pH 4.5. We conducted the same experiment on a DOX-liposome, which had no pH-stimulated property. As expected, the zeta potential of the DOX-liposome remained consistent when pH dropped (Figure S1). To determine whether the phase transition of the LPNs is a quick or gradual process, we tested the particle potential changes with time. The zero point was recorded as soon as the LPNs were added to the buffer at pH 7.4, 6.5, or 5.5. As shown in Figure 3B, the potential of the particle changes at the first time point at pH 6.5 or 5.5, indicating a very fast transition process. At pH 6.5, the potential remains stable after the transition; this further proves that the particle can remain in this stage until pH further drops. To summarize, the results suggest that the LPNs undergo a two-step phase transition at acidic pH, as illustrated in Figure 1A. First, at pH 7.0−6.5 (pHe), PHIS in the core of the LPNs partially protonates and turns from hydrophobic to amphiphilic, which drives the particle swelling. Simultaneously, the hydrophilic and cationic part of the PHIS chain moves to the 283

DOI: 10.1021/acsami.7b15978 ACS Appl. Mater. Interfaces 2018, 10, 278−289

Research Article

ACS Applied Materials & Interfaces

Figure 4. Modification of LPNs with iNGR. (A) Synthesis scheme of DSPE-PEG-iNGR; (B) matrix-assisted laser desorption ionization time-offlight mass spectrum of DSPE-PEG-iNGR.

surface of the particle, resulting in an increase in the zeta potential. The LPNs do not dissociate at this pH because of the protection of the lipid layer. Second, at pH 6.5−4.5, the PHIS completely protonates and interrupts the structure of the lipid layer, making the LPNs dissociated. We hypothesized that the special two-step pH-responsive phase transition property of LPNs may facilitate the cellular uptake and intracellular drug release at the tumor site. We conducted the following cellular uptake and cytotoxicity test to prove the hypothesis. 3.3. Modification of LPNs with the iNGR Ligand. To further improve the tumor accumulation, iNRG was modified on the surface of LPNs. First, iNRG was reacted with NHSDSPE-PEG to form the iNGR-DSPE-PEG conjugate (Figure 4A). The final product was identified by an Axima-CFR plus (Kratos Analytical Ltd, Manchester, UK) mass spectrometer. As shown in Figure 4B, the peak of 3961 indicates the molecular weight of iNGR-DSPE-PEG. The DOX-loaded iNGR-modified LPNs (iNGR-LPNs) were prepared by a protocol similar to that of DOX-loaded LPNs, except substitution of a portion of DSPE-PEG with iNGR-DSPEPEG. As shown in Table 1, compared to nonmodified LPNs, the iNGR-LPNs exhibit a similar size distribution, potential, as well as EE and LE. 3.4. pH-Stimulated Drug Release of LPNs. The in vitro drug release test of DOX-loaded LPNs, iNGR-LPNs was performed at pH 7.4, 6.5, and 5.5 for 24 h. As shown in Figure 5A, iNGR-LPNs show a pH-stimulated drug release profile. At pH 7.4, DOX gradually and slowly releases from iNGR-LPNs, and the accumulated released drug is only 19.32% during 24 h. On changing the medium pH to 6.5 (pHe), a quick release of drug is observed in the first 4 h and 53.83% of the drug is released during 24 h, which can be attributed to the swelling of the nanoparticle, as described in part 3.2. The nature of the DOX molecules also contributes to the drug release here. Because DOX is loaded into the LPNs in the form of a hydrophobic DOX base, it protonates and turns into a more

Figure 5. pH-dependent drug release of LPNs. The profiles of DOX released from iNGR-LPNs (A) and LPNs (B) at pH 5.5, 6.5, and 7.4.

hydrophilic molecular at a slight acidic pH, making it more easily to release from the swelling particles. On further decreasing the medium pH to 5.5 (pHendo), iNGR-LPNs show 284

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ACS Applied Materials & Interfaces a burst drug release in the first 2 h (over 50% of drugs released), and over 80% of the drug is released at 24 h because of the particle dissociation. Additionally, LPNs exhibit a similar pH responsive release profile, as shown in Figure 5B, indicating that the modification of iNGR does not influence the pHsensitive releasing property of the nanoparticles. 3.5. Cellular Uptake of Various DOX Formulations. The cellular uptake of free DOX and DOX-loaded LPNs, iNGR-LPNs was evaluated by flow cytometry analysis on breast tumor 4T-1 cells. We chose the 4T-1 cell line because it represents the late-stage aggressive breast carcinoma in clinic, which is difficult to treat in clinic by conventional chemotherapeutics. Moreover, it overexpresses the aminopeptidase N (APN/CD13) receptor that can be recognized by iNGR. The pH of the culture medium was adjusted to 7.4 or 6.8 to simulate the physiological or TME. As shown in Figure 6A−C, by comparing the cellular uptake of LPNs at pH 7.4 and 6.8, we can conclude that the cellular uptake of LPNs at pH 6.8 is significantly higher than that at 7.4 (P < 0.001), indicating the effect of acid-triggered enhancement of cellular uptake at the tumor site. Moreover, the internalization of iNGR-LPNs is about 1.5 times of LPNs at pH 7.4. Because iNGR-LPNs and LPNs have the same formulation expect the modification of iNGR, the enhanced cellular uptake is attributed to the interaction of iNGR with the CD31 receptor on the tumor cell surface. The iNGR-LPNs at pH 6.8 show the highest cellular uptake among all groups, indicating a combined effect of pHtriggered and receptor-mediated cellular uptake. The acidtriggered increment of cellular uptake at the TME might be attributed to the phase transition of the LPNs that induced multiple changes of the systems. First, the protonated segments of the polyhistine chain moved to the surface of the particle, turning the particle potential from negative to neutral (as shown in part 3.2), which might decrease the electrical repulse between the negatively charged particles and the cellular membrane, thus facilitating the nanoparticle’s internalization. Also, the polyhistine moving to the surface of the particles pocesses the cellular-penetrating property, which may further increase the cellular uptake.20 On the other hand, a portion of DOX base protonates at acid pH, leaks from the particle as demonstrated in the drug-release study (part 3.3), and passively diffuses across cell membranes, which may also add to the increment of the internalization. Surface PEG hinders the nanoparticle internalization, limiting the antitumor efficacy of nanoparticles. Herein, the iNGR-LPNs can profoundly improve the tumor-specific cellular uptake via acid-triggered phase transition and ligand−receptor interactions. 3.6. Cytotoxicity of Various DOX Formulations. The in vitro cytotoxicity assay of various DOX formulations, including free DOX, LPNs, and iNGR-LPNs was performed via an MTT assay against 4T-1 cells. DOX is a well-known chemotherapy medication used to treat cancer, and it can bind to DNA, thereby preventing DNA replication and ultimately inhibiting protein synthesis.33 As shown in Figure 6D and Table 2, iNGR-LPNs show an IC50 value similar to that of free DOX. Free DOX can easily pass through cellular membranes via passive diffusion. Many reported DOX-loaded nanoparticles exerted less cytotoxicity against tumor cells than free DOX because of the inefficient cellular uptake and intracellular drug release.34 Herein, we demonstrated that the DOX-loaded iNGR-LPNs exhibited cytotoxicity similar to that of free DOX, indicating the efficient intracellular drug-delivery potential of the nanoparticle. Moreover, compared to LPNs, iNGR-LPNs

Figure 6. Cellular uptakes and cytotoxicity of DOX-loaded iNGRLPNs against 4T-1 cells. 4T-1 cells were incubated with free DOX, DOX-loaded LPNs, and iNGR-LPNs for 2 h at 37 °C. Flow cytometric curves of intracellular DOX at pH 7.4 (A) and 6.8 (B); (C) graphs of fluorescence intensity quantification of intracellular DOX for each group; (D) in vitro cytotoxicity assay. 4T-1 cells were incubated with different concentrations of free DOX, LPNs, and iNGR-LPNs for 48 h at 37 °C. *p < 0.05; **p < 0.01.

Table 2. IC50 Value of Different DOX Formulations against 4T-1 Cells (n = 6) LPNs iNGR-LPNs free DOX

IC50 (μg/mL)

95% confidence intervals

1.926 1.140 1.127

1.231 to 3.012 0.6417 to 2.026 0.5564 to 2.089

further increase the cytotoxicity; this is attributed to the target effect of iNGR. Totally, the results demonstrate that the pHsensitive iNGR-LPNs promote cellular uptake and intracellular drug delivery, thus exerting profound antitumor activity on 4T1 cells. 3.7. Targeted Delivery to 4T-1 Tumor In Vivo. To evaluate the tumor target delivery of LPNs and iNGR-LPNs, an in vivo fluorescence imaging test was performed on the 285

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model represents late-stage aggressive breast carcinoma in clinic, which is highly metastatic and responds poorly to traditional chemotherapy such as free DOX.36 In the previous research, an increment in DOX dosage was used to obtain therapeutic efficacy.36,37 Herein, mice in the free DOX group were treated with 5 mg/kg DOX each time, as it is the maximum tolerated dose (MTD) of free DOX.36 Nanoparticles alleviate the systemic toxicity of their encapsulated drugs, correspondingly increasing their MTD.18 According to the pre-experiment, mice treated with iNGR-LPNs or LPNs (10 mg/kg in DOX) did not show any obvious weight loss. Therefore, we used 10 mg/kg DOX in LPN and iNGR-LPN groups to improve the therapeutic responses. To evaluate the antitumor efficacy, the tumor volume and survival rate were recorded after the first treatments. As shown in Figure 8A,C, free DOX failed to inhibit the tumor growth and prolong the median survival time as compared to the PBS group. On the contrary, both iNGR-LPNs and LPNs showed significant suppression of tumor growth (p < 0.01 vs control and free DOX groups). As shown in Table 3, the median survival time of iNGR-LPNs (40 days) and LPNs (36 days) was extended for 9.5 and 5.5 days as compared to the control group (30.5 days). Moreover, tumor immunohistochemical studies of CD31 and Ki67 were conducted on day 33 that helps to determine the nature of the tumors. CD31 is an angiogenesis-related marker to identify tumor vascular. As shown in Figure 8D, the tumor microvessel density in iNGRLPN and LPN groups is significantly lower than that in free DOX and control group. Previous studies have confirmed that angiogenesis plays a central role in breast cancer progression, and high level of angiogenesis vascular in tumor tissue relates to poor prognosis.38 The decreased microvessel density in iNGR-LPN and LPN groups suggested a less progressive tumor and better prognosis of the treatments. Ki67 was a proliferation biomarker correlated with breast cancer prognosis.39 The density of Ki67-positive cells in iNGR-LPN and LPN groups is much lower than that in free DOX and control group, indicating that DOX-loaded LPNs have a lower proliferative activity of cancer cells and better prognosis of the treatment than free DOX. Altogether, the results suggest that pH-sensitive LPNs exert potent antitumor efficacy against late-stage aggressive breast carcinoma, which responds poorly to clinically available free DOX. Additionally, iNGR-LPNs present comparatively slower tumor volume growth and longer survival time than LPNs, indicating that modification of iNGR further promotes the antitumor efficacy of DOX-loaded LPNs. On the other hand, the drug-related toxicity was evaluated via mice weight variation during the treatment. As shown in Figure 8B, free DOX at the dosage of 5 mg/kg does not induce any obvious body weight loss. Although exceeding the MTD of DOX, LPN and iNGR-LPN groups (DOX: 10 mg/kg) have a constant animal weight after treatments. The cardiotoxicity of the various DOX formulations was further evaluated by observing the myocardial pathology on day 33 after injection for five times. Figure 8D shows the typical images of mice heart after H&E stain. No apparent pathological variations are observed in the control group. Free DOX causes the most severe myocardial lesions compared to other formulations, histologically classified as single cell necrosis, cytoplasmic vacuolation, large area of myofibrillar disorganization, and inflammatory infiltration. LPNs and iNGR-LPNs did not induce pronounced changes in heart tissues except minor inflammatory infiltration. In conclusion, the tumor weight and

female BALB/c mice bearing 4T-1 tumors. iNGR-LPNs or LPNs encapsulating a near-infrared fluorescent indicator DiR was administrated to the mice via the tail vein. Free DiR was tested as a control group, in which no signal was detected in the tumor tissues during the whole test (data not shown). In both iNGR-LPN and LPN groups, the fluorescent signal of tumors gradually intensifies during the first 20 h and remains constant until 48 h (Figure 7A), indicating a long circulation property and passive target effect induced by the PEGylated surface and EPR effect. Modification of iNGR on the surface of LPNs further improves the target efficiency to 4T-1 tumors, as seen in the picture. iNGR-LPNs show significant stronger fluorescence tumor accumulation than LPNs at each time point and in ex vivo images after 48 h (Figure 7B). Moreover, no fluorescent signal was detected in the heart section in both iNGR-LPN and LPN groups after 48 h. Free DOX distributes

Figure 7. In vivo fluorescent images. BALB/c nude mice bearing 4T-1 tumors were administrated with the fluorescent probe DiR-loaded LPNs and iNGR-LPNs via the tail vein and photographed by an in vivo imaging system. (A) Biodistribution imaging of LPNs and iNGRLPNs during 48 h; (B) ex vivo images of tumors and major organs (heart, liver, spleen, lung, and kidney) captured at the end of the test. The white arrows point out the tumor sites of the mice.

in the heart, inducing severe cardiotoxicity. The iNGR-LPNs or LPNs might reduce the cardiotoxicity via avoiding the drug distribution to heart.1 To summarize, LPNs effectively change the biodistribution of free DOX, presenting a special long circulation and tumorspecific distribution property and simultaneously avoiding heart distribution of the drug. CD13, the receptor of iNGR, is overexpressed on both tumor neovascular and 4T-1 cells.23 Hence, when iNGR-LPNs were administrated into mice via the tail vein, it might first recognize tumor neovascular, inducing a higher level of particle accumulation in the tumor site, then promoting the tumor-specific cellular uptake via CD13 receptor-mediated endocytosis along with the pH-triggered phase transition of the particles.35 3.8. In Vivo Antitumor Efficacy and Systemic Toxicity. For the in vivo test, DOX-loaded LPNs, iNGR-LPNs, free DOX, and PBS (control group) were intravenously injected into 4T-1 breast tumor bearing BALB/c mice. 4T1 tumor 286

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Figure 8. In vivo antitumor activity and systemic toxicity. 4T1 tumor-bearing BALB/c mice were intravenously administrated with PBS (pH 7.4), free DOX (5 mg/kg), LPNs/DOX (DOX: 10 mg/kg), and iNGR-LPNs (DOX: 10 mg/kg); tumor volume (A) and body weight (B) changes during the test; (C) survival curves; (D) typical images of the tumor tissue by Ki67 and CD31 stains and the mice heart after H&E staining. *p < 0.05; **p < 0.01.

efficacy of PEGylated nanoparticles. Herein, we provided a novel drug-delivery system to solve both of the problems and proved its potent antitumor efficacy against late-stage aggressive breast carcinoma, which responds poorly to clinical available free DOX. The excipients used in preparing LPNs are totally biocompatible. Also, the preparation of LPN was via a simply single-step nanoprecipitation method, which may have advantages in future manufacture for clinical trials and clinical use. For all these reasons, the LPN might serve as a promising vector for the delivery of antitumor agents.

Table 3. Median Survival Time of Different DOX Formulations median survival (days)

PBS

DOX

LPNs

iNGR-LPNs

30.5

31.5

36

40

myocardial pathology observation consistently demonstrate that DOX-loaded LPNs with or without iNGR modification exhibit less systemic toxicity than free DOX.

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4. CONCLUSIONS In this study, we designed a tumor-targeted smart LPN that holds a special two-step phase transition in the acidic tumor environment. The LPNs are around 100 nm with a narrow distribution and negative charge at pH 7.4. The in vitro studies demonstrated that the LPNs underwent two-step pHresponsive phase transition at the tumor site, facilitating particle internalization and intracellular drug release. The in vivo studies demonstrated that pH-sensitive LPNs exerted potent antitumor efficacy against late-stage aggressive breast carcinoma, and at the same time, it exhibited less systemic toxicity than free DOX. Further conjugating tumor-specific ligand iNGR on the surface of the LPNs improved the tumorspecific cellular uptake and in vivo accumulation, thus achieving better in vivo antitumor efficacy than nonmodified LPNs. Inefficient intracellular drug delivery and release at the tumor site are major clinical obstacles that limit the antitumor

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b15978. Zeta potential of DOX-loaded PEGylated liposomes at different pH values (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/Fax: 17347735423. ORCID

Victor C. Yang: 0000-0003-0971-5996 Wei Gao: 0000-0002-5233-3734 Notes

The authors declare no competing financial interest. 287

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ACKNOWLEDGMENTS This study was supported by the National Science Foundation of China grant 81402857. This project is partially supported by the National Science Foundation of Tianjin City (grant no 15JCZDJC36300) and the National Science Foundation of China A3 program (grant 813611403), in which W.G. is a participating faculty member.

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