Protein Nanoparticle Fabrication for Optimized Reticuloendothelial

Mar 7, 2019 - Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for ...
0 downloads 0 Views 5MB Size
Article Cite This: Langmuir XXXX, XXX, XXX−XXX

pubs.acs.org/Langmuir

Protein Nanoparticle Fabrication for Optimized Reticuloendothelial System Evasion and Tumor Accumulation Yeong Gon Roh,† Seung Won Shin,† Sun-Young Kim,‡ Sohyun Kim,‡ Yong Taik Lim,†,‡ Byung-Keun Oh,§ and Soong Ho Um*,†,‡ School of Chemical Engineering and ‡SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea § Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, South Korea Downloaded via EAST CAROLINA UNIV on March 7, 2019 at 21:36:49 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.



ABSTRACT: Nanoparticles (NPs) of protein-based materials have become one of the most promising candidates for drug carriers in drug-delivery systems because of their in vivo nontoxicity, biodegradability, compatibility with hydrophilic drugs, and adaptability to the human body. Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for these NPs to remain in the blood until they reach their therapeutic target to achieve the desired effect; the quicker the clearance of drugs from the body, the shorter is the residence time of drugs in the body, which eventually reduces drug efficacy. Macrophage uptake is a major mechanism for clearance of NPs from the body, so, reducing the degree of macrophage uptake is a major challenge in drug-delivery systems. Original studies of HSA NP uptake by macrophages showed that denatured HSA and HSA NPs synthesized with 80% (v/v) ethanol showed a high degree of macrophage uptake. We found that HSA NPs synthesized with lower ethanol content at pH 7 showed lower macrophage uptake in in vitro macrophage cellular uptake experiments. The effects of the preparation parameters of ethanol concentration, pH, and glutaraldehyde on the macrophage uptake of NPs were thoroughly studied. This newly developed protein NP with lower macrophage uptake has potential application as a drug carrier for many delivery systems.



pressure and to transport nutrients to cells.9 HSA is therefore intrinsically biocompatible and highly soluble in blood. Additionally, HSA has high specific affinity with drugs because of its amphiphilicity.10 Owing to these properties, HSA is widely used for many delivery systems in the form of NPs.11 To manufacture protein NPs, a combination of emulsion and desolvation methods are used. Bovine serum albumin NPs made by an emulsion method was established by Müller et al.11 There are, however, serious disadvantages to this emulsion method as removal of surfactants used in the procedure is a complex process. To solve this problem, a simple desolvation method for protein NPs was developed by Marty et al.12 and Weber et al.13 and finally optimized by Langer et al.14 In these desolvation methods, HSA NPs were synthesized by continuous addition of ethanol into an HSA solution, followed by cross-linking with glutaraldehyde. The sizes of the synthesized HSA NPs were in the target range of 100−200 nm. Problematically, HSA NPs synthesized by desolvation at 80% (v/v) ethanol avidly bound to macrophages.14 Although this property has advantages for other applications, it is greatly disadvantageous for targeting tumors for drug delivery because of clearance via the RES. Methods such as PEGylation have been used to prevent opsonization and RES;15−17 however,

INTRODUCTION Currently, cancer is one of the most malignant diseases. In the past few decades, several chemotherapies have been investigated and implemented to effectively treat cancers.1 Various drugs have been used as chemotherapy agents. However, drugs cannot be administered alone because of their unexpected side effects, sizes, and/or physicochemical properties, such as hydrophobicity and solubility. Therefore, to prevent side effects and increase drug efficiency, carriers are necessary for cancer treatment in vivo.2−6 There are some essential requirements for the drug carrier in a delivery system. To minimize side effects, the intrinsic biocompatibility of drug carriers is one of the principal consideration factors for drug delivery systems. To ensure accurate dosing, the shells of the drug-containing carriers should be soluble in blood.2−6 Carrier size is also critical for preventing premature clearance from the body. Nanoparticles (NPs) sized less than 20 nm are usually removed via renal clearance. NPs 20−100 nm in size will enter lymphatic capillaries, resulting in selective elimination from the body.7 NPs larger than 250 nm in size are easily removed by the reticuloendothelial system (RES),8 which operates by macrophage phagocytosis with the help of opsonization. Therefore, to avoid these clearance mechanisms, NP drug carriers 100− 200 nm in size would be ideal for delivery systems. Human serum albumin (HSA) with a molecular weight of 66 kDa is the most abundant protein in the circulatory system. The primary roles of HSA are to maintain blood osmotic © XXXX American Chemical Society

Received: November 9, 2018 Revised: February 19, 2019

A

DOI: 10.1021/acs.langmuir.8b03776 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Density Gradient Centrifugation of F-HSA NPs. To accomplish density gradient centrifugation of the F-HSA NPs, a sucrose density gradient solution with 12, 24, 36, 48, and 60 wt % sucrose solutions was prepared. After adding the F-HSA NP solution on top of the sucrose gradient solution prepared by ice-stacked sucrose gradient solution, the solution was centrifuged at 7000g for 120 min. The centrifuged sucrose gradient solution containing F-HSA NPs was frozen, and fluorescence images of the frozen solution were collected by a Fluorescence-Labeled Organism Bioimaging Instrument (FOBI, FM08-BR, NEO science, Korea). Characterization. The size and shape of the HSA NPs were visualized using transmission electron microscopy (TEM, JEM-2100F, JEOL, Japan) operated at 300 kV and scanning electron microscopy (SEM, JSM-7600F, JEOL, Japan) operated at 15 kV. To capture the TEM images, purified HSA NP solution was dropped onto a carbon film-covered copper grid (200-mesh pure carbon, TED PELLA Inc., USA) and dried at room temperature. For SEM analysis, HSA NP solution was dropped onto a silicon wafer and dried at room temperature. The hydrodynamic size, which indicates the size of the NPs in solution including the hydration layer, was measured by dynamic light scattering (DLS, ZETASIZER NANO ZS90, Malvern Panalytical, UK). For such measurements, the purified HSA NP solution was diluted to 1 mg/mL with purified water. Measurement of the Yield of HSA NPs. The yield of HSA NPs was measured by extracting unreacted single HSA molecules using a 100 kDa NMWL centrifugal filter. The concentration of unreacted single HSA molecules was measured by the Bradford assay. Measurement of Fluorescence Intensity of F-HSA NPs. Fluorescence intensities of F-HSA NPs synthesized at various ethanol concentrations were measured by a UV−vis spectrometer (SpectraMax M5 Microplate Reader, Molecular Devices, USA). The measurements were performed with 25 μg of F-HSA NPs suspended in 200 μL of purified water placed in 96-well plates. In Vitro Macrophage Uptake. The macrophage cell line J774A.1 was cultured in DMEM, supplemented with 10% FBS and 1% penicillin and streptomycin at 37 °C under 5% CO2, humidified air. The fluorescence intensity of macrophages that had absorbed F-HSA NPs was investigated by flow cytometry (MACSQuant VYB, Miltenyi Biotec, Germany). F-HSA NPs (25 μg) were suspended in 1 mL of the J774A.1 culture medium and applied to 2 × 105 cells in a 24-well plate. Treated cells were incubated for 3 h, after which they were washed with PBS three times to remove unattached NPs. Macrophages were detached using trypsin and moved to a 1.5 mL tube. After washing two times using PBS, the cells were stored in 200 μL of 2% paraformaldehyde solution to fix the cells. Fluorescence intensity of the fixed cells was calculated by flow cytometry. Fluorescence of the fixed cells was observed using a fluorescence microscope (Axiovert 200M, ZEISS, Germany). The fluorescence images of macrophages that had absorbed F-HSA NPs were visualized by confocal microscopy (TCS SP8 HyVolution, Leica, Germany). Cells (2 × 105) attached to the cover glass of a 24well plate were treated with 25 μg of F-HSA NPs. The treated cells were incubated for 3 h and then washed with PBS three times to remove the unattached NPs. Washed cells were fixed in 4% paraformaldehyde for 10 min. After a single wash with PBS, cells were stained by adding 300 nM DAPI dissolved in PBS. After 5 min, the cells were washed two times with PBS. The stained cells were stored in PBS. Fluorescence images of stained macrophages that had absorbed F-HSA NPs were visualized by confocal microscopy. HSA NP Distribution in Vivo. BALB/c-nude mice were used for analysis of in vivo tumor accumulation of F-HSA NPs. Twelve female mice aged 10−16 weeks were used for the analysis, and the weight of the mice was approximately 25−30 g. For the in vivo distribution analysis, we used PC3, one of the representative prostate cancer cell lines. The mice were subcutaneously inoculated on the right thigh with 107 PC3 cells in 100 μL of PBS. After allowing the tumors to grow for 3 weeks, 100 μL of the 10 μg/mL FITC-HSA NP solution was injected intravenously. For treatment, the mice were anesthetized with 2,2,2-tribromoethanol. After 24 h of injection, the tumors were

additional modification of HSA NPs would require additional, potentially complicated synthesis steps. One reason for the strong affinity of macrophages to HSA NPs synthesized in this manner relates to the use of ethanol in the synthesis. The denaturation of HSA is strongly affected by ethanol,23 and denatured HSA is strongly absorbed into macrophages, more so than native HSA.10,18 After ingestion by macrophages, which is due to scavenger receptors gp18 and gp30 in many cells, including macrophages, endothelium, fibroblasts, and MDA-MB-453 breast cancer, the denatured NPs are degraded even more through removal of older and damaged albumins.19−22 To minimize denaturation, HSA NPs were synthesized at lower ethanol concentration under physiological pH conditions. Here, we compared the degree of macrophage cellular uptake for NPs synthesized at a variety of ethanol concentrations and pHs. The results showed that HSA NPs synthesized at lower ethanol concentration and neutral pH were less susceptible to macrophage cellular uptake. Strikingly, this suggests that the circulation time of HSA NPs can be increased without additional chemical modification steps, such as PEGylation.



EXPERIMENTAL SECTION

Materials. HSA (>96%), 25% glutaraldehyde solution, fluorescein isothiocyanate isomer (FITC, >90%), formaldehyde solution, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from SigmaAldrich (USA). Centrifugal filters with 30 000 and 100 000 nominal molecular weight limits (30 and 100 kD NMWL) were purchased from Merck Millipore (USA). Fetal bovine serum (FBS), 0.25% trypsin−EDTA solution, penicillin streptomycin solution, Dulbecco’s modified Eagle’s medium (DMEM), and phosphate-buffered saline (PBS) were purchased from Gibco (USA). All chemicals were used as-received without further purification. Deionized water (18.2 MΩ cm−1) and ethanol (99.9%) were used in various experimental steps. Preparation of HSA NPs at Different Ethanol Concentrations. HSA NPs were synthesized by a desolvation method. For this, HSA (25 mg) was added to 0.5 mL of purified water in a glass vial to make a 50 mg/mL HSA solution. To titrate the solution to pH 7, 1 M sodium hydroxide (NaOH) was added. While stirring this pHadjusted HSA solution at 700 rpm at room temperature, ethanol was continuously added at 1 mL/min using a syringe pump. Final ethanol amounts for the samples in this experiment were 0, 0.0625, 0.088, 0.125, 0.167, 0.214, 0.269, 0.333, 0.409, 0.5, 0.611, 0.75, 0.929, 1.167, 1.5, and 2 mL, which correspond to ethanol concentrations of 0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80%, respectively. While stirring continued at 700 rpm, 14.7 μL of 8% glutaraldehyde solution was added to each sample to create crosslinking, which proceeded over the next 12 h under stirring at room temperature. To remove ethanol from the HSA NP solution, the solution was purified by 30 000 NMWL centrifugal filters over three cycles of centrifugation (14 000g, 30 min) and redispersion to the original volume in water. Redispersion was accomplished using a vortex mixer and a sonication bath. The final solutions were stored in 4 °C. To prepare fluorescent HSA NPs, FITC-conjugated HSA (F-HSA) NPs were synthesized. HSA was dissolved in a pH 9 borate buffer at a concentration of 10 mg/mL, and FITC was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL. FITC is unstable in DMSO solution, so the solution must be used immediately after the FITC is solubilized. The FITC solution (1.17 mL) was added to 4 mL of the HSA solution, and the solution was immediately protected from light to prevent photobleaching of the FITC. The solution was then incubated at 37 °C for 1 h. After adding the solution to a 30 000 NMWL centrifugal filter, the solution was purified by seven cycles of centrifugation (14 000g, 30 min) and redispersion to 50 mg/mL of PBS. F-HSA NPs were synthesized with 3 wt % of F-HSA by the desolvation method described above. B

DOI: 10.1021/acs.langmuir.8b03776 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir harvested, and fluorescence images of the tumors were visualized by CCD camera (ORCA-ER, C4742-80, Hamamatsu, Japan) using an FITC/EGFP filter (HQ480/40x, Chroma, USA). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to measure the relative fluorescence intensity. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University School of Medicine, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and abides by the Institute of Laboratory Animal Resources (ILAR) guide.

centrifugation (Figure 1). The synthesis of protein NPs can be separated into two ways depending on the amount of the



RESULTS AND DISCUSSION To synthesize HSA NPs, a desolvation method was used following the protocols proposed by Langer et al.14 In brief, ethanol was used as a desolvative agent. Cross-linking was accomplished by the use of glutaraldehyde. In previous studies, producing HSA NPs with the desired size and uniformity was accomplished by controlling several synthetic conditions, including concentration of HSA, glutaraldehyde, and ethanol.14,15 Even with well-defined protocol regarding the synthesis of HSA NPs, it is important for HSA NPs to be effective drug carriers by reaching targeted disease sites in vivo and to remain in the blood for significant time. To increase the efficiency of drug-delivery with HSA NPs, the synthesis conditions of the HSA NPs were studied to optimize circulation times in vivo by avoiding macrophage cellular uptake (Scheme 1). Scheme 1. Schematic Diagram of Protein NP Fabrication for Optimized RES Evasion and Tumor Accumulation; (a) High-Density HSA NPs Composed of Denatured HSAs Were Synthesized at High (>55%, v/v) Ethanol Concentrations by HSA Denaturation; (b) High-Density HSA NPs Were Absorbed by Macrophages and (c) Transferred to Liver for Degradation; (d) Lower Density HSA NPs Were Synthesized at pH 7 and Low (