Hemoglobin as a Smart pH-Sensitive Nanocarrier To Achieve

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Hemoglobin as a smart pH-sensitive nanocarrier to achieve aggregation enhanced tumor retention Huan Li, Yangjun Chen, Zuhong Li, Xu Li, Qiao Jin, and Jian Ji Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00241 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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Biomacromolecules

Hemoglobin as a smart pH-sensitive nanocarrier to achieve aggregation enhanced tumor retention Huan Li, Yangjun Chen, Zuhong Li, Xu Li, Qiao Jin and Jian Ji* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China KEYWORDS: hemoglobin, pH-sensitivity, aggregation, tumor retention, IR780.

ABSTRACT: Natural proteins have been greatly explored to address unmet medical needs. However, few work has treated proteins as natural pH-sensitive nanoplatforms that make use of the inherent pH gradient of pathogenic sites. Here, hemoglobin is employed as a smart pHsensitive nanocarrier for near infrared dye IR780, which disperses well at normal tissue pH and exhibits aggregation at tumor acidic milieu. The pH-sensitive hemoglobin loaded with IR780 shows higher uptake by cancer cells at tumor acidic pH 6.5 than normal tissue pH 7.4. In vivo and ex vivo studies reveal that the hemoglobin nanocarrier exhibits distinct retention kinetics with remarkably prolonged residence time in tumor. Hemoglobin is then proved to be a potent pH-sensitive nanocarrier for cancer diagnosis and treatment.

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1. INTRODUCTION Nanocarriers such as liposome1-3, polymersome4,5, micelle6-9, and nanogel10 have received enormous attention for the delivery of therapeutic agents. Among them, natural proteins have been considered as strong candidates due to the container-like shape, wide accessibility, biocompatibility, biodegradability and feasibility for further modification.11-16 Protein-based nanoplatforms, especially those developed from albumin, have shown great potential in diagnosis and treatment of cancer, diabetes, rheumatoid arthritis and infectious diseases.11,12,15,17,18 For example, Liu’s group19 employed human serum albumin as a nanocarrier for near-infrared dye IR 825 to achieve fluorescence imaging and photothermal cancer therapy. Folate-conjugated bovine serum albumin has shown efficiency in delivering etoricoxib with targeting ability to rheumatoid arthritis.20 However, most of protein nanocarriers are only treated as inert carriers, which have no sensitivity to physiological characteristics of pathogenic sites. Retention in pathogenic sites is crucial for therapeutics to realize enhanced bioavailability to exert treatment effects.21-26 Also, it’s the prerequisite for imaging agents to be used in long-term imaging, which is of great use in monitoring ongoing biological process and therapeutic outcomes.27,28 Aggregation enhanced retention has been proved to be an effective way for nanocarriers to achieve long-term retention in tumor tissues.21,24,29-32 In our previous work24, tumor pH-sensitive mixed charge gold nanoparticles were prepared which dispersed at physiological pH and aggregated at tumor acidic microenvironment (pH 7.0-6.5). The aggregation of gold nanoparticles resulted in a dramatic increase in size, which cut off backflow to bloodstream and realized efficient retention in tumor. After 72h post-injection, ~80% of the nanoparticles were held up in tumor of that at 24h. Ruan et al. 30 developed a nanoplatform that consisted of Ala-Ala-Asn-Cys-Lys modified gold nanoparticles and 2-cyano-6-


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Biomacromolecules

aminobenzothiazole modified gold nanoparticles. The two kinds of gold nanoparticles could undergo enzyme-triggered click cycloaddition which led to aggregate formation in glioma site. The enzyme-induced aggregation of gold nanoparticles led to significantly higher retention level than PEG modified nanoparticles which resulted in positive therapeutic outcome. In fact, proteins can be deemed as natural pH-sensitive nanoplatforms due to the presence of acidic or basic amino acid residues. It’s demonstrated that proteins exhibit distinct colloidal stability at different pH, as aggregation is observable around the isoelectric point (pI).33,34 Here, we explored bovine hemoglobin (Hb) as a pH-sensitive nanocarrier for near infrared dye IR780 iodide (Figure 1). It’s expected that the tumor pH-triggered aggregation of hemoglobin may enhance the retention of IR780 at tumor site. IR780 was chosen here since it was considered as a versatile therapeutic agent for cancer imaging35,36, photothermal therapy36-40 and photodynamic therapy39,41. Bovine serum albumin (BSA) that without tumor acidic pH-induced aggregation behavior was chosen as a control. The biological fate of the Hb-IR780 complex was carefully examined by in vitro, in vivo and ex vivo investigations, which provided new insight in utilizing natural proteins as pH-sensitive nanocarriers for enhanced tumor retention.


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Figure 1. Schematic illustration of aggregation enabled retention of the Hb-IR780 complex in tumor tissue. The pH-sensitive Hb-IR780 complex was well-dispersed at normal tissue pH and formed aggregates after extravasated into tumor acidic microenvironment. The aggregation of the Hb-IR780 complex resulted in enhanced uptake by cancer cells and an increase in size, which prevented the complex from re-entrance to bloodstreams and prolonged the retention time in tumor site.

2. EXPERIMENTAL SECTION 2.1. Materials. Bovine Hb and IR-780 iodide were bought from Sigma-Aldrich. BSA was purchased from Life Science Products & Services. KB (human epidermoid carcinoma) cells were purchased from China Center for Typical Culture Collection. All other chemicals and materials


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were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China) and used as received. 2.2. Preparation of the Hb-IR780 complex and the BSA-IR780 complex. 5 mg ml-1 DMSO solution of IR780 was added dropwisely to 10 mg ml-1 aqueous solution of Hb or BSA under vigorous stirring. The molar feed ratio between IR-780 and the protein was 1:1. The solution was kept away from light and stirred for 10 minutes. Sterilized 450 nm pore size filter was used to purify the above protein solution. The Hb-IR780 complex and the BSA-IR780 complex were subjected to UV-Vis spectrometry to study the loading efficiency (IR780 loading efficiency = [Weight of loaded IR780/Weight of feeded IR780] ×100%) utilizing the characteristic peak of the proteins and IR780. TEM (HT7700, HITACHI) and DLS (Zetasizer Nano-ZS, Malvern Instruments, 633 nm He-Ne laser, 25 °C with a detection angle of 173°) assay was employed to study the morphology and hydrodynamic size of the Hb-IR780 complex and the BSA-IR780 complex. 2.3. pH sensitivity. The pH sensitivity was studied by incubating the complex in 100 mM phosphate buffer of different pH. TEM images, hydrodynamic size and zeta potential were recorded to analyze the pH-sensitivity of the Hb-IR780 complex and the BSA-IR780 complex. The NIR fluorescence images of the Hb-IR780 complex were taken by Maestro optical imaging system. 2.4. Cell Uptake. Cell uptake of free IR780, the Hb-IR780 complex and the BSA-IR780 complex was studied by observing the fluorescence signal of IR780 in KB cells. Briefly, KB cells were seeded at a density of 400,000 per dish in glass bottom cell culture dish and incubated at 37 °C under 5% CO2 for 24h. The cell culture medium used here was pH 7.4 RPMI 1640


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medium with 10% fetal bovine serum and 1% penicillin and 1% streptomycin. Then, free IR780, Hb-IR780 and BSA-IR780 in pH 7.4 or pH 6.5 RPMI 1640 medium (IR780 dosage being 1 µg ml-1) was used to incubate KB cells for 6h. After 6h incubation, cells were washed three times by PBS and fixed by 4% paraformaldehyde for 30 min. The fixed cells were further dyed by DAPI and observed by confocal laser scanning microscope (Leica TCS SP5) with excitation wavelength of 633 nm for IR-780. 2.5. In vivo imaging. Healthy male nude mice (3-4 weeks old) were bought from the animal center of Zhejiang Academy of Medical Sciences. The Guidelines for Animal Care and Use Committee, Zhejiang University was followed to conduct animal experiments. Mice were subcutaneously injected with 100 µL PBS of 1.5×106 KB cells into right thigh. Mice were all ~20 g weight and tumors were allowed to grow to ~65 mm3 for further experiments. Mice were randomly divided into three groups and treated with free IR780 (DMSO/H2O solution, DMSO

Hemoglobin as a Smart pH-Sensitive Nanocarrier To Achieve

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