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Bacteria-Responsive Nanoliposomes as Smart Sonotheranostics for Multidrug Resistant Bacterial Infections Xin Pang, Qicai Xiao, Yi Cheng, En Ren, Lanlan Lian, Yang Zhang, Haiyan Gao, Xiaoyong Wang, Wingnang Leung, Xiaoyuan Chen, Gang Liu, and Chuanshan Xu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09336 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019
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Bacteria-Responsive Nanoliposomes as Smart Sonotheranostics for Multidrug Resistant Bacterial Infections Xin Pang,†‡ Qicai Xiao,‖ Yi Cheng,‡ En Ren,‡ Lanlan Lian,‡ Yang Zhang,‡ Haiyan Gao,‡ Xiaoyong Wang,‡ Wingnang Leung,§ Xiaoyuan Chen,¶ Gang Liu,*‡ Chuanshan Xu,*† †
Key Laboratory of Molecular Target and Clinical Pharmacology, State Key Laboratory of
Respiratory Disease, School of Pharmaceutical Sciences & Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, China ‡
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for
Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China ‖School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Guangzhou 510006,
China §
Division of Chinese Medicine, School of Professional and Continuing Education, The
University of Hong Kong, Hong Kong ¶
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical
Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA
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ABSTRACT: Rapid emergence of multidrug resistant (MDR) “superbugs” poses a severe threat to global health. Notably, undeveloped diagnosis and concomitant treatment failure remain highly challenging. Herein, we report a sonotheranostic strategy to achieve bacteriaspecific labeling and visualized sonodynamic therapy (SDT). Using maltohexaose-decorated cholesterol and bacteria-responsive lipid compositions, a smart nanoliposomes platform (MLP18) was developed for precise delivery of purpurin 18, a potent sonosensitizer proved in this study. Taking advantages of bacteria-specific maltodextrin transport pathway, the prepared MLP18 can specifically target to bacterial infection site and accurately distinguish the foci from sterile inflammation or cancer with highly selective fluorescence/photoacoustic signal on the bacteria-infected site of mice. Moreover, the bacteria-responsive feature of MLP18 activated an efficient release and internalization of high concentration sonosensitizer into bacterial cell, resulting in effective sonodynamic elimination of MDR bacteria. In situ MRI monitoring visualized such potent sonodynamic activity and indicated that MLP18-mediated SDT could successfully eradicate inflammation and abscess from mice with bacterial myositis. In view of the above advantages, the developed nanoliposomes may serve as a promising sonotheranostic platform against MDR bacteria in the areas of healthcare.
KEYWORDS: bacterial infections, sonodynamic therapy, multidrug resistant, optical imaging, theranostic
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Multidrug-resistant (MDR) bacteria are an ever-growing global crisis, with devastating consequences to public health care. We are now approaching a post-antibiotic era in which most existing antibiotic treatments are no longer functional but the new drug discovery has essentially frozen. In the quest for novel alternatives, physical methods (e.g., irradiation,1, 2 heat,3, 4 high pressure,5, 6 etc.) gain great popularity because they are amenable to combat MDR pathogens, and have less potential to induce resistance and systemic toxicity. Especially ultrasonic wave-driven sonodynamic therapy, it now emerges as a highly promising technology for infection eradication.7 Antimicrobial sonodynamic therapy (aSDT) depends on the interaction between low-frequency ultrasound (US) and a nontoxic sonosensitizer to generate reactive oxygen species (ROS), which are highly cytotoxic in virtually all bacteria without concerns about resistance.8, 9 Compared to photo/hyperthermia-induced antibacterial therapies that are limited to skin lesion,10-12 aSDT takes advantages of superior tissue penetrability and non-invasiveness of ultrasound, showing great potential in deeply-seated diseases.13-15 In the past decade, we and many researchers are active in this field, proving efficient aSDT with various sonosensitizers, including curcumin,16, 17 rose bengal,18 hypocrellin B,19, 20 and some porphyrin-based compounds.21,
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However, given the undeveloped methods for infection
detection, coupled with high heterogeneity and adaptability of bacterial infections,23-25 misdiagnosis and mismanagement continuously increase. Approaches with only therapeutic function cannot always offer timely and effective antibacterial stewardship. A more efficacious method is to develop visualized theranostic platforms that allow accurate diagnosis of the causative microorganism, potent eradiation of infection lesion, and real-time monitoring of treatment progress.
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Optical imaging, which relies on the detection of fluorescence (FL) or photoacoustic (PA) signals at a target site, provides golden opportunities for aSDT-based visualization of bacterial infections, since the unique luminescent properties of sonosensitizers endow themselves not just bactericidal drugs also powerful optical imaging agents. By contrast to current in situ diagnosis methods of [18F] FDG or radio-labelled leukocytes whose clinical practice are plagued by high charge, short shelf-life and radiation-related risks,26 in vivo optical bacterial imaging, although relatively new, is rapidly improving in cost, safety, simplicity, and speed.27 However, current sonosensitizers usually suffer from high optical interference by intrinsic chromophores in living subjects, which significantly hinder their potential application in clinical imaging. To address this issue, purpurin 18 (P18), a porphyrin structural molecule, was exploited in this study and proven to be a potent sonosensitizer for aSDT. Different from conventional sonisensitizers with UV-visible light excitation, P18 works in the near infrared (NIR, 700–900 nm) spectral region. When imaging in vivo, its NIR FL and PA signal can potently overcome the tissue absorption and scattering. Despite such progress, some fundamental limitations of sonosensitizers, including high hydrophobicity, ease of auto quenching, and poor bacterial specificity, still remain unsolved. Inspired from the advances in sonodynamic cancer therapy, nano-engineering aSDT by clinically effective materials may be a potential solution, but to date, the first example is still awaited. Herein, an attempt to couple nanotechnology with aSDT was made. Responsible for more than 50% of the total nanoformulations commercially available, nanoliposomes have been considered as promising candidate for drug delivery,28 and are selected as the carrier for P18. Due to short lifetimes and mobility of ROS, the sensitizers that localize inside the cells are
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recognized mostly more efficient.29-31 Consequently, an ideal nanoliposome system should ensure close contact with pathogenic bacteria, controllable sonosensitizer release, and subsequent bacterial internalization. If possible, it should accumulate specifically to pathogens with low binding affinity for human cells. In this regard, the versatile formula composition of nanoliposomes offers unparalleled flexibility to regulate the drug delivery. For example, 1,2dioctadecanoyl-sn-glycero-3-phospho-(1′-racglycerol) (DSPG) has been found susceptible to bacteria-oversecreted phospholipase A2 (PLA2).32, 33 As a result, employing DSPG as the lipid composition of nanoliposomes is expected to realize bacteria-activated sonosensitizer delivery via PLA2-mediated degradation. Further modification with piloting agents, such as maltohexaose which can selectively target to bacteria but not mammalian cells through the bacteria-specific maltodextrin transporter pathway,34,
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the proposed nanoliposomes will
facilitate precise diagnosis and sonodynamic treatment of bacterial infections. Based on such principles, we report a novel MDR bacterial theranostic strategy by encapsulating P18 into nanoliposomes (MLP18) prepared with maltohexaose-modified cholesterol and DSPGcontained lipid compositions. The bacteria-responsive feature of MLP18 activated an efficient release and internalization of high concentration P18 into bacteria. Selective NIR FL and PA signals from infection area of mice demonstrated that MLP18 with strong bacteria-targeting can specifically distinguish the infection from sterile inflammation and cancer. For in situ visualization of aSDT, magnetic resonance imaging (MRI) was introduced to monitor the therapeutic progression and picturized potent bactericidal effects of MLP18 in mice with bacterial myositis. RESULTS AND DISCUSSION
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Preparation and Characterization of MLP18. The MLP18 nanoliposomes were prepared by decorating cholesterol with bacteria-targeting maltohexaose, and then the maltohexaosemodified cholesterol combined with DSPG-contained lipid compositions were mixed to physically encapsulate P18 through a thin-film hydration method (Figure S1 and 1). The anomeric carbon of maltohexaose was selected for cholesterol conjugation because maltodextrin transporters specifically recognize the non-reducing end of maltodextrins and should therefore tolerate substitutions at the reducing end. The prepared MLP18 were measured with dynamic light scattering, showing an average diameter and zeta potential of 150 nm and -27 mV, respectively (Figure 2A, B). By contrast, nanoliposomes without maltohexaose modification (LP18) yielded a reduced uniformity of size distribution. This may be attributed to the absence of hydration shell formed by abundant hydroxyl groups in maltohexaose with surrounding water molecules, which can potentially stabilize and disperse the nanoformulation. The loading efficiency and drug content of P18 in MLP18 nanoliposomes were 94.63% and 4.84%, respectively. Observed by transmission electron microscopy (TEM), the MLP18 had a well-defined spherical structure (Figure 2C). The morphology of MLP18 that were stored for almost 6 weeks was not changed, which demonstrates their high stability (Figure 2I). From the UV-vis absorption spectrum of MLP18 (Figure 2D), it was found that the nanoliposomes exhibited the characteristic absorption peaks of P18 at around 410, 550 and 710 nm, indicating the successful encapsulation of P18. As shown in Figure 2E, the fluorescence of free P18 was almost quenched totally in PBS due to the self-aggregation effect. Conversely, MLP18 nanoliposomes with good P18 dispersion exhibited an obvious fluorescent emission around 710 nm. We further evaluated the ability of MLP18 as a PA imaging contrast agent. As
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expected, the PA signal of MLP18 steadily enhanced with P18 concentration increasing, which can be visualized in clear photographs (Figure 2F). Potential Sonodynamic Effect of MLP18. Singlet oxygen (1O2) is considered as the dominant active species of ROS, mediating the cytotoxic mechanism for most of sonosensitizers, especially those based on porphyrin structure. In this study, the ROS generation upon USactivation of P18 was first evaluated using commercially available, 9,10-dimethylanthracene (DMA) as a singlet oxygen (1O2) probe. The detector can irreversibly react with 1O2, causing a fluorescence quenching effect. Therefore, the high fluorescent decay of DMA generally means high production of 1O2. As shown in Figure 2G, the fluorescent signals of DMA presented a gradual reduction as increased P18 molecules were exposed to US irradiation. Further compared with commonly-investigated sonosensitizers, including curcumin, rose bengal, and chlorin e6, the P18 showed the highest ROS generation after ultrasound activation (Figure S7), suggesting its great potential as sonosensitizer for disease treatment. Quantitative comparison of the DMA fluorescence at 434 nm revealed that nanoliposomes after P18 encapsulation could still efficiently generate ROS in a concentration-dependent manner, whereas no significant fluorescent decrease was observed in comparison to groups only exposed to US or MLP18 (Figure S8). Such fluorescent results confirmed that MLP18 could act as potential nanosonosensitizers to eradicate bacterial infections. PLA2 Enzyme-Responsive Drug Release from MLP18. The sonosensitizer release from MLP18 nanoliposomes was evaluated in high PLA2 enzyme condition which is a characteristic feature of bacterial infection microenvironment and can degrade the DSPG lipid in the skeleton of MLP18 nanoliposomes. After 24 h incubation, more than 90% P18 was liberated in the
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presence of PLA2 enzyme, while the nanoliposomes were stable with only 40% P18 release in media without PLA2 (Figure 2H). Considering the PLA2-responsive release of P18, MLP18 nanoliposomes are expected to maintain good integrity during shipping condition (bloodstream, low PLA2), guaranteeing the improved biosafety and controlled biodegradation by bacterial infection microenvironment in vivo. Antibacterial Activity and Mechanism of MPL18-Mediated aSDT. Clinically isolated methicillin-resistant Staphylococcus aureus (MRSA, gram-positive bacterium) and extendedspectrum beta-lactamase Escherichia coli (ESBL-EC, gram-negative bacterium) were used to estimate the aSDT efficacy of MLP18 using colony counting method. In the absence of ultrasound irradiation, no bactericidal activity was found in all treatments, while groups containing P18 sonosensitizer showed obvious growth inhibition upon ultrasound activation (Figure 3B, C). Taking advantages of targeting nanotechnology, the MLP18 nanoliposomes performed the most efficient aSDT and such antibacterial efficacy was significantly weakened when a PLA2 inhibitor (0.13 μM) was added to the culture medium (Figure 3B, C). Flow cytometry results showed that the presence of PLA2 inhibitor remarkably attenuated the uptake of P18 by bacteria, suggesting that enzyme-enhanced drug release and internalization may be the key for great bactericidal effect of MLP18-mediated aSDT (Figure S9). Additionally, the viability of bacteria gradually decreased as the P18 concentration in MLP18 increased (Figure 3D). By contrast to Gram-negative ESBL-EC, the sonodynamic toxicity of MLP18 to Grampositive MRSA was statistically higher (P
