Sequentially responsive biomimetic nanoparticles with optimal size in combination with checkpoint blockade for cascade synergetic treatment of breast cancer and lung metastasis
Graphical abstract
Introduction
With the rapid development of nanotechnology, researchers nowadays have developed kinds of nanomaterials to deliver anti-cancer drugs to tumor sites. However, little progress was made as the heterogeneous and complicated tumor microenvironment hinders the efficacy of drug delivery. The enhanced permeability and retention (EPR) effect, which is caused by the abnormal leaky tumor vasculature [1,2], is the basic strategy that mostly used to passively deliver nanodrugs to tumor sites [3]. Nevertheless more and more studies found that nanoparticles with relatively large sizes tend to distribute around blood vessels rather than penetrate into tumor parenchyma [4], while smaller nanoparticles penetrate deeply into tumor but are cleared rapidly at the same time [5,6], showing a contradict size-dependent EPR effect. As a key character of nanoparticles, size is considered to play pivotal role in biodistribution [7], tumor accumulation [8,9], penetration [10,11], cellular internalization [12,13], exocytosis [14,15] and circulation in vivo [16,17]. However, recent studies investigated the size impact based on the nanomaterials with untunable sizes [18,19], which couldn't ensure the consistence of intratumoral accumulation and penetration behaviors among nanoparticles with variable sizes because of the existing paradox in size-dependent EPR effect. And with the emergence of intelligent size-reducible nanocarriers which exhibited enhanced anti-tumor efficacy [20], the influence of original diameter of size-reducible nanoparticles on the tumor drug delivery is urgently needed to be explored. Here, we constructed series of size-reducible nanoparticles with varied initial sizes (CAuNCs@HA). The hyaluronic acid (HA) outlayer could specifically respond to hyaluronidase (HAase) [[21], [22], [23]], followed by the exposure of the same cationized gold nanoclusters (CAuNCs) with small sizes. The designed nanoparticles could be ideal model for studying the optimal initial size of size-reducible nanodrugs in cancer treatments.
As an exogenous system, nanoparticles are easily recognized and cleared by the mononuclear-phagocyte system (MPS), failing to concentrate in target sites. Classical strategy is to modify the surface of nanoparticles with PEG, and the hydrated outlayer acts as a shield to reduce clearance by MPS. However, with the deepening of research, it was found that PEGylated nanoparticles could stimulate body to produce anti-PEG antibodies, and the particles would be rapidly cleared after the second injection [24,25]. Zhang's group reported that red blood cell (RBC) membrane can be engineered as artificial biomaterials for long circulation [26,27]. As a natural long-circulation delivery vehicle, RBC has a life span of 120 days [28], and CD47 on the membrane can be identified as a self-marker preventing from macrophage uptake [29]. The stealth RBC membrane cloaking strategy exhibited enhanced circulation time of nanoparticles without impacting function of nanodrugs [[30], [31], [32]]. In this study, we coated size-reducible nanoparticles with RBC membrane (mCAuNCs@HA) for longer circulation and better delivery to tumor site.
Paclitaxel (PTX) is a well-known first line drug for breast cancer. However, the premature drug release always attenuates the clinical therapeutic efficacies and brings systemic drug-oriented side effects. Recently, several kinds of dimeric prodrugs were developed [33]. By controlling the linker with different substrates, the dimeric prodrug was supposed to specifically respond to different stimuli (eg: GSH, ROS) and hydrolyze to the corresponding monomeric drugs to exert efficacy [34]. Pei and colleagues synthesized reactive oxygen species (ROS)-responsive PTX dimer (PTX2-TK) with thioketal (TK) as the linker, which exhibited specifically release of PTX avoiding cytotoxicity to normal cells [35]. Though the ROS level of tumor is relatively higher than normal organs, it's still not enough to stimulate the rapid release of large amounts of monomeric drugs. Therefore, the ROS-responsive dimer drugs were usually combined with photodynamic therapy (PDT). However, due to the consumption of ROS in the ROS-responsive hydrolysis process, the ROS level might not be high enough which would even affect the PDT efficacy. To overcome this problem, we chose the cinnamaldehyde and thioacetal based PTX dimer (PXTK) [36]. Besides specifically responding to ROS under irradiation and photosensitizer, the produced cinnamaldehyde in the hydrolysis process could also stimulate mitochondria to produce ROS, maintaining the ROS at a certain level.
PDT has emerged as a novel treatment with prospect for its noninvasive and target specificity [37]. Under the existence of photosensitizer and irradiation, the surrounding oxygen can be transformed to ROS such as singlet oxygen (1O2), which is cytotoxic and leads to cell apoptosis and necrosis. Furthermore, the PDT-induced cell death undergoes damage-associated molecular patterns (DAMPs) [38], which is the characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), secreted ATP and released high mobility group protein B1 [39] (HMGB1). The dying cancer cells undergo DAMPs can further stimulate the presentation of tumor antigens and operate as vaccines to provoke tumor-specific immune response. Besides killing cells in a direct way, PDT has also been found to stimulate the host antitumor immune response by up-regulating transcription factor nuclear factor kappa B (NF-κB) and heat shock protein 70 (HSP-70) [40], activating dendrite cells (DCs) through tumor cell residues [41], and promoting cytokine secretion [42]. However, the host immune boost was very limited with PDT alone, and we reputed it was because of the immunosuppressive environment in tumor. We therefore combined PDT with immune check point inhibitor and deduced that the combination therapy could relieve the immunosuppressive environment in tumor and exert prosperous anti-tumor effect.
Many studies have demonstrated that tumors are in an immunosuppressed environment, which is the reason why some immunotherapy didn't exhibit expected antitumor efficacy. Among them, the immune checkpoint pathway is followed with much interests, immune checkpoint inhibitors such as cytotoxicity T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death receptor 1 (PD-1) and its corresponding ligand (PD-L1) gained great success in relieving the immunosuppressive environment of tumor [43,44]. Though several PD-1/PD-L1 immune checkpoint inhibitor antibodies have been approved in clinical treatments, there still exists the problem like other common macromolecular antibodies such as immunogenicity, awful tumor penetration, low stability and high manufacturing costs [45]. To overcome these shortages, a hydrolysis-resistant D-peptide antagonist (dPPA) was developed to target the PD-1/PD-L1 pathway [46]. Therefore, dPPA was utilized in this study for immunosuppressive tumor cell targeting and cancer immunotherapy.
In this study, we constructed HAase-triggered size reducible nanoparticles with diameters of 150, 200 and 300 nm respectively by controlling the mass ratio of HA and CAuNCs. And then RBC membrane was coated outside. The as synthesized size-reducible RBC membrane coated nanoparticles with variable initial sizes could degrade into the same small cores after hydrolysis of HAase. We evaluated the cytotoxicity, cellular uptake, biodistribution and pharmacokinetic profiles of these particles with and without RBC membrane coating, and screened out an optimal size for further evaluation. The optimal nanoparticles were then loaded with photosensitizer pheophorbide A (PheoA), ROS-responsive prodrug PXTK and anti-PD-L1 peptide dPPA to explore the antitumor and anti-metastasis efficacy of the combination of chemotherapy, PDT and immunotherapy (Scheme 1).
Section snippets
Synthesis of AuNCs
5 mL aqueous HAuCl4 solution (10 mM) was added to 5 mL BSA solution (50 mg/mL, 37 °C), and after vigorously stirring for 2 min, 0.5 mL NaOH aqueous solution (1 M) was introduced to the system to maintain the alkaline environment. This reaction was needed for at least 12 h for completion until the color of the mixture went from yellow to reddish brown [47]. And the whole process was carried out in 37 °C oil bath in dark. The obtained AuNCs were purified through dialysis (MW = 3500) for 48 h.
Synthesis of CAuNCs
Preparation and characterization of mCAuNCs-PTX@HA
AuNCs were prepared via a green chemical procedure as described previously [50], the as synthesized AuNCs consisted of 25 gold atoms with BSA outside for stabilization, and intense red fluorescence can be seen at a wavelength of 365 nm under UV light (Fig. 1A). In order to absorb HA outside the AuNCs, we chose cationized BSA for better electrostatic interaction [51,52]. Cationization of albumin has been reported early in 1967 [53], here we chose the classic protocol [54]. By using EDC as an
Conclusions
In summary, we prepared size-reducible CAuNCs-PTX@HA with diameter of 150, 200, and 300 nm respectively to study the influence of original size on tumor targeting. Besides, RBC membrane was cloaked outside to facilitate the nanocarriers with long circulation time and enhanced tumor targeting in vivo. By controlling the consistency of particle size after degradation, we found that the original size of 150 nm exhibited the best tumor targeting and accumulation effect, and RBC membrane coating
Funding
This work was supported by National Natural Science Foundation of China [grant numbers 81872806, 31571016]; the Young Elite Scientists Sponsorship Program by CAST [grant number 2017QNR001].
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
We are grateful to the West China School of Public Health, Research Center for Public Health and Preventive Medicine. And we would also be grateful to Doctor Chenghui Li (Analytical & Testing Center, Sichuan University) for her help of taking images.
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