Full length articlePeptide-guided resiquimod-loaded lignin nanoparticles convert tumor-associated macrophages from M2 to M1 phenotype for enhanced chemotherapy☆
Graphical abstract
Introduction
Several nanoplatforms have been employed for cancer immunotherapy, where biomaterials with immunostimulatory properties can be used to formulate cancer vaccines, or other nanomaterials can load and deliver different immunomodulatory compounds [1], [2], [3], [4], [5]. Additionally, cancer nanomedicines represent a promising alternative over the conventional therapies to overcome their limitations, including the poor water solubility, the lack of anti-tumor specificity, and consequent systemic side effects [6], [7], [8]. For this purpose, different types of nanomaterials have been developed, such as inorganic nanoparticles (NPs) [9,10], lipid-based nanosystems [11], [12], [13], and polymeric NPs [14], [15], [16], [17], [18]. Lignocellulosic materials, such as cellulose, hemicellulose, and lignin, have gained increased attention, as they are derived from natural sources, are largely abundant in nature, and are biocompatible and biodegradable [19,20]. In particular, lignin has been recently used as a drug delivery vehicle, after transforming the raw material into lignin NPs (LNPs), using different approaches like antisolvent precipitation, solvent exchange or sonication [19,[21], [22], [23]]. Additionally, the functional groups on the original lignin polymer can be chemically modified before preparing the LNPs in order to be functionalized with different targeting moieties, and thus, increase their potential application [19,24,25].
Triple-negative breast cancer (TNBC) is characterized by the lack of molecular targets/receptors, such as estrogen, progesterone, or human epidermal growth factor receptor 2 (HER2), which comprise important molecular targets for different therapeutic agents, and therefore, it is associated to a poor prognosis compared to other breast cancer subtypes [26,27]. Additionally, tumor-associated macrophages (TAMs) resembling a pro-tumor M2-like phenotype comprise up to 50% of the tumor mass in breast cancer [28], being usually associated with a worse prognosis, tumor progression and recurrence, higher risk of distant metastasis, and suppression of other effector cells like CD8+ T cells [28], [29], [30]. In the tumor microenvironment (TME), TAMs exhibit either an immunosuppressive M2-like phenotype, characterized by the expression of distinctive cell surface markers, including CD163, CD204, and mannose receptor (CD206/MRC1), and increased production of anti-inflammatory signals (e.g., IL-10 and transforming growth factor (TGF)-β, or arginase 1 (ARG 1)), or an anti-tumor M1-like phenotype, in which macrophages can express MHC-II, CD68, CD86, and CD80 cell surface markers, release pro-inflammatory cytokines (e.g., interleukin (IL)-12, IL-1β, IL-6 and tumor necrosis factor (TNF)-α), and produce reactive oxygen species (ROS) [31], [32], [33], [34]. Targeting therapeutics to TME components like TAMs can be seen as a complementary approach to chemotherapy, the primary established systemic treatment choice for patients with both early-stage and advanced-stage TNBC, to improve the therapeutic outcome [35]. Emerging therapies that aim to target TAMs comprise the inhibition of monocyte/macrophage recruitment to the tumor tissue, after blocking colony-stimulating factor 1 (CSF-1) receptor or CCL2-CCR2 signaling pathway [36], [37], [38], elimination or depletion of TAMs in the TME, by triggering their apoptosis and delay the tumor progression [34,39], and reversion or reprogramming the immunosuppressive M2-like phenotype towards a tumoricidal M1-like TAMs, by manipulating the environmental stimuli [34,[39], [40], [41], [42], [43]]. However, these approaches are usually associated with a lack of specific targeting and consequent systemic side effects, which limit their application in the clinic [34,39]. In order to circumvent these limitations, nanomedicines can be tailored to carry therapeutics and effectively target them to the TAMs at the tumor site [44,45].
In this study, we took advantage of the lignin biopolymer as starting material to prepare LNPs, and employ them as a drug delivery vehicle to carry resiquimod (R848), a dual agonist of the toll-like receptors TLR7/8 that can promote the repolarization of M2-like into M1-like TAMs, yielding R848@LNPs [46]. The R848@LNPs were further functionalized with 5(6)-carboxyfluorescein (FAM)-labeled hexapeptide (sequence: CSPGAK, “mUNO”) that targets the mannose receptor (CD206), typically overexpressed in the M2-like macrophages [[47], [48], [49]]. The in vitro cytocompatibility of both non-targeted and targeted R848@LNPs, hereafter referred to as R848@LNPs-P-FAM and R848@LNPs-P-F-mUNO, respectively, was evaluated towards mouse derived M2-like macrophages, and their repolarization effect was assessed by evaluating the expression levels of the cell surface markers CD86 and CD206, and the production of ROS, such as hydrogen peroxide (H2O2), and also TNF-α. In vivo biodistribution studies were conducted to analyze the homing ability of R848@LNPs towards the M2-like TAMs in orthotopic 4T1 tumor-bearing mice, an aggressive TNBC preclinical model. Finally, a chemotherapeutic compound (Vinblastine, Vin) was co-administered with free R848 or R848@LNPs for synergetic anticancer effect in the same TNBC model, and the immunological profile of the cell population in the tumor tissues was evaluated.
Section snippets
Materials
BioPiva™ softwood kraft lignin was acquired from UPM Biochemicals (Finland). Maleimide-poly(ethylene glycol)-amine (Mal-PEG-NH2, 2K) was obtained from Biochempeg Scientific Inc. (Watertown, MA, USA). 5(6)-carboxyfluorescein (FAM)-Ahx-Cys-NH2 and FAM-Ahx-CSPGAK-COOH (mUNO) (hereafter referred to as FAM and FAM-mUNO, respectively) were purchased from TAG Copenhagen (Denmark). Resiquimod (R848) was purchased from DC Chemicals (Shanghai, China). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Preparation and characterization of the LNPs
Firstly, the empty LNPs and R848@LNPs were prepared via solvent exchange, using the dialysis method described elsewhere (Fig. 1A) [50]. During this process, the carboxylated lignin polymer dissolved in THF, with or without R848, self-assembled into colloidal NPs as the THF was gradually replaced by MilliQ-water. Acting as a non-solvent, the water reduces lignin's degrees of freedom and causes the segregation of hydrophobic regions to compartments within the forming NPs, which also allowed the
Conclusion
In this study, we successfully developed an effective lignin-based nanosystem to target a potent TLR7/8 agonist (R848) to the TME, in order to revert the tumor supportive (M2-like) macrophages into an anti-tumor (M1-like) phenotype, for enhanced chemotherapy. The use of mUNO to target the R848@LNPs to M2 macrophages showed an improved efficiency of the R848 in different ways, such as by modifying the biodistribution of the R848 and enhancing its accumulation and efficacy in shifting the
Declaration of Competing Interest
None.
Acknowledgements
P. Figueiredo acknowledges the Finnish Cultural Foundation for a research grant (decision no. 00190246), and the financial support from the Orion Research Foundation and the NordForsk for the Nordic University Hub project #85352 (Nordic POP, Patient Oriented Products). P. Scodeller acknowledges a personal research grant from the Estonian Research Council (PUT PSG38). M. Tiboni acknowledges the Italian Ministry of Education, University and Research for the “Bando Leonardo da Vinci – Azione 2”
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Part of the Special Issue on Immunomodulatory Biomaterials, guest-edited by Professor Georg Duda and Doctor Taimoor Qazi.