CrossRef (0)
AbstractCancer is a major disease that poses challenges for the development of therapeutics. Immunotherapy offers a unique cancer elimination strategy that identifies immune evasion and induces a response (Li et al., 2018). Over the past few decades, cancer immunotherapies have evolved by utilizing T cells, cytokines, vaccines, antibodies, and immune check-point inhibitors (Riley et al., 2019). Blocking programmed cell death protein 1 (PD-1) on T cells enhances immune activation within the tumor microenvironment (TME). Programmed death-ligand 1 (PD-L1), a membrane protein on tumor cells, interacts with PD-1, suppresses T-cell activity, and causes immune evasion (Granier et al., 2017; Chen et al., 2021; Sanmamed and Chen, 2018). Monoclonal antibody-based treatments targeting PD-1 or PD-L1 have proven effective in clinical trials; however, antibody-based immune checkpoint blockade (ICB) does not extend the anticancer response and poses a risk of cancer recurrence (Martins et al., 2019). Additionally, these treatments are difficult to implement and require high costs and time. Thus, combining immune-activating drugs or alternative ligands is crucial to restoring suppressed anticancer responses and addressing issues related to therapeutic production (Kennedy and Salama, 2020; Lv et al., 2022).
Histone deacetylases (HDACs) cause dysregulation and abnormal histone mutations in various diseases, resulting in cancer development and an unfavorable prognosis. Histone deacetylase inhibitors (HDACi) restore cellular acetylation homeostasis and related gene expression and improve tumor immunogenicity by increasing the expression of major histocompatibility complex (MHC) molecules (Conte et al., 2018; West and Johnstone, 2014). Various chemokines, including CXC motif chemokine ligand 10 (CXCL10) secreted by several HDACis, induce antitumor immune responses (Zheng et al., 2016; Terranova-Barberio et al., 2017). However, suberoylanilide hydroxamic acid (SAHA), which was approved by the Food and Drug Administration (FDA) in 2006, is an HDACi that is used as an anticancer agent but has low permeability and bioavailability; therefore, a targeted delivery system is needed to overcome these problems (Mann et al., 2007; Le et al., 2021).
Liposomes, which are used as drug delivery carriers for diverse disease treatments, encapsulate drugs and exhibit amphiphilic characteristics that enable the transport of both hydrophilic and hydrophobic molecules (Large et al., 2021). With excellent biocompatibility, efficient cellular uptake, and the ability to escape endosomes, liposomes have enhanced therapeutic effects in cancer treatment (Akbarzadeh et al., 2013). While clinical trials often use liposomal delivery systems based on enhanced permeation and retention (EPR), enhancing
Aptamers, which are short single-stranded DNA or RNA oligonucleotides that can bind to target molecules, are promising ligands for targeted drug delivery and can accurately and efficiently deliver drugs through high affinity and specific binding to receptors or antigens on tumor (Liu et al., 2014; Alshaer et al., 2018). The disadvantages of currently used antibody treatments are that they are expensive and may cause inflammatory side effects due to non-specific false positive binding. However, when aptamosomes using aptamers are used for treatment, side effects can be minimized due to the structural stability, low toxicity, and low immunogenicity of the aptamers (Lv et al., 2022; Agnello et al., 2021). The advantage of these aptamer-linked liposomes is that they can target drugs more effectively, increasing the possibility of selective drug delivery to cancer cells (Gao et al., 2021).
In this study, anti-PD-L1 aptamer-conjugated liposomes were used for the targeted delivery of the anticancer agent SAHA to PD-L1-highly expressed cancer cells. First, we formulated a liposome [SAHA] containing SAHA in a neutral liposome containing SAHA based on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Subsequently, we evaluated aptamosome [SAHA] designed for specific cancer cell targeting by combining liposome [SAHA] with an anti-PD-L1 DNA aptamer in comparison with liposome [SAHA] lacking the anti-PD-L1 aptamer. Furthermore, we evaluated the potential of aptamosome [SAHA] to induce an anticancer immune response, including cytotoxicity and secretion of T-cell-activating chemokines, both
DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N[amino(polyethylene glycol)-2000]), DSPE-PEG2000-Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene-glycol)-2000]), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), Rho-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoenthanol-amine), and cholesterol were purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). Liposome-sized extruders and polycarbonate membranes (100, 200, 400, and 800 nm) were purchased from Whatman (Maidstone, UK). Suberoylanilide hydroxamic acid (SAHA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Amicon tube (30 k) was purchased from Merck Millipore (Billerica, MA, USA). The anti-PD-L1 DNA aptamer (sequence: 5'-ACGGGCC-ACATCAACTCATTGATAGACAATGCGTCCACTGCC-CGTTTTTTTTTT-3') was synthesized and modified with FAM (6-fluorescein amidite) and thiol at the 5' and 3' ends, respectively (Bioneer, Daejeon, Korea).
LL/2 Lewis lung carcinoma and HepG2 hepatocellular carcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbeccós Modified Eaglés Medium (DMEM) (Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco/Invitrogen, Carlsbad, CA, USA), and 1% penicillin/streptomycin (Hyclone, Logan, UT, USA) in an incubator in 5% CO2 at 37℃.
POPC, cholesterol, PEG-DSPE2000, and DOPE were mixed at a molar ratio of 4:1:0.4:1 in chloroform and methanol (2:1, v/v). The mixtures were evaporated using N2 gas to form a thin lipid film and desiccated using a vacuum pump for a minimum of 1 h to remove the residual organic solvent. The dried lipid film was hydrated in HEPES buffer (20 mM HEPES and 150 mM NaCl, pH 7.5) containing 500 μM SAHA. Then the hydrated solution was incubated for 2 h at 60℃ with stirring to form liposomes containing SAHA. To prepare small unilamellar vesicles, liposomes were sonicated for 3 min in a bath-type sonicator (Branson Inc., Danbury, USA). Finally, the liposome solution was extruded through 800-, 400-, 200-, and 100-nm polycarbonate membranes to adjust the liposome size.
To prepare the anti-PD-L1 DNA aptamer for conjugation with micelles, 1 nM anti-PD-L1 aptamers were treated with 10 μL of 100 mM TCEP (tris(2-carboxyethyl) phosphine) for 1 h at 25℃ and then mixed with 3 M NaOAc and 100% cold ethanol. Micelles were prepared using DSPE-PEG2000 /DSPE-PEG2000-maleimide (1:4 molar ratio). Afterward, DNA aptamers were conjugated to the maleimide groups of micelles by incubation for 2 h at 25℃, producing aptamer-conjugated micelles. After the incubation, anti-PD-L1 aptamer-conjugated micelles were mixed with liposomes containing SAHA and incubated for 1 h at 60℃. Finally, the cells were incubated for 1 h at 25℃ to complete the formation of anti-PD-L1 aptamer-conjugated liposomes containing SAHA (aptamosomes [SAHA]).
The cytotoxic effects of free drugs and formulations were evaluated using the MTT assay. LL/2 cells (1 × 104 cells/ well) were seeded in a 96-well plate and incubated overnight at 37℃. The cells were treated with various concentrations of free SAHA (0.39~50 μM) or different formulations (SAHA, liposome [SAHA], and aptamosome [SAHA]) in 100 μL medium and incubated for 24 and 48 h. After incubation, the cells were washed with DPBS and incubated with a fresh medium containing MTT reagent (5 mg/mL) for 3 h at 37℃. The media were discharged, and the cells were incubated with DMSO to solubilize the purple formazan crystals for 15 min at 37℃. Absorbance was measured at 570 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The cytotoxic effects of formulations: free SAHA, liposome [SAHA], and aptamosome [SAHA] containing the equivalent amount of SAHA (10 μM) were analyzed by the same method described above.
LL/2 cells (4 × 104 cells/well) were seeded on a 24-well plate and grown overnight at 37℃. The cells were treated with SAHA (0, 2, 4, 6, 8, 10, and 12 μM) or various formulations (SAHA, liposome [SAHA], and aptamosome [SAHA]) in 500 μL medium and incubated for 24 and 48 h at 37℃. After incubation, the cells were harvested using trypsinization, and pellets were redispersed in 100 μL of the annexin-binding buffer. The cells were co-stained with a combo of 5 μL of Annexin-V/FITC and 1 μL of PI working solution (Invitrogen, Carlsbad, CA, USA) and incubated for 20 min at room temperature. The stained cells were analyzed using a NovoCyte Flow Cytometer (ACEA Bioscience, San Diego, CA, USA).
LL/2 cells (4 × 104 cells/well) were seeded on a 24-well plate and grown overnight at 37℃. The cells were treated with various concentrations of SAHA (0, 1, 3, 10, 25, and 50 μM) or formulations (SAHA, liposome [SAHA], and aptamosome [SAHA]) in 500 μL medium and incubated for 48 h at 37℃. After incubation, cell lysates and supernatants were harvested from each well and assayed to determine the secretion levels of CXCL10 using a Mouse CXCL10 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader.
LL/2 cells and HepG2 cells (8 × 104 cells/well) were cultured on cover glass (Ф12 mm) placed in 24-well plates and grown overnight at 37℃. The cells were treated with various formulations: liposome [SAHA], aptamosome [SAHA] labeled with rhodamine, respectively; containing equivalent lipids (30 μg). After incubation times (10 min, 30 min, 6 h, and 12 h) at 37℃, the cells were washed three times with cold PBS and fixed with 100% chilled methanol. Nuclei were stained with the mounting medium with DAPI (Invitrogen, Carlsbad, CA, USA) overnight at 4℃. The images were captured using a fluorescence microscope. The fluorescence from the rhodamine-conjugated DOPE lipids was monitored using red fluorescence.
For all experiments, eight-week-old male C57BL/6N mice were purchased from Koatech (Pyeongtaek, Korea). All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC approval no. KU22232). The tumor-bearing models were established by subcutaneous administration of 100 μL of PBS containing LL/2 cells (7 × 105) into the flank of the mice. To establish lung experimental metastasis models, LL/2 cells (4 × 105) in 100 μL PBS were intravenously injected into the mice.
The antitumor efficacies of SAHA, liposome [SAHA] and aptamosome [SAHA] were investigated in tumor-bearing mice. When the tumors reached a volume of approximately 100 mm3, the mice were randomly allocated to five groups based on their tumor volumes. All mice were treated by intravenous injection twice a week with different formulations (diluted in 200 μL saline): free SAHA, liposome [SAHA], aptamosome [SAHA] which contain an equivalent amount of SAHA (0.25 mg/kg) in each liposome formulation. Body weight and tumor volume were measured every two days over a period of 28 days. Tumor volume (mm3) = (length × width2)/2.
Mice injected with different formulations were euthanized 28 days after inoculation for histological analysis. Tumors were harvested and fixed in a 10% formalin solution. The tissues were dehydrated with 70~100% ethanol and embedded in paraffin. The sections were deparaffinized and stained with hematoxylin and eosin. The stained slides were observed under a light microscope.
The experiment results are expressed as the means ± standard deviation (SD). Statistical analyses of
To evaluate the anticancer effect of SAHA, we analyzed the cytotoxicity and apoptosis of LL/2 cells treated with SAHA. After cells were treated with doubly increasing concentrations of SAHA from 0.39 μM to 50 μM for 24 and 48 h, the cytotoxicity of LL/2 was measured using the MTT assay (Fig. 1A). As a result, cell death gradually increased with increasing concentration of SAHA, and the IC50 value of SAHA was measured to be 10 μM when cultured in LL/2 for 48 hours. Additionally, SAHA-induced apoptosis in LL/2 cells was measured using flow cytometry using Annexin V-FITC/PI staining (Fig. 1B and 1C). These results indicate that SAHA can be used as an anticancer agent by inducing cytotoxicity and apoptosis in lung cancer cells.
Fig. 1. Anticancer efficacy of SAHA against LL/2 mouse lung carcinoma cells.
(A) LL/2 cells were treated with SAHA at 2-fold increasing concentrations from 0.3 μM to 50 μM for 24 and 48 h. Cell viability was measured via MTT assay at 570 nm and expressed as % relative to the negative control. (B and C) LL/2 cells were treated with the indicated concentration of SAHA for 24 and 48 h. Apoptosis was analyzed via flow cytometry using Annexin V-FITC/PI staining. The sum of early apoptotic cells and late apoptotic cells indicates total apoptosis. The data are represented as mean ± SD (n = 4).
Specific binding of the aptamosome [SAHA] to the aptamer was measured by identifying the lipid fluorescence of rhodamine. Liposome [SAHA] and aptamosome [SAHA] with rhodamine-labeled DOPE were treated with LL/2 and HepG2 cells for 10 and 30 min, respectively. After incubation, fluorescence images were captured with a fluorescence microscope (Fig. 2A), and the binding affinity was measured using flow cytometry (Fig. 2B). No noticeable uptake efficiency was observed of aptamosomes [SAHA] in PD-L1-negative control HepG2 cells. However, LL/2 cells expressing PD-L1 showed rhodamine fluorescence signals that were approximately 1.8-fold higher than those in the cells treated with liposomes [SAHA]. These results indicate that the aptamosome [SAHA], through the anti-PD-L1 DNA aptamer, can selectively target cancer cells and effectively deliver SAHA.
Fig. 2. Cell binding affinity of aptamosome[SAHA] by anti-PD-L1 aptamer.
(A) LL/2 cells and HepG2 cells were incubated for 10 and 30 min with liposome [SAHA] and aptamosome [SAHA] at 37℃. The intercellular localization of the liposome was visualized through fluorescence microscopy with rhodamine attached to the lipids. The fluorescent images were captured at 40× magnification. DAPI (blue): nuclei, Rhodamine (red): liposomes. (B) In LL/2 cells, the binding affinities of liposome [SAHA] and aptamosome [SAHA] were analyzed using flow cytometry. Red: liposome [SAHA] 10 min, Green: liposome [SAHA] 30 min, Blue: aptamosome [SAHA] 10 min, Brown: aptamosome [SAHA] 30 min.
The anticancer effect of the aptamosome [SAHA] on the mouse lung cancer cell line LL/2 was examined via the MTT assay (Fig. 3A). The cells were incubated for 48 h after treatment with SAHA, liposome [SAHA], and aptamosome [SAHA] containing an equivalent concentration of SAHA at 10 μM. The cell viability graph shows that cells treated with aptamosomes [SAHA] exhibited higher cytotoxicity than those treated with SAHA or liposomes [SAHA]. Additionally, apoptosis was measured using flow cytometry (Fig. 3B). Apoptosis levels showed the same trend as cytotoxicity levels (Fig. 3C). These results indicate that aptamosomes [SAHA] induce greater cytotoxicity in lung cancer cells than that in liposomes [SAHA] or free SAHA.
Fig. 3. Anticancer efficacy by aptamosome [SAHA] in LL/2 cells.
(A) LL/2 cells were treated with different formulations for 48 h. The cell viability is evaluated via MTT assay and measured at 570 nm. The cell viability is indicated as % compared to an untreated control group. (B and C) LL/2 cells were treated with different formulations for 48 h. Apoptosis assays were performed with the annexin-V-FITC/PI flow cytometry analysis to measure total apoptosis levels. Data are represented as the mean ± SD for triplicate experiments. A; ***To determine whether T cell-derived chemokine CXCL10 expression was induced by SAHA, it was measured using ELISA. LL/2 cells were treated with 1, 5, 10, 25, and 50 μM SAHA for 48 hours, and CXCL10 expression was confirmed to be increased in a dose-dependent manner in SAHA cells (Fig. 4A). Next, to confirm whether aptamosome [SAHA] induces immune activity, cells were treated with SAHA, liposome [SAHA], and aptamosome [SAHA] for 48 hours, respectively, and then the secretion of CXCL10 was examined. As a result, CXCL10 levels were higher in cells treated with aptamosomes [SAHA] than in cells treated with other agents (Fig. 4B). These results suggest that aptamosomes [SAHA] have a high potential to recruit cytotoxic T cells and induce immune activity.
Fig. 4. Secretion of chemokine CXCL10 by SAHA and aptamosome [SAHA] in LL/2 cells.
(A) The Secretion level of CXCL10 was confirmed using the ELISA method in LL/2 cells treated with SAHA (0~50 μM) for 48 h. (B) LL/2 cells were treated with SAHA, liposome [SAHA], and aptamosome [SAHA] for 48 h, respectively, and the CXCL10 secretion level was evaluated using ELISA. The data are represented as the mean ± SD (n = 3). A; **Based on these
Fig. 5. Apoptotic cells within a tumor can be identified by distinct features such as cell shrinkage, condensed nuclei, and hypereosinophilic cytoplasm. Accordingly, we inoculated the LL/2 tumor-bearing mouse model with various formulations for 28 days, and isolated tumor tissues were stained with H&E reagent for histological analysis (Fig. 6). In tumor tissues treated with various agents, progressive changes were observed that induce apoptosis in tumor cells. Tumors treated with free SAHA appeared similar to those treated with untreated controls. However, changes were observed in liposomes [SAHA] and aptamosomes [SAHA]. PD-L1 aptamer-binding aptamosomes [SAHA] showed a significant reduction in tumor cell numbers. These results indicate that aptamosome [SAHA] has antitumor efficacy
Fig. 6. Histopathological variation of tumor tissues in LL/2 tumor-bearing models treated by aptamosome [SAHA].
Mouse tumor tissues treated with different formulations were stained with hematoxylin and eosin (H&E) staining and observed under a light microscope. The untreated group represented LL/2 tumor-bearing mice that were not treated with any drugs. The regions in the red boxes indicate images at 40× magnification. (Scale bar, 20X: 200 μm, 40X: 100 μm).
In recent years, the blockade of immune checkpoints has led to advances in cancer immunity therapeutics. Among ICB therapies, PD-1/PD-L1 blockade has been widely used for cancer treatment. Numerous methods, such as anti-PD-1 or anti-PD-L1 drugs and monoclonal antibodies, have been proposed to block PD-1/PD-L1 (Melosky et al., 2019; Li et al., 2021). Although these methods are less toxic than chemotherapy, negative effects related to immune activation such as diarrhea, fatigue, and rashes have been reported (Abdel-Rahman et al., 2016). Accordingly, developing more effective therapeutic approaches to avoid negative immune-related effects and increase the immune response in solid cancers is a challenge for cancer immunotherapy (Baxi et al., 2018). Therefore, ICB therapies may enhance anticancer effects when combined with other immune-activating agents.
In this study, SAHA, an HDAC inhibitor used as an anticancer drug, was selected and its immune activation ability was analyzed. First, to determine whether SAHA has anticancer and apoptotic effects in LL/2 cells, we performed cytotoxicity and apoptosis assays and confirmed that SAHA is effective (Fig. 1). Next, the immune activity of SAHA was demonstrated through the secretion of CXCL10, a chemokine that recruits T cells (Fig. 4A).
Accordingly, the main purpose of this study was to utilize the liposome delivery system to increase the efficacy of SAHA drug delivery. Liposome delivery systems have been considered an ideal delivery vehicle for clinical applications due to their excellent biocompatibility and loading capacity for hydrophobic and hydrophilic drugs. Liposomes can encapsulate drugs in lipid bilayers, and the EPR effect of liposomes gives the encapsulated drugs a long circulation time (Peer et al., 2020). Based on these advantages, it was expected that using liposomes to deliver SAHA would improve the drug delivery effect. Liposomes have excellent delivery capabilities, but the need for combination with antibodies, aptamers, etc. has been raised to complement affinity and specificity (Bagalkot et al., 2006). The primary objective of this study was to establish an approach for cancer immunotherapy by conjugating aptamers to liposomes and delivering SAHA. In this study, we designed POPC-based liposomes to encapsulate SAHA. Subsequently, PD-L1 aptamers were conjugated with liposomal carriers to specifically target cancer cells (aptamosomes). The binding affinity of the aptamosomes was evaluated in LL/2 cells
In Fig. 3, the cancer cell inhibition effects of the SAHA-treated group and the aptamosome-treated group were similar, but aptamosomes with targeting ability had a higher cancer cell inhibition effect than non-targeting liposomes.
Anticancer immune responses to aptamosomes were evaluated
Finally, tumor shrinkage was confirmed via histopathological analysis. H&E images of tumor tissues treated with aptamosome [SAHA] showed an increase in eosinophilic cytoplasm and more cell shrinkage than the other treatment groups (Fig. 6). These results suggest that there was a significant occurrence of tumor apoptosis, as indicated by the observed cell shrinkage and hypereosinophilic cytoplasm in apoptotic tumor cells (Nayak et al., 2016; Jain et al., 2009).
In summary, we have developed aptamer-conjugated liposomes for drug delivery to induce anticancer immune responses. Liposomes containing SAHA were conjugated to PD-L1 aptamers to target the surface of PD-L1-expressing cancer cells (aptamosome [SAHA]). These aptamer-linked liposomes were delivered to LL/2 cells
However, there are research reports that aptamers can cause immune plaques and rarely cause allergic reactions when introduced into
Schematic
The authors have no conflict of interest to declare.
