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. 2024 Apr 12;7(5):1612–1623. doi: 10.1021/acsptsci.4c00121

Liposomes-Encapsulating Double-Stranded Nucleic Acid (Poly I:C) for Head and Neck Cancer Treatment

Vidit Singh †, Anna Chernatynskaya †, Lin Qi †, Hsin-Yin Chuang †, Tristan Cole †, Vimalin Mani Jeyalatha †, Lavanya Bhargava †, W Andrew Yeudall ‡, Laszlo Farkas §, Hu Yang †,*
PMCID: PMC11092114  PMID: 38751634

Abstract

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Polyriboinosinic acid–polyribocytidylic acid (Poly I:C) serves as a synthetic mimic of viral double-stranded dsRNA, capable of inducing apoptosis in numerous cancer cells. Despite its potential, therapeutic benefits, the application of Poly I:C has been hindered by concerns regarding toxicity, stability, enzymatic degradation, and undue immune stimulation, leading to autoimmune disorders. To address these challenges, encapsulation of antitumor drugs within delivery systems such as cationic liposomes is often employed to enhance their efficacy while minimizing dosages. In this study, we investigated the potential of cationic liposomes to deliver Poly I:C into the Head and Neck 12 (HN12) cell line to induce apoptosis in the carcinoma cells and tumor model. Cationic liposomes made by the hydrodynamic focusing method surpass traditional methods by offering a continuous flow-based approach for encapsulating genes, which is ideal for efficient tumor delivery. DOTAP liposomes efficiently bind Poly I:C, confirmed by transmission electron microscopy images displaying their spherical morphology. Liposomes are easily endocytosed in HN12 cells, suggesting their potential for therapeutic gene and drug delivery in head and neck squamous carcinoma cells. Activation of apoptotic pathways involving MDA5, RIG-I, and TLR3 is evidenced by upregulated caspase-3, caspase-8, and IRF3 genes upon endocytosis of Poly(I:C)-encapsulated liposomes. Therapeutic evaluations revealed significant inhibition of tumor growth with Poly I:C liposomes, indicating the possibility of MDA5, RIG-I, and TLR3-induced apoptosis pathways via Poly I:C liposomes in HN12 xenografts in J:NU mouse models. Comparative histological analysis underscores enhanced cell death with Poly I:C liposomes, warranting further investigation into the precise mechanisms of apoptosis and inflammatory cytokine response in murine models for future research.

Keywords: liposomes, poly I:C, microfluidics, head and neck cancer, apoptosis

The incidence of head and neck squamous carcinoma cells (HNSCCs) ranks the sixth most common human cancer globally, and over 600,000 cases are newly diagnosed annually. HNSCC is a type of cancer that originates in the mucosal lining of the upper aerodigestive tract, including the mouth, throat, and larynx.1 HNSCC is strongly associated with tobacco and alcohol use, as well as infection with human papillomavirus subtypes.1−4 Treatment options for HNSCC include surgery, radiation therapy, chemotherapy, and targeted therapy.5

HNSCC is a common malignancy that often presents with locoregional disease and can spread to distant sites.6 Recent studies have shown that HNSCC cells express elevated levels of Toll-like receptor-3 (TLR-3), which is involved in the recognition of viral RNA and the activation of antitumor immunity.6−9 TLR3 is one of 10 members of the TLR family, which contains an extracellular domain that recognizes and binds to pathogen-associated molecular patterns and also a cytoplasmic tail which acts as residual for recruitment of TLR3 signal mediators.6,9,10

Poly I:C is a synthetic dsRNA which is employed as an antiviral strand. Poly I:C when delivered into the cytoplasm of cancer cells is known to induce apoptosis via generation of interferons (IFNs) which lead to apoptosis in carcinoma cells.6,11 Poly I:C activation in HNSCC cells has been shown to induce apoptosis, leading to the inhibition of tumor growth and the activation of the immune system against cancer cells.12,13 In another study, the combination of a Poly I:C and a chemotherapeutic agent was found to enhance antitumor activity in HNSCC cell lines and animal models.14 These findings suggest that Poly I:C may be a promising therapeutic approach for HNSCC, either alone or in combination with other treatments.

Encapsulating Poly I:C in liposomes delivers Poly I:C directly to the cytoplasm and enhances their activity.15,16 Liposomes are spherical vesicles composed of a phospholipid bilayer that encloses an aqueous core.17 Liposomes can be used as drug and gene delivery vehicles, and as model membranes for studying biological processes.17,18 They have many advantages over traditional drug delivery methods including increased drug efficacy and decreased toxicity. Poly I:C, when delivered to cytoplasm by liposomes, is amassed by TLR3-expressing endosomes.19,20 It also activates melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene-I protein (RIG-I) apoptotic pathways21,22 (Figure 1). Since TLR3, MDA5, and RIG-I strongly influence the production of IFNs, treatment with Poly I:C encapsulated in liposomes, is predicted to reduce the growth of carcinoma cells.20,23,24 This makes Poly I:C liposomes a promising tool for developing novel therapies for a wide range of diseases, including cancer, autoimmune disorders, and infectious diseases.18,25

Figure 1.

Figure 1

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Diagrammatic representation of liposome manufacturing via HDF method and the mechanism of action of liposomes-encapsulating Poly I:C in HN12 cells.

The objective of this study was to enhance the effectiveness of Poly I:C in combating HNSCC carcinoma by encapsulating the synthetic dsRNA in liposomes for the intravenous route of administration. We established that encapsulating Poly I:C in liposomes enhanced the cytoplasmic delivery of Poly I:C in HN12 cells and induced the apoptotic pathways, as seen by increased caspase cascade and interferon regulatory factor 3 (IRF3) signals. To access the antitumor efficacy, we employed the xenograft model and administered Poly I:C-encapsulated liposomes via intravenous delivery and intratumoral injection. We observed that the Poly I:C-encapsulated liposomes are able to endocytose Poly I:C in HN12 cells. Poly I:C released after endocytosis induces the apoptotic pathways leading to the arrest of HN12 cell growth in vitro and suppressing tumor xenografts in J:NU nude mice model.

Experimental Section

Materials

1,2-stearoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), L-α-phosphatidylcholine (95%) (Egg, Chicken) Egg PC, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) DSPE-PEG-2000 were purchased from Avanti Polar Lipids (Alabama), while cholesterol was purchased from Sigma-Aldrich.

Cell Lines

HN12 human metastasis-derived HNSCCs were extracted, as previously described26 and were cultured in Dulbecco’s Modification of Eagle’s Medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Thermo Fisher Scientific) and maintained in a humidified incubator in an atmosphere of 5% CO2 /95% air at 37 °C.

Animals

Female (J:NU) nude mice 6 weeks old were purchased from Jackson Laboratories (Bar Harbor, ME) and used in this work. The mice were given free access to food and water and 12-h light and dark cycles in a temperature-controlled room (18–22 °C). The procedures conducted were approved by the Institutional Animal Care and Use Committee of the University of Missouri (Missouri, USA).

Synthesis of Poly I:C-loaded Cationic Liposomes

The cationic liposomes loaded with Poly I:C were synthesized by hydrodynamic focusing (HDF), thin film hydration (TFH), and vigorous mixing (VM) methods to compare the physical characteristics of the particles. For liposomes made by the HDF method, the lipids were mixed in a 3:2:2:0.3 molar ratio of DOTAP:Egg PC:cholesterol:DSPE-PEG 2000, while Poly I:C was mixed in phosphate-buffered saline (PBS) at a mass ratio of 20:1 of DOTAP to Poly I:C.16,27 A Y-shaped microfluidic chip with a 60-μm channel width was attached to the syringe pump via Teflon pipes. The lipid dissolved in ethanol stream and Poly I:C in PBS stream were mixed in the microfluidics in a 1:5 volumetric flow ratio and at a total volumetric flow rate of 1 mL/min. The resultant mixture was dialyzed in 2000 excess volumes of PBS, using 3.5 kDa MWCO dialysis tubing (Sigma) for 3 h with a magnetic stirrer to decrease the concentration of ethanol (to less than 0.5%). The resultant formulation was passed through a 220-nm filter (Sigma) and used for further analysis of the particle size and zeta potential. After dialysis, the final concentration of Poly I/C in the formulation was maintained at 0.4 mg/mL.

For the TFH method, lipids dissolved in ethanol in the same ratio as for the HDF method were placed in a round-bottomed flask and evaporated in a rotary evaporator. Then, the formulation was placed in a vacuum oven overnight to completely remove ethanol, thus generating a thin film of lipids. Thereafter, Poly I:C mixed in PBS was added to the thin film in the same ratio as for the HDF method and ultrasonicated for 30 min. The formulation was sequentially extruded five times through 800, 400, 200, and 100 nm polycarbonate membranes. The liposomes made by the VM method had ethanol with dissolved lipids at the same molar ratio as the above methods. The mixture was added into PBS and Poly I:C solution at the same ratio as the previous methods and mixed vigorously by vortexing for 30 min. The formulation was then dialyzed in 2000 excess volumes of PBS, similar to the HDF method.

Particle Characterization

The size distribution, polydispersity index (PDI), and zeta potential were measured with a Zetasizer (Malvern, UK). For size and zeta potential analyses, the formulation was diluted 40-fold in deionized water. The liposomes for in vivo and in vitro experiments were prepared fresh and used within 24 h. A stability test was also performed for the particles.

The morphology of liposomes was completed by transmission electron microscopy (TEM) analysis using JEOL Transmission Electron Microscopy (JEOL TEM) (University of Missouri, Columbia). For TEM analysis, the liposomal formulation was diluted 50-fold in deionized water. Ten microliters of the diluted formulation was added onto the TEM grid and left to room temperature for 10 min. An equal volume of 2% (w/v) phosphotungstate (PTA) was added to the grid and left until it was dried.28 The negatively stained liposomes were then observed by TEM at 80 kV. Microscopic analysis was carried out at 50,000× magnification.

Encapsulation Efficiency

Gel retardation assays were performed to analyze the encapsulation of Poly I:C by the cationic lipids. For quantitative analysis of the encapsulation efficiency (EE), the formulation was subjected to ultrafiltration at 3000×g at 4 °C for 1 h (Vivaspin 2, 100 kDa MWCO).16 The filtrate was then used to measure free Poly I:C content by absorbance at 250 nm using a microplate reader (Tecan, GMI, Switzerland). The EE % was determined by the following formula:

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Cytotoxicity Test

Cells (5 × 104 cells/well) were seeded in 96-well plates and cultured overnight. Subsequently, cells were treated with 100 μL of free Poly I:C, free liposomes, or Poly I:C-encapsulated liposomes at a final concentration of 1, 5, and 10 μg/mL for 24 h. Liposomes concentration was 20 times as Poly I:C. After treatment, cytotoxicity was accessed by the addition of Cell Counting Kit-8 reagent (GLPBIO, Montclair, CA) according to the manufacturer’s protocol. The cells were incubated for 4 h, and absorbance was measured by a microplate reader at 450 nm. Cell viability was expressed as the absorbance of the contents of a given well divided by that of the mean absorbance measured for the control group (untreated cells).16,29,30

Cellular Uptake Studies

HN12 cells were seeded in a 24-well plate (5 × 104 cells/well), and 24 h later, each well was incubated with a nontoxic concentration of Poly I:C-encapsulated liposomes labeled with DiI dye (550/565 nm) (Biotium, San Francisco, California) for 24 h.31 After treatment, cellular uptake was observed using a fluorescent microscope (Zeiss, USA) and quantified by flow cytometry (Accuri C6 plus, BD, USA). The uptake of DiI labeled liposomes was analyzed by using the FL-2 channel (λex = 488 nm and λem = 585 nm).

Apoptotic Pathway Analysis

Cells (4 × 105 cells/well) were seeded in a six-well plate. At 24 h, the cells were dosed with PBS, Poly I:C (1 μg), blank liposomes, and Poly I:C liposomes (1 μg Poly I:C). The cells were collected 24 h later and pelleted for RNA extraction. The RNA extraction process was done by using Qiagen Mini kit (Qiagen, Germany) while following the manufacturer’s instructions. Reverse transcription was conducted by employing a Taqman RNA-CT 1-step kit (Applied Biosystems-Thermo Fisher Scientific). Real-time PCR was carried out using QuantStudio real-time RT-PCR (Thermo Fisher Scientific). The quantification of Cas-3, Cas-8, and IRF3 was performed thereafter. Normalization of gene expressions was performed relative to GAPDH (Applied Biosystems Taqman, Thermo Fisher Scientific) as an internal gene control. Data analysis for PCR results was conducted using the standard 2–ΔCt method.

In Vivo Biodistribution Studies

Three groups of 6–8-week-old female (J:NU) nude mice (Jackson Laboratory, Bar Harbor, ME) (n = 4) were used in the biodistribution tests. For the xenograft model, 5 × 106 HN12 cells suspended in PBS were injected subcutaneously in the right flank of each mouse. After 2 weeks of xenograft and tumor growth (average tumor volume = 100 mm3), DiR (745/810 nm excitation/emission)-labeled liposomes (0.1 mol % of lipids) were injected by IV through the lateral tail vein (1st group) and IT (2ndgroup).31 In the third group—control, mice were injected via IV route with PBS. The tumor size was determined by the empirical formula V = 0.5 × length × width2.32

The AmiHTX (Spectral Instrument Imaging Inc., Tucson, Arizona) imager was used to monitor formulation distribution in live animals at 6 and 24 h after injection of DiR-labeled liposomes. The mice were imaged at 24 h and then euthanized. Major organs (heart, liver, kidney, spleen, and lungs) and the tumor were harvested and imaged.32 The image was taken at excitation/emission of 745/810 nm with a binning setting at 8, excitation power at 5, and exposure time at 1 s. Relative fluorescence was quantified for each major organ in every mouse for each group.

In Vivo Efficacy Assessment

Female nude mice (6–8 weeks old) were used for the therapeutic studies, using HN12 xenografts as previously described using 2 × 106 cells per mouse.32 The tumor volume and body mass were monitored daily. The mice were divided into four groups (n = 5) PBS, liposomes alone, Poly I:C alone, and Poly I:C-encapsulated liposomes. Treatments were first administered when the tumor volume reached a 40-mm3 volume. The injections were on the 3rd, 5th, 9th, and 11th day. The animals were sacrificed on the 15th day, and the tumors were excised and fixed in 4% PFA at 4 °C for 48 h. The tissues were subsequently immersed in 15% sucrose for 6 h and 30% sucrose for 12 h and embedded in optimal cutting temperature compound (Sakura-Finetek, CA, USA) and frozen at −80 °C until use. Tissue sections of 6 μm thickness were cut using a cryostat, and the slides were fixed in 95% ethanol, stained with hematoxylin and eosin and mounted with DPX mounting media.

Statistical Analysis

All results are expressed as the mean ± standard error of the mean (SEM) unless otherwise noted. A two-way ANOVA test was used for the analysis of biodistribution data in Figure 10. These tests were performed using the Origin 2023 software for Windows (Northampton, MA, USA). p values of < 0.01 were considered statistically significant.

Figure 10.

Figure 10

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(A) Tumor size in euthanized mice on the 15th day for (from left to right) PBS (control), blank liposomes (Blank Lip), Poly I:C, and Poly I:C liposomes (Poly I:C Lip) groups. (B) Tumors extracted from mice on the 15th day with a scale on the left for (from left to right) PBS (control), blank liposomes, Poly I:C, and Poly I:C liposomes groups. (C) Relative tumor volume at each day average quantified for each group describing the therapeutic efficiency of Poly I:C liposomes complex. Statistically significant difference in tumor size was found on the 15th day. The Poly I:C liposomes group showed statistically significant difference between the PBS, blank liposomes, and Poly I:C group. (* p < 0.1, ** p < 0.05, *** p < 0.005, **** p < 0.0001). (D) Relative mass of each mice in respective groups as compared to their mass on the day of tumor inoculation with respect to the days of observance.

Results

Structure Identification and Stability of the Poly I:C Liposome Complex

The liposomal nanoparticle generated at high concentration by the HDF method resulted in a small size of particles (100 nm) and low PDI (0.21) with high EE of Poly I:C (98%).

Figure 2A and Table 1 show the size distribution of the liposomes loaded with Poly I:C generated/produced by three different methods. Although the characteristics of the particles made by extrusion and microfluidics methods are similar, the microfluidics method was chosen because of the ease of manufacturing and the scalability of the process.

Figure 2.

Figure 2

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(A) Size distribution of DOTAP Poly I/C liposomes by different methods: VM, TFH, and HDF. Comparison of the methods indicates the variation in size of the particles made and the distribution (PDI) of the particles. HDF offers the best particle size and distribution among the three methods. (B) Stability test of DOTAP Poly I:C liposomes across 21 days made by microfluidics. The particles generated by the HDF method and maintained at 4 °C demonstrate that the size of the particles does not change significantly over 21 days, and hence, they can be used for therapeutic purposes for an extended period of time. All the samples were tested in triplicates.

Table 1. Poly I:C Liposome Particle Characterization Results via Zetasizer for the Three Methods of Manufacturing: HDF, TFH, VMa.

methods poly-dispersity index hydrodynamic diameter (nm) zeta potential (mV) encapsulation efficiency %
HDF 0.21 96.07 ± 1.18 48.65 ± 0.42 97.63 ± 0.87
TFH 0.27 112.90 ± 0.57 45.26 ± 1.09 97.51 ± 0.96
VM 0.68 284.40 ± 23.61 47.46 ± 1.40 96.45 ± 0.72
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a

The results indicate the hydrodynamic diameter (HDD), PDI, and zeta potential of each formulation. Particles generated by HDF have the best distribution and smallest size and were hence used for further experiments.

Figure 2B and Table 2 describe the stability of the liposomes made by microfluidics (HDF method) across 21 days after the particles were made. The morphology of liposomes observed by TEM imaging shows that Poly I:C-loaded liposomes are spherical in shape and bind Poly I:C inside the bilayer of the lipids (shown by the bright core in Figure 3). The TEM image also confirms the size distribution by dynamic light scattering (DLS) analysis. The TEM analysis also shows that using the VM method, there is no control over generating particles of the same/similar size and morphology. In contrast, the TFH method produces particles of spherical morphology; however, a tail-like structure is also observed in the liposomes, which can lead to leakage of Poly I:C. Therefore, as a result of the spherical morphology and intactness of the Poly I:C inside the liposomes, the microfluidics method was chosen for further analysis. EE of Poly I:C was found to be 97.50 ± 0.67%, and binding efficiency was confirmed by the gel electrophoresis (Figure 4).

Table 2. Stability Test of the Poly I:C Liposomes Made by the HDF Method Across 21 Daysa.

days since formulation made PDI HDD (nm) zeta potential (mV) EE %
0 0.21 96.07 ± 1.18 48.65 ± 0.42 97.63 ± 0.87
7 0.22 101.80 ± 0.47 51.17 ± 1.13 97.42 ± 0.71
14 0.22 104.00 ± 1.90 53.09 ± 1.31 96.91 ± 0.92
21 0.24 108.3 ± 1.30 47.98 ± 1.64 96.51 ± 0.85
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a

Particles size, distribution, and EE did not change significantly across 21 days.

Figure 3.

Figure 3

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TEM image for particle morphology of Poly I:C-encapsulated liposomes made by HDF method (left), TFH (middle), and VM (right). HDF method image indicates the spherical morphology and evenly distributed size of the Poly I:C liposomes particles. The white contrast at the center of the particles indicates the presence of Poly I:C. THF method has a similar size and distribution as HDF but shows a tail-like structure of Poly I:C escaping the liposomal structure. VM method image shows that the particles are of irregular size and too widely distributed.

Figure 4.

Figure 4

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Encapsulation confirmation of Poly I:C in the DOTAP Poly I:C liposomes. The agarose gel (0.8%) was run at 100 V for 40 min. The 2nd lane was loaded with Poly I:C liposomes (Poly I:C concentration was 400 μg/mL) and the 4th lane with just Poly I:C (400 μg/mL), and the 7th lane was the RNA ladder. The gel was premixed with ethidium bromide, and the Poly I:C was mixed with Millenium RNA marker Kit (Thermo Fischer) according to the manufacturer’s instruction.

Increased Cytotoxicity of Liposomes Poly I:C in HN12 Cells

Cytotoxicity studies carried out in HN12 cells showed that liposomes loaded with Poly I:C have greater cytotoxicity than the blank liposomes and free Poly I:C, with an IC50 value between 1 and 5 μg/mL of Poly I:C with a relative concentration of 20–137.5 μg/mL of lipid (Figure 5). Using HN12 cells, we found that DiI-labeled liposomes were successfully taken up by the cells (Figure 6A), and flow cytometry analysis confirmed that 100% of live cells have endocytosed the liposomes (Figure 6B).

Figure 5.

Figure 5

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Cell viability was analyzed by the CCK-8 assay. HN12 cells (5 × 104 cells/well) were seeded on 96-well plates and treated with PBS (C), free Poly I:C (P), blank liposomes (L), or Poly I:C-encapsulated liposomes (L + P) at a final concentration of 1, 5, and 10 μg/mL for 24 h (n = 5). The height of the columns indicates mean viability. IC50 value for the blank liposomes was reached at a concentration higher than 5 μg/mL, while Poly I:C liposomes had an increased cytotoxicity at the same concentration, indicating higher HN12 cell death at 5 μg/mL.

Figure 6.

Figure 6

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Cellular uptake of DiI-labeled liposomes (DiI-lip) was assessed by fluorescence microscopy (A) and flow cytometry (B). HN12 cells were incubated with liposomes (control-top) or with DiI-labeled liposomes (DiI-lip-bottom) 3 mg/mL for 24 h. BF-bright Field, DiI-DiI (DiIC18(3); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine). n = 4, ****p < 0.0001.

Poly I:C Liposomes Induces Elevated Apoptosis via IRF3, Cas-8, and Cas-3

It was seen that Poly I:C-encapsulated liposomes may have increased apoptosis of HN12 cells compared to Poly I:C or blank liposomes through the IRF3 apoptotic pathway. The RT-qPCR study demonstrated that HN12 cells indeed have increased the expression of caspase-3 (cas-3), caspase-8 (cas-8), and IRF as compared to PBS, blank liposomes, and Poly I:C by almost a factor of 2 (Figure 7). The mRNA expression levels were measured with respect to GAPDH which is an internal control gene.

Figure 7.

Figure 7

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(A) Relative mRNA quantification performed by RT-qPCR analysis for Caspase-8, Caspase-3, and IRF3 for apoptotic pathway determination. Real-time PCR was carried out using on QuantStudio- Real Time RT-PCR (Thermo Fisher Scientific). The relative mRNA expression quantification of Cas-3, Cas-8, and IRF3 was performed. Normalization of gene expressions was done relative to GAPDH (internal gene control), showing almost 2-fold increase in gene expression when treated with Poly I:C liposomes.

Improved Liposome Collection in Tumors via IV Route of Administration

The objective of the study was to identify the preferred route of administration by evaluating the DiR when the liposomes were administered via the IV and IT routes.

The images of live mice were taken using an AmiHTX imager at 6 and 24 h after injection of the labeled formulation (Figure 8). It was observed that the circulation of the IV-injected fluorescent particles increased over a period of 24 h, while we found the opposite distribution for IT administration route. The fluorescence decreased from 6 to 24 h, which suggests that particles slowly leave the local site of injection and redistribute, mainly into the liver, whereas IV-injected formulations circulate in the body for a longer time.

Figure 8.

Figure 8

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Whole body image of DiR-labeled liposomes in HN12 cell xenograft model of J:NU mice. DiR-labeled liposomes were injected in live mice through IV (at 6 and 24 h) and IT (at 6 and 24 h) (n = 4). AmiHTX imager was used to image the live mice in an unconscious condition by continuous isoflurane inhalation. Fluorescence intensity measurement was done by excitation/emission at (745/810 nm), and ROI’s (region of interest) were created for measurement. ROI fluorescent intensity for the first mouse (control-no liposomes) was used as a background ROI for the other four mice.

At 24-h postinjection, the animals were imaged and euthanized. Major organs and tumors were harvested. It was observed (Figure 9A) that the IV injected particles via tail vein had better accumulation in the tumor site more than at the liver as compared to IT injections. The quantification of the fluorescence intensity (Figure 9B) was used as a basis for analysis of the distribution in the major organs and tumors.

Figure 9.

Figure 9

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(A) Liposomal biodistribution in tumor and major organs (heart, lungs, liver, kidneys, and spleen) seen after extraction from the xenograft model (average tumor size = 100 mm3) 24 h after the IV and IT injections (groups = 3 Control, IV, and IT route of administration) (n = 4). (B) Fluorescent efficiency was quantified for the tumor and major organs via IT and IV injection. ROIs were created and fluorescence efficiency was quantified. Control mice's organs were used for the background ROI reduction. Percentage fluorescence for each organ was calculated by dividing individual organ fluorescence with the total fluorescence intensity of all organs.

Poly I:C Liposomes Complex Significantly Affects the Size of Tumor

The in vivo tumor therapeutic efficiency was observed in the nude mice (NU/J heterozygous) with the HN12 xenografttumor model. The mice were injected with HN12 cells (2 × 106) on the right flanks. The mice were divided into four groups (n = 5 per group) when the tumor volume reached 40 mm3 volume. The four groups were given IV injections of PBS, blank liposomes, Poly I:C only, and Poly I:C-encapsulated liposomes on days 3, 5, 9, and 11. The mice were sacrificed on day 15, and the tumor volume was measured. The tumor size in the Poly I:C encapsulated liposome group was significantly reduced compared to those of other groups. The average size of the three control groups—PBS, blank liposomes, and only Poly I:C were approximately 4.5 times, 4.3 times, and 4.1 times bigger than Poly I:C-encapsulated liposomes group (Figure 10A–C). There was no significant difference between tumor size in each of the control (PBS, blank liposomes, and Poly I:C) groups. This indicated that Poly I:C-encapsulated liposomes reduced the growth of HN12 xenografts. The mice were monitored for the entire duration of the experiment, and weight was measured and no significant change was observed across all groups (Figure 10D). Comparative histological examination of the excised tumors from the control and treated groups provided insight into the positive outcome of the Poly I:C-encapsulated liposome treatment. The tumor histology of PBS and blank liposomes groups revealed distinct tumor nests, intact viable atypical cells with pleomorphic nuclei, and neovascularization, and this was similar in the Poly I:C-treated group. In contrast, considerable enhancement of cellular apoptosis and lymphocytic reaction were observed in the Poly I:C-encapsulated liposome-treated group, which contributed to tumor regression (Figure 11).

Figure 11.

Figure 11

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Histological images for tumors extracted from mice treated with (in clockwise direction from top left) PBS, blank liposomes, Poly I:C, and Poly I:C liposomes. (A, B) PBS and Liposome only treatment showing moderately differentiated viable tumor nest presenting characteristic neoplastic cells with pleomorphic nuclei, signs of invasion, (C) Poly I:C-treated tumor showing enhanced neovascularization and active tumor cells. (D) Lymphocytic reaction post Poly I:C liposome treatment and activated apoptosis of tumor cells.

Discussion

Liposomal nanoparticles encapsulating TLR-3 agonist Poly I:C were formulated by employing DOTAP, Egg PC, DSPE-PEG-2000, and cholesterol. DOTAP is a cationic lipid that is used to encapsulate nucleic acids since the nucleic acids are negatively charged. Egg PC is a phospholipid used to build the structural unit of these liposomal nanoparticles, and cholesterol improves the rigidity of the nanoparticle structure. DSPE-PEG-2000 has been used as the PEG lipid for stealth property improvement of liposomes.

Microfluidics is often used as a chip that utilizes the HDF technique to implement the manufacturing of liposomes. HDF using microfluidics is a technology that has emerged as a promising tool for the precise control and manipulation of fluids at the microscale level.33 In the field of liposome production, microfluidics has enabled the production of highly uniform and monodisperse liposomes, which have several advantages over traditional liposome production methods. One of the key advantages of microfluidics-based liposome production is the ability to precisely control the size and composition of the liposomes. This is achieved by controlling the flow rate, concentration, and mixing of the liposome components, which lead to a high degree of reproducibility and consistency in liposome production.33,34 In the present study, microfluidics enables the high-throughput production of liposomes, with the ability to produce higher number of liposomes in a short amount of time similar to those findings in the literature.25,33−35 DOTAP as a cationic lipid showed the capability to transfect nearly all of the HN12 cells in vitro, as suggested by the DiI uptake in the cells (Figure 6A,B).

Liposomes also facilitate the interaction of Poly I:C with TLRs as the primary receptors. Upon the phagocytosis of the Poly I:C liposome complex, Poly I:C binds to TLRs, leading to the recruitment of adaptor proteins, including myeloid differentiation primary response 88, Toll/interleukin-1 receptor domain-containing adapter-inducing IFN-β, and translocating chain-associated membrane protein. This cascade induces the phosphorylation of IRF3 via NF-κB signaling, thereby activating type I IFN signaling.15,36−39 Salaun et al. were the first to demonstrate that the activation of TLR3 induces apoptosis in vitro across different breast cancer cell lines.23,40 They also found that while IFN type I is necessary for this process, it alone is not sufficient to activate the apoptotic pathway.23 Similarly, studies conducted in vitro using melanoma models have shown that in cancer cells expressing TLR3, activation of the receptor by its agonist directly suppresses cell proliferation. Moreover, when combined with type I IFN, the agonist induces apoptosis. The primary mechanism by which TLR3 mediates apoptosis in cancer cells relies on the activation of caspase-8.23,40

Poly I:C is recognized by RIG-I-like receptors, specifically MDA5 and RIG-I, which then interact with IFN-β promoter stimulator 1 (IPS-1).41,42 Subsequently, IPS-1, also known as Mitochondria Anti-Viral Signaling protein (MAVS), an adaptor protein localized in the mitochondria recruits and phosphorylates NF-κB activator-binding kinase 1 (TBK1), leading to the phosphorylation of IRF3.15,41,43−45 Chattopadhyay et al. proposed a novel mechanism involving RIG-I-induced IRF3-mediated apoptosis, suggesting that cytoplasmic IRF3 interacts with B-cell lymphoma 2-associated X protein (BMX), resulting in the activation of caspase-9 and subsequent cas-3 activation, primarily initiating intrinsic apoptosis.36,37

Quantitative RT-PCR results in this study showed the liposomes Poly I:C increased the trend of the cas-3 and IRF3 by almost a factor of 2 in HN12 cells, which indicate the MDA5 and RIG-I apoptosis pathway. Cas-8 also increased in a similar manner as compared to only Poly I:C and blank liposomes treatment, which indicated the apoptotic pathway via TLR3 receptors.

In the current study of biodistribution in vivo, it was observed that a part of the injected particles got absorbed into the bloodstream and collected into the liver easily. This might have reduced the uptake of the particles into the tumor directly compared to the IV route of administration. Although IT injections offer a direct delivery of the particles at the tumor site, the xenograft model being a solid tumor has negligible space for the formulations to collect within the tumor.46,47 As a result, the formulation forms a sag around the tumor. In the realm of cancer therapy, the delivery of gene-loaded liposomes via intravenous injection emerges as a compelling approach, offering unique advantages over intratumoral injection. The systemic reach achieved through IV delivery facilitates the widespread distribution of gene-loaded liposomes throughout the circulatory system, enabling their access to both primary tumors and distant metastatic sites. This multifocal targeting capability proves advantageous for cancers with diverse manifestations.48,49 Moreover, IV injections provide a versatile and less invasive administration method, overcoming the challenges posed by tumors in anatomically intricate locations. In contrast, while IT injections excel at delivering a concentrated payload directly to the tumor site, their scope is confined to accessible lesions. The localized concentration achieved through IT injection is particularly beneficial for tumors amenable to direct injection, ensuring a focused therapeutic effect with minimized systemic exposure.48,49

Role of protein corona is an important issue which needs to be accessed when injecting a nanoparticle formulation in biological fluid.50,51 Protein corona patterns around nanoparticles, influenced by their properties and biological environment, pose challenges in therapeutic nanomedicine. Mechanistically, these patterns can mask particle surfaces, hinder uptake, protect against immune responses, limit circulation, or enhance toxicity. Understanding and controlling protein corona formation are crucial for optimizing nanomedicine efficacy.50−52 In order to counter such a risk of fouling the particles, multiple approaches are used: protein-repellent coatings like zwitterionic compounds, antifouling polymers, such as polyethylene glycol or poly(ethylene oxide), which mitigate corona shielding based on coating characteristics like density, size, and heterogeneity.51−53 In the current study, we have employed PEG 2000 as an antifouling agent, which increases the steric hindrance and downplays the affinity of protein corona on the liposomes surface. PEG also increases the stealth property by dodging the macrophages in the blood.51,54,55

The efficacy of Poly I:C liposomes in vivo setup was done in xenograft J:NU model wherein the inoculation with Poly I:C liposomes resulted in significantly smaller tumor and had delayed tumor delay growth as compared to other treatments. This is a consistent observation with other TLR3 tumor-bearing in vivo setup.56 Poly I:C as a stand-alone treatment in such an in vivo setup does not result in a tumor suppression effect (Figure 10A–C) unless it is delivered into the tumor cells cytoplasm directly, wherein the apoptotic cascade of the HN12 cancer cell is triggered. Qu et al.57 observed the effect of Poly I:C delivered in the cytoplasm of tumor cells with the lack of T cells in a nude mice model. They suggested that the intracellular RNA receptors like TLR3, MDA5, and RIG-I would be responsible for the carcinoma cells apoptosis by introduction of Poly I:C into the cytoplasm.57−59 Finally, comparative histological analysis showed that Poly I:C liposomes enhanced cell death.

Apoptosis is an efficient self-protection response to eliminate dysfunctional cells and avoid pathogen infection. Interestingly, we observed that the mRNA level of the apoptotic genes Cas-3, Cas-9, and IRF3 was upregulated by the presence of liposomes encapsulating Poly I:C; indicating that MDA5, RIG-I and TLR3 pathways were triggered.57−59 Conversely, Poly I:C as a stand-alone treatment did not show a significant upregulation of the apoptotic genes.

Systematic Poly I:C administration is known to have some side effects to the body.60,61 However, Poly I:C liposomes at same dosages have less inflammatory cytokines production (TNF, IL, and IFN-γ) developed in J:NU nude mice.62,63 These cytokines are mostly produced by macrophages, dendritic cells, and natural killer cells in the nude mice.62−64 Although the inflammatory cytokines study was not done in this research, the general effect on the health of the mice was observed by regular monitoring of the weight of the mice. No significant change in the body response within the mice was observed when Poly I:C liposomes were injected across 15 days, as there was no significant change in the body weight. However, in vivo pro-inflammatory cytokine study and toxicity on normal cells at high dosages of Poly I:C liposomes warrant further study in future.

These results suggested that delivery of Poly I:C to the cytoplasm via liposomes endocytosis is a must for apoptosis in HN12 cells; however, the in-depth mechanisms will need to be clarified by future studies.

Conclusions

The current study confirmed that HDF is a robust methodology to generate Poly I:C-encapsulated liposomes, with small size, uniform distribution, and low PDI by DLS analysis as compared to VM methods. Although the thin layer hydration method gave a similar result, HDF offers a flow-based continuous approach to make the DOTAP liposomal nanoparticles, which can be used to encapsulate different genes and drugs for efficient delivery into the tumor. IV injection of Poly I:C liposomes showed a significant effect on tumor growth inhibition, with more than 4-fold reduced tumor growth compared to controls (Poly I:C, Blank liposomes, and PBS). This study proves that Poly I:C liposomes are a viable therapeutic pathway for HN12 xenograft in J:NU mouse model even without innate immune system in the body. Further in-depth study into the exact mechanisms of apoptosis and inflammatory cytokines for the body response in the mouse model needs to be done in the future.

Acknowledgments

This work was supported, in part, by National Institutes of Health (R01EY035088, R01HL140684, and R01HL139881).

Author Contributions

V.S., A.Y., L.F., and H.Y. proposed and designed the research. V.S. performed most of the experiments. A.C. worked on cellular uptake studies. L.B. and T.C. worked on particle characterization of TFH and VM methods. V.S., L.Q., and H.-Y.C. worked on in vivo experiments. V.S. and V.M.J. conducted histological and RT-qPCR analysis analysis. All of the authors have read and approved the final manuscript.

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Pharmacology & Translational Sciencevirtual special issue “Nucleosides, Nucleotides, and Nucleic Acids as Therapeutics”.

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