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. 2023 Dec 27;103(2):147–155. doi: 10.1177/00220345231216110

CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma

QJ Ding 1, MT Remy 1, C Upara 1, J Hu 1, AV Mora Mata 2, AJ Haes 2, E Lanzel 3, H Sun 1, MR Buchakjian 4, L Hong 1,✉
PMCID: PMC10915176  PMID: 38149503

Abstract

MicroRNA (miR)–200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c–based gene therapy to inhibit OSCC growth has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA (pDNA) encoding miR-200c delivered via nonviral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were used to transfect pDNA encoding miR-200c into OSCC cells, and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c, were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line–derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and improve clinical outcomes in OSCC treatment, while more comprehensive examinations to confirm the safety and efficacy of the CaCO3/pDNA miR-200c system using various preclinical models are needed.

Keywords: miR-200c; OSCC; CaCO3-based nanoparticles; transfection efficiency; tumor growth, xenograft

Introduction

Oral squamous cell carcinoma (OSCC) is a highly invasive cancer and accounts for approximately 90% of oral cavity malignancies (Bagan et al. 2010). Although postoperative adjuvant radiotherapy or chemoradiotherapy is used for advanced OSCC tumors, nearly 40% of patients with advanced stage cancer will experience a recurrence. The 5-y survival rate for advanced stage OSCC is less than 50% (Wang et al. 2013; Chinn and Myers 2015; Adel et al. 2016).

MicroRNAs (miRNAs) are small noncoding RNAs that play crucial roles in OSCC initiation and progression via epigenetically regulating cellular processes (Yoshizawa and Wong 2013; Momen-Heravi and Bala 2018; Troiano et al. 2018; Aali et al. 2020; Emfietzoglou et al. 2021; Manzano-Moreno et al. 2021). miR-200 family members are highly involved in OSCC (Arunkumar et al. 2018; Kim et al. 2019; Hsieh et al. 2021). Notably, the level of miR-200c in OSCC tumor tissues is significantly less than in adjacent normal tissues, and this downregulation is associated with a poorer prognosis in OSCC treatment (Song et al. 2020; Aghiorghiesei et al. 2022). miR-200c also exerts multifaceted effects in OSCC. Specifically, miR-200c suppresses OSCC stemness and tumor-initiating properties by targeting the Sox2/Wnt signaling (Liu et al. 2017). In addition, miR-200c attenuates OSCC-related inflammation and progression by targeting NF-ĸB signaling and inhibiting the epithelial–mesenchymal transition (EMT) process (Brabletz et al. 2011; Tamagawa et al. 2014; Johnson et al. 2016; Sztukowska et al. 2016). Furthermore, miR-200c has been shown to inhibit the proliferation of OSCC cells (Yan et al. 2018). In addition to its tumor-suppressive functions, miR-200c plays a crucial role in reducing chemoradiation resistance in oral cancer treatment by targeting hub genes associated with cisplatin and docetaxel resistance in OSCC (Brabletz et al. 2011; Wu et al. 2019; Cui et al. 2020). While it was controversial that miR-200c alone is sufficient for treating cancer patients (Kumar et al. 2015), the cumulative evidence from these studies strongly suggests that enhancing miR-200c expression could significantly enhance the effectiveness of OSCC treatment and reduce the risk of tumor recurrence. However, it is noteworthy that miR-200c–based gene therapy for OSCC treatment remains unexplored.

CaCO3 is a well-studied, mineral biomaterial with excellent osteoconductivity, biocompatibility, and biodegradability in bone formation. Nanosized Food and Drug Administration–approved CaCO3/DNA coprecipitates were also developed for gene delivery (Sharma et al. 2015). Notably, protamine sulfate (PS), a clinically used antidote for heparin-induced anticoagulation, has been reported to substantially improve the transfection efficiency of CaCO3-mediated plasmid DNA (pDNA) into cells (Wang et al. 2014; He et al. 2018). Our previous studies have demonstrated that pDNA miR-200c delivered by CaCO3 effectively improves transfection efficiency and alveolar bone formation (Remy et al. 2022). CaCO3-based nanoparticles have also been developed for cancer therapy (Chen et al. 2016; Poojari et al. 2016). In the present studies, the transfection efficiencies of pDNA miR-200c into OSCC cells were investigated by varying ratios of CaCO3 and PS, and anti-OSCC capacities of pDNA encoding miR-200c were evaluated subsequently in vitro and in vivo. It is demonstrated that CaCO3-based nanoparticles effectively transfect pDNA miR-200c into OSCC cells and significantly inhibit the viability and migration of OSCC cells and reduce tumor growth in mice. This evidence strongly indicates that the nanoparticles composed of CaCO3 and PS may deliver pDNA miR-200c to prevent tumor recurrence in clinical OSCC treatment.

Materials and Methods

Preparation and Characterization of pDNA miR-200c and CaCO3 Nanocomplexes with Different Ratios of CaCO3 and PS

pDNA encoding miR-200c, empty vector (EV), and scramble vector (SV) were prepared as in our previous studies (Hong et al. 2016). CaCO3/pDNA miR-200c nanocomplexes at CaCO3/PS ratios of 1:0.03125, 1:0.125, 1:0.25, and 1:0.5 were prepared as previously described (Remy et al. 2022). The nanocomplexes were visualized using scanning electron microscopy, and the hydrodynamic particle diameter and zeta potential of the CaCO3/pDNA nanocomplexes were measured using dynamic light scattering (DLS) as previously described (Remy et al. 2022).

Transfection of pDNA miR-200c into OSCC Cells Using CaCO3-Based Nanoparticles with Different Ratios of CaCO3/PS

To determine the influence of the CaCO3/PS ratio on miR-200c transfection efficiency, 2 × 104 OSCC cells (SCC 193 cells; Millipore Sigma) were treated with 1 µg pDNA miR-200c/CaCO3 nanocomplexes at different ratios of CaCO3/PS in Opti-MEM. To determine the transfection efficiency of CaCO3/miR-200c compared to other delivery systems, we transfected SCC 193 cells with 1 μg pDNA EV or miR-200c using naked pDNA alone, branched polyethylenimine (PEI, MW 25 kDa; Sigma-Aldrich) (Hong et al. 2016), or CaCO3/PS coprecipitated nanoparticles.

Cell Viability, Migration, and Oncogenic Marker Measurements after pDNA Encoding miR-200c Transfection

To determine the viability of SCC 180 and 193 cells (Millipore Sigma), 2 × 103 cells were seeded in 96-well plates and then treated with 0.2 μg miR-200c/CaCO3. The cell viability was documented via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Biotium).

To investigate cell migration, 2 × 104 cells were cultured to confluency in 12-well plates and switched to the media without fetal bovine serum (FBS). After 2 μg pDNA miR-200c/CaCO3 or the same amount of EV and SV was added, a pipette tip was used to create a linear wound scratch across the confluent cell monolayer. The cell migration motility was measured using the repaired area (ImageJ; National Institutes of Health).

To investigate the inhibitory function of miR-200c transfection on OSCC cells, 104 cells were treated with 1 μg plasmid miR-200c/CaCO3 nanocomplexes. Total RNA of the cells transfected with miR-200c and the same amount EV and SV were extracted after 3 days. Most commonly altered genes for OSCC (AACR Project GENIE: Powering Precision Medicine through an International Consortium; AACR Project GENIE Consortium 2017), including Notch1 (neurogenic locus notch homolog protein 1), FAT1 (FAT atypical cadherin 1), CDKN2A (cyclin-dependent kinase inhibitor 2A), CCND (cyclin D1), FADD (Fas associated via death domain), p53 (tumor protein P53), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), and Zeb 1 (zinc finger E-box binding homeobox 1), were quantitatively measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

In Vivo Tumor Growth of OSCC Cells Pretreated with pDNA miR-200c/CaCO3 Nanocomplexes

All proposed animal studies were performed following a protocol approved by the Institutional Animal Care and Use Committee at the University of Iowa. This animal study followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) 2.0 guidelines. In total, 2 × 106 SCC 193 cells were treated with 20 μg miR-200c/CaCO3 nanocomplexes or the same amount of EV/CaCO3 and SV/CaCO3 complexes overnight. The cells suspended in 100 μL Dulbecco’s modified Eagle medium (DMEM) were then subcutaneously injected into flanks of 8-wk-old male BALB/c nude mice (Jackson Lab) (n = 4). After 5 wk, harvested tumor samples with different treatments were weighted. Half of the tumor tissues was used to measure oncogenic markers and the remaining was stained with hematoxylin and eosin (H&E) and immunofluorescence (IF). The histological analysis of the explant section was performed blindly by a pathologist.

Local Administration of pDNA miR-200c/CaCO3 Nanocomplexes Inhibiting OSCC Tumor Growth In Vivo

In total, 2 × 106 SCC 193 cells were suspended in 50 μL serum-free DMEM and injected subcutaneously into the flanks of nude mice. After 24 h, 20 μg pDNA miR-200c/CaCO3 complexes or EV/CaCO3 controls were suspended in 100 μL solution and injected into the site of OSCC cell injection (n = 4). Mice were sacrificed after 3 wk, and the tumors were analyzed using qRT-PCR and immunohistochemistry. The histological analysis of explant sections after H&E staining was performed blindly by a pathologist. In a supplemental study, 20 μg pDNA EV/CaCO3 complexes, SV/CaCO3, or phosphate-buffered saline (PBS) controls were injected to test the function of SV and EV on OSCC tumor suppression (n = 3−7).

qRT-PCR

Total RNA was extracted with the miRNeasy microKit (Qiagen). MirX (Takara) cDNA synthesis kits were used to detect miRNA expression. Quantitative PCR was performed with the SYBR Premix Ex TaqTM II (Takara). Comparative real-time PCR was performed in replicate, and relative expression was obtained using the comparative Ct (ΔΔCt) method.

IF and H&E Staining

Tumor tissues were fixed in 4% paraformaldehyde overnight, embedded in optimal cutting temperature compound, and cut into 7-μm sections. For IF staining, the slides were incubated with primary antibody (1:50 dilution) and with fluorescently labeled secondary antibody goat anti-rabbit 488 antibody (1:100 dilution) (Elabscience). Samples were then mounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired using a microscope (Eclipse Ts2; Nikon). The frozen sections were also stained with H&E.

Statistical Analysis

Descriptive statistics were conducted for both in vitro and in vivo investigations. A one-way analysis of variance (ANOVA) with post hoc Tukey’s honestly significant difference (HSD) test was used to determine whether there were significant differences between treatment groups for nanoparticle size, the in vitro miR-200c transfection, proliferation, migration, and oncogenic makers in vitro. For the in vivo study, a one-way ANOVA with post hoc Tukey’s HSD test was used to evaluate tumor size and oncogenic markers of various pretreated OSCC cells and functions of local administration of EV, SV, and PBS on OSCC tumor growth. A Student’s t test was used to assess differences of local administration of pDNA miR-200c/CaCO3 and EV. The Shapiro–Wilk test was also applied to verify the assumption of normality. All statistical tests completed for the in vitro and in vivo quantifications used a significance level of 0.05, and each graphic depicts mean values and associated standard deviations. Statistical analyses and associated figures were created via GraphPad Prism (GraphPad Software).

Results

CaCO3-Based Nanoparticles Enhance Transfection Efficiencies of pDNA miR-200c in OSCC Cells

Figure 1A summarizes the expression level of miR-200c after pDNA miR-200c delivery by CaCO3-based nanoparticles and other delivery systems. Notably, naked pDNA miR-200c increased expression of miR-200c more substantially than the EV and untreated controls, yet CaCO3/pDNA miR-200c significantly increased miR-200c expression in comparison to naked pDNA transfection. Although PEI transfection also increased miR-200c more than naked pDNA and untreated controls, miR-200c expression after PEI transfection was significantly lower than pDNA miR-200c delivered by CaCO3 nanoparticles at a CaCO3/PS ratio of 1:0.25. However, EV delivered by CaCO3 had no ability to increase miR-200c expression. The expression level of miR-200c was dose-dependently based on the doses of CaCO3/pDNA miR-200c nanocomplexes (Fig. 1B) and continuously increased after 6 d of transfection (Fig. 1C). Among the different ratios of CaCO3/PS, miR-200c expression was significantly highest when delivered by CaCO3/PS at 1:0.25 than at other CaCO3/PS ratios (Fig. 1D).

Figure 1.

Figure 1.

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CaCO3-based nanoparticles improve the transfection efficiency of plasmid DNA (pDNA) miR-200c in oral squamous cell carcinoma (OSCC) cells. (A) Normalized miR-200c transcript in SCC 193 treated with pDNA miR-200c and empty vector (EV) at 1.0 µg delivered by naked pDNA, polyethylenimine (PEI), and CaCO3 nanoparticles. (B) Normalized miR-200c transcripts in SCC 193 cells with CaCO3 nanoparticle delivered pDNA miR-200c and EV at different concentrations (from 0.1 to 1.0 μg). (C) Normalized miR-200c transcripts in SCC 193 cells with CaCO3 nanoparticle delivered 1.0 µg pDNA miR-200c and EV after different times. (D) Normalized transcripts of miR-200c delivered by CaCO3-based nanoparticles with different ratios of CaCO3/protamine sulfate (PS). *P < 0.05, ****P < 0.0001. Performed in triplicate.

CaCO3/PS Ratios Affect Characteristics of CaCO3/pDNA miR-200c Nanocomplexes

Figure 2A, B summarize the standard error of the mean (SEM) images and quantitative diameter measurements of the nanocomplexes at different CaCO3/PS ratios. There is notably an inverse relationship between size and PS concentration, where the diameter of the nanocomplexes decreases with increasing amounts of PS. The size of CaCO3/PS at 1:0.25 (128 ± 32 nm) and 1:0.5 (74 ± 33 nm) is significantly smaller than the CaCO3/PS at 1:0.125 (433 ± 92) and 1:0.03125 (3312 ± 1055 nm) (Fig. 2B). In addition, the nanoparticles at the 1:0.03125 and 1:0.125 ratio of CaCO3/PS exhibit irregular crystalline morphologies, while the particles at a higher ratio of CaCO3/PS are structurally round (Fig. 2A). Under hydrodynamic conditions, we also confirm that the diameters of nanocomplexes made by CaCO3/PS at 1:0.25 (176 ± 50 nm) and 1:0.5 (178 ± 16 nm) are significantly smaller than the CaCO3/PS at 1:0.125 (341 ± 0 nm) and 1:0.03125 (367 ± 56 nm) (Fig. 2C). However, the zeta potential surface was not statistically different among the nanocomplexes at different CaCO3/PS ratios (Fig. 2D).

Figure 2.

Figure 2.

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Characterization of plasmid DNA (pDNA) miR-200c/CaCO3 nanocomplexes. Standard error of the mean (SEM) images (A), diameters measured by SEM images (B) and dynamic light scattering (DLS) (C), and zeta surface potential of pDNA miR-200c/CaCO3 with different ratios of CaCO3/protamine sulfate (PS) (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Performed in triplicate.

Transfection of pDNA Encoding miR-200c Using CaCO3-Based Nanoparticles Reduces Viability, Migration, and Oncogene Expression of OSCC Cells In Vitro

Two OSCC cell types were treated with CaCO3/pDNA miR-200c at a CaCO3/PS ratio of 1:0.25. The viabilities of OSCC cells measured by MTT assay after treatment with pDNA miR-200c are significantly lower than that of untreated OSCC cells. The same dose of EV and SV has no inhibitory effect on the viability of OSCC cells (Fig. 3A). The treatment of CaCO3/miR-200c significantly inhibited cancer cell migration. The noncell scratch areas remained open after 1 and 2 d for SCC 193 and SCC 180 cells, respectively, while untreated cells and cells treated with EV and SV quickly migrated and covered the scratched area (Fig. 3B, C). Notch1, FAT1, and CDKN2A were significantly downregulated in cells treated with miR-200c (Fig. 3E). In addition, transcripts of Zeb1, PIK3CA, p53, FADD, and CCND were also downregulated by miR-200c overexpression, while the statistical analysis for Zeb1 was not significant (Appendix Fig. 1).

Figure 3.

Figure 3.

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Oncogenic characterization of oral squamous cell carcinoma (OSCC) cells treated with plasmid DNA (pDNA) miR-200c/CaCO3 nanocomplexes and controls. (A) Viabilities of SCC 180 and SCC 193 cells transfected with 0.1 µg pDNA miR-200c, empty vector (EV), and scramble vector (SV) delivered by CaCO3-based nanoparticles. (B) Photographs of wound-healing/scratch assays of OSCC cells transfected with 0.1 µg pDNA miR-200c and controls. (C) Quantitative measurement of un-cell area percentages after a wound-healing/scratch assay of OSCC cells with different treatment. (D) Normalized fold changes of OSCC associated oncogene transcripts of SCC 193 transfected with pDNA miR-200c and controls after 3 d. *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated and EV/SV. Scale bars: 100 µm. Performed in triplicate.

Overexpression of miR-200c with CaCO3/miR-200c Nanocomplex Treatment Reduces OSCC Cell-Derived Tumor Growth In Vivo

After 5 wk of subcutaneous injection of SCC 193 cells, the harvested tumor weight induced by SCC 193 pretreated with CaCO3/pDNA miR-200c was significantly smaller than those with EV and SV (Fig. 4A, B). The transcripts of CDKN3A, FAT1, and NOTCH1 were significantly downregulated in tumors induced by cells with miR-200c overexpression, compared to the cells pretreated with EV (Fig. 4C). The transcripts of FADD and CCND1 were also downregulated (Appendix Fig. 2). IF stains using antibodies against CDKN2A, FAT1, and NOTCH1 showed the positive stains in sections of tumor cells pretreated with EV and SV. However, limited positive staining was found in cells pretreated with CaCO3/pDNA miR-200c (Fig. 4D). In addition, according to histological examination by pathologists, the morphology of cells in explants of OSCC cells pretreated with miR-200c showed no evidence of SCC cells, while the cells treated with EV or SV were well-differentiated or moderately differentiated SCC cells.

Figure 4.

Figure 4.

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Transfection of plasmid DNA (pDNA) miR-200c by CaCO3-based nanoparticles suppresses tumor growth of oral squamous cell carcinoma (OSCC) in vivo. (A, B) Photographs of OSCC tumor explants (A) and the weight of tumors (B) induced by SCC 193 cells pretreated with CaCO3/miR-200c complexes, CaCO3/empty vector (EV), or CaCO3/scramble vector (SV) and subcutaneously implanted into the flank of nude mice for 5 wk. (C) Normalized transcript fold changes of OSCC-related oncogenes of tumor explants with different treatments. (D) Microphotographs of sections of OSCC tumor explants induced with the OSCC cells with different treatments, hematoxylin and eosin stain, and immunofluorescence stain with antibodies of CDKN2A, FAT1, and NOTCH. All biostatistical analysis was performed by analysis of variance with post hoc Tukey’s honestly significant difference test or Student’s t test. *P < 0.05, **P < 0.01 vs EV and SV. n = 4. Scale bars: 50 µm.

Local Application of CaCO3/pDNA miR-200c Complexes Inhibits OSCC Tumor Growth in CDX Model

After a cell-derived xenograft of OSCC using SCC 193 cells was created, CaCO3/miR-200c nanocomplexes were injected to test whether local application of pDNA miR-200c delivered by CaCO3-based nanoparticles inhibits OSCC growth in vivo. pDNA containing green fluorescent protein (GFP) was used to track the transfection following the local injection. After 3 d, positive GFP expression in the harvested tumor mass was observed (Fig. 5A). GFP expression was also significantly higher in tumor tissue with miR-200c treatment than untreated controls (Fig. 5B). Figure 5C, D summarize the tumor growth size after different treatments. Notably, after 3 wk, the weight of harvested tumors treated with local injection of CaCO3/miR-200c nanoparticles was significantly smaller than those treated with CaCO3/EV. In addition, significantly upregulated miR-200c expression and downregulated transcripts of OSCC-associated genes in tumors treated with CaCO3/miR-200c were confirmed (Fig. 5E and Appendix Fig. 3). Well-differentiated SCCs were detected in the explants with different treatments. In our supplemental study, no significant body weight difference was observed among the mice after treatment with CaCO3/EV, CaCO3/SV, and untreated controls. The tumor sizes treated with CaCO3/EV were comparable to the CaCO3/SV and untreated control after 3 wk (Appendix Fig. 4).

Figure 5.

Figure 5.

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Local application of CaCO3/plasmid DNA (pDNA) miR-200c effectively improves the transfection efficiency of miR-200c and inhibits oral squamous cell carcinoma (OSCC) tumor growth in a CDX model. (A, B) Immunofluorescence images and quantitative measurements of green fluorescent protein (GFP) expression in OSCC tumors treated with local injection CaCO3/pDNA encoding GFP and untreated control. (C–E) Photographs of OSCC tumor explants (C), the weight of tumors (D), and transcript fold changes of miR-200c and OSCC-related oncogenes within tumors (E) 3 wk after local administration of 20 μg CaCO3/miR-200c or CaCO3/empty vector (EV). (F) Microphotographs of sections of OSCC tumor explants induced with the OSCC cells with different treatments, hematoxylin and eosin stain, and immunofluorescence stain with antibodies of CDKN2A, FAT1, and NOTCH. *P < 00.05, **P < 0.01 vs EV/control. n = 4. Scale bars: 50 µm.

Discussion

Previous studies have reported that CaCO3-based nanoparticles can effectively improve transfection efficiencies of pDNA miR-200c and effectively enhance bone formation in alveolar bone defects (Remy et al. 2022). In the present studies, it is confirmed that the system of CaCO3 with PS coprecipitation nanoparticles effectively improves transfection efficiencies of pDNA miR-200c into OSCC cells. Local administration of pDNA using CaCO3-based nanoparticle delivery also enhances the pDNA transfection and increases the expression of miR-200c in tumor tissues in a mouse model. Because of its biocompatibility and biodegradability, CaCO3 with coprecipitation of PS may potentially be developed for clinical application as a nonviral nanoparticle delivery system. In this study, it is also observed that the transfection efficiency of pDNA miR-200c varies in the ratio of CaCO3/PS in OSCC cells, and the ratio of CaCO3/PS at 1:0.25 is optimal to enhance the transfection efficiency for pDNA miR-200c to the highest degree. These findings were partially consistent with a previous report claiming that a higher concentration of PS is associated with a higher transfection efficiency (Wang et al. 2014). We identified that the lower PS concentrations have relatively lower transfection efficiencies probably due to the relatively large size of the nanocomplexes, where increasing nanoparticle size decreases transfection efficiency (Ota et al. 2013). In this study, the diameters of nanocomplexes at lower PS concentrations are ~400 and 3,000 nm, which are significantly larger than the ideal size of nanoparticles (~100 nm) for enhancing transfection efficiency. We found that a higher concentration of PS (CaCO3/PS ratio 1:0.25) can effectively reduce the size of nanoparticles and reach the ideal size to enhance transfection efficiency. However, the small-diameter nanoparticles at the highest PS concentration did not enhance transfection efficiency. While the underlying mechanism(s) remain unknown, overdoses of PS in the CaCO3 nanoparticles changes the electrical environment of the nanoparticle system, likely causing decreased pDNA encapsulation efficiency. Therefore, future in vivo experiments to optimize the ratio of CaCO3/pDNA miR-200c for enhanced in vivo transfection are needed.

In the current study, we have demonstrated that local application of pDNA miR-200c delivered using CaCO3-based nanoparticles can effectively transfect pDNA and upregulate miR-200c expression locally. This approach reduced the growth and gene expression of OSCC cell-derived tumors. This evidence strongly supports the idea that overexpressing miR-200c by local administration at the time of reconstructive surgery will improve OSCC treatment effectiveness and prevent tumor recurrence and metastasis. Although application of chemically synthesized miRNA mimics may simulate the inhibitory function of miR-200c on OSCC, transient transfection of miRNA mimics may lead to the accumulation of high molecular weight RNA species (Jin et al. 2015). To achieve mature miRNA functional levels, miRNA mimics require a high concentration, which easily induces nonspecific alterations in gene expression as a side effect. In addition, sustaining a high concentration with effective overexpression level using miRNA mimics is challenging due to its relative instability. In addition, the supraphysiological levels of mature miRNAs and the artifactual RNA species may induce the potential nonspecific changes in gene expression. However, transfection of pDNA encoding miRNAs can simulate the biogenesis of endogenous miRNAs and not lead to high molecular weight RNA species. Although the level of mature miRNA increased by pDNA is relatively less than the miRNA mimics, many studies demonstrated that they sufficiently suppress the target genes. In addition, our previous studies have demonstrated that cells with overexpression of miR-200c after transfection of pDNA miR-200c may upregulate miR-200c levels in surrounding cells by secreting miR-200c enriched exosomes (Krongbaramee et al. 2021). Therefore, transfection of pDNA miR-200c may effectively sustain the overexpression period and enhance the inhibitory function on OSCC.

In conclusion, we have demonstrated that pDNA miR-200c using a CaCO3-based delivery system effectively increases miR-200c expression in OSCC cells and suppresses oncogenic activities, thereby reducing tumor growth in a preclinical animal model. In addition to its capacities to promote bone regeneration and inhibit inflammation-associated bone resorption, miR-200c may serve as an adjuvant tool in reconstructive surgeries of patients with advanced OSCC to enhance bone healing and decrease tumor recurrence. Nevertheless, several critical questions must be addressed before considering clinical applications. First, although we did not observe any significant changes in body weight following local administration, it is essential to investigate the clearance rates of the plasmid/CaCO3 system in vivo and conduct systemic toxicity assessments. Furthermore, we must acknowledge the limitations of our current in vivo studies. This study could not provide insights into the tumor microenvironment or potential host immune responses due to the inherent constraints of animal models. The variations of observation time points may also contribute to the inconsistent pathologies of in vivo tumors between pretreated OSCC cells and local administration of miR-200c. Future research should prioritize more advanced investigations that delve into the influence of immune responses and microenvironment dynamics on tumor growth when miR-200c is locally overexpressed. Such comprehensive studies will be instrumental in further assessing the feasibility and safety of this delivery system for clinical use.

Author Contributions

Q.J. Ding, A.V. Mora Mata, contributed to design, data acquisition, analysis, and interpretation, drafted the manuscript; M.T. Remy, contributed to data acquisition and analysis, drafted and critically revised the manuscript; C. Upara, contributed to data acquisition, analysis, and interpretation, critically revised the manuscript; J. Hu, contributed to design, data acquisition and analysis, critically revised the manuscript; A.J. Haes, contributed to design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; E. Lanzel, contributed to data analysis and interpretation, drafted and critically revised the manuscript; H. Sun, contributed to conception, data interpretation, critically revised the manuscript; M.R. Buchakjian, contributed to conception, drafted and critically revised the manuscript; L. Hong, contributed to conception, design data analysis and interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

sj-pdf-1-jdr-10.1177_00220345231216110 – Supplemental material for CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma
sj-pdf-1-jdr-10.1177_00220345231216110.pdf (141KB, pdf)

Supplemental material, sj-pdf-1-jdr-10.1177_00220345231216110 for CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma by Q.J. Ding, M.T. Remy, C. Upara, J. Hu, A.V. Mora Mata, A.J. Haes, E. Lanzel, H. Sun, M.R. Buchakjian and L. Hong in Journal of Dental Research

Acknowledgments

The authors thank Brad A. Amendt and Steven Eliason for their assistance in plasmid preparations used in this study.

Footnotes

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the public–private partnership (P3) of the University of Iowa Strategic Initiatives Fund and the National Institute of Dental and Craniofacial Research (NIDCR) (grant R01DE026433 [L. Hong]; R01DE029159 [H. Sun]) of the National Institutes of Health (NIH). This research was further facilitated by the IR/D (Individual Research and Development) program associated with Amanda J. Haes’s appointment at the National Science Foundation. Support was also received from the NIH/NIDCR (grant F31DE031153 [M.T. Remy] and T90DE 023520 [M.T. Remy, Q.J. Ding]).

A supplemental appendix to this article is available online.

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Supplementary Materials

sj-pdf-1-jdr-10.1177_00220345231216110 – Supplemental material for CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma
sj-pdf-1-jdr-10.1177_00220345231216110.pdf (141KB, pdf)

Supplemental material, sj-pdf-1-jdr-10.1177_00220345231216110 for CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma by Q.J. Ding, M.T. Remy, C. Upara, J. Hu, A.V. Mora Mata, A.J. Haes, E. Lanzel, H. Sun, M.R. Buchakjian and L. Hong in Journal of Dental Research

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