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July 5, 2025

Boosting Hydroxyl Radical Generation with Nitrogen Vacancies Modified

Introduction

Oral squamous cell carcinoma (OSCC) is a major global health issue, with over 350,000 new diagnosed cases and a high mortality rate each year.¹ Conventional surgical treatment based on removing primary lesions causes serious side effects, such as loss of facial function (chewing, swallowing, and speaking) and different levels of aesthetic change.² In addition, the much higher levels of DNA-damage repair in cancer cells than in normal cells can easily cause tumor resistance during radiotherapy and chemotherapy.³

Photodynamic therapy (PDT), with its advantages of light-controlled selectivity, minimal invasiveness, and low side effects, has garnered significant attention.⁴ FDA-approved photosensitizers, such as chlorin e6 (Ce6) and 5-aminolevulinic acid, have been used to treat superficial tumors.^5,6 However, almost all solid cancers are characterized by hypoxia, and the hypoxic tumor microenvironment can reduce effectiveness of the two abovementioned agents.⁷ Other photosensitizers, such as MnO₂ and Fe-MOF, can catalyze the production of highly toxic •OH with H₂O₂,^8,9 inducing multiorganelle damage in tumors. While these can be modified for targeted •OH generation, the potential release of metal ions poses a threat to health.^10,11 In the ongoing search for anticancer photocatalysts that can inflict oxygen-independent tumor damage, inhibit DNA-damage repair, and prevent organ damage from metal residues, researchers have set their sights on polymeric carbon nitride (PCN), a nanomaterial composed exclusively of carbon and nitrogen elements. Unfortunately, the PDT anticancer effect of pristine PCN is not satisfactory due to the limited •OH caused by surface inertness and severe carrier recombination.¹²

In this study, nitrogen vacancy–modified PCN (N_V-PCN) was synthesized to improve the cell accessibility of carbon nitride by increasing the content of –NH₂ and provide more reactive sites for H₂O₂ reduction. As presented in Figure 1, N_V-PCN leads to a synchronous explosion of •OH around the nuclei and mitochondria of Cal-27 cells under illumination, which synchronously leads to nucleus DNA damage (increased expression of double-strand break marker γH₂AX) and mitochondrial dysfunction (decreased mitochondrial membrane potential [MMP]). Mitochondrial dysfunction triggers an ROS storm to intensify DNA damage, and the ATP energy chain is attenuated to inhibit DNA-damage repair (reduced expression of 53BP1 and GADD45A). Compared to Ce6, N_V-PCN exhibits excellent anticancer performance in vitro and in vivo, opening up new avenues for carbon nitride-based cancer PDT.

Figure 1 Schematic illustration of (a) the preparation process and (b) the therapeutic process of N_V-PCN.

Results and Discussion

Material Characterization of N_V-PCN

The morphologies of pristine PCN and N_V-PCN were observed by TEM. N_V-PCN exhibited an irregular flaked structure with a diameter of approximately 100 nm (Figure 2a), half that of PCN (Figure S1). XRD and FTIR tests were conducted to reveal the crystal phase and surface functional groups. In Figure 2b, the pristine PCN exhibits two typical diffraction peaks at 13.0° and 27.4°, which are assigned to the periodic stacking of tri-s-triazine rings in plane (100) and graphitic layer (002), respectively.¹³ The XRD pattern of N_V-PCN exclusively presents the diffraction peaks of the PCN component, revealing that the crystal structure of N_V-PCN is similar to that of PCN. However, all peaks of N_V-PCN weaken compared to PCN, suggesting a distorted structure and smaller dimensions of unit cells of N_V-PCN than PCN.¹⁴ As shown in Figure 2c, PCN and N_V-PCN possess similar FTIR spectra. Specifically, the peaks at 3500–3000, 1650–1245, and 810 cm⁻¹, are attributable to the stretching vibrations of N–H group, stretching modes of tri-s-triazine rings in plane, and the out-of-plane bending mode of heptazine rings, respectively.¹⁵ This indicates that the basic structure of carbon nitride was preserved during the secondary roasting process.

Figure 2 Characterization of as-prepared N_V-PCN. (a) TEM image of N_V-PCN. (b) XRD patterns, (c) FTIR spectra, and (d) N1s XPS signals of PCN and N_V-PCN. (e) Structure diagram of N_V-PCN. (f) UV-vis diffuse reflectance spectra. Inset: Tauc plots. (g) Band structures of PCN and N_V-PCN. h) In situ DRIFT spectra of H₂O₂ on PCN and N_V-PCN under illumination for 10 min at 5-min intervals. (i) In situ EPR signals of H₂O₂ over PCN and N_V-PCN under illumination.

XPS was used to detect the type and position of vacancies formed in the secondary roasting process. As shown in Table S1, N_V-PCN (71.66%) had higher C/N (at.%) than PCN (71%), which proved that nitrogen vacancies (N_V) were introduced in carbon nitride after secondary roasting. For PCN, the three characteristic peaks at 288.30, 285.90, and 284.72 eV in C_1s XPS spectra (Figure S2) are attributable to N–C=N, C–NHx, and C=C (hybridized carbon in heptazine ring), respectively. The three characteristic peaks at 398.62, 399.30, and 400.85 eV in N_1s XPS spectra of PCN (Figure 2d) are attributable to N–C=N, N3C, and NH_X, respectively.¹⁶ The corresponding characteristic C_1s and N_1s N–C=N peaks of N_V-PCN are shifted to lower binding energies compared with those of PCN, which can be attributed to the negative electron enrichment around N_V. To verify the position of the N vacancy in N_V-PCN, the relative content of different N species was analyzed, with results shown in Figure 2d. The peak-area ratio of N3C decreased from 35.62% in PCN to 28.84% in N_V-PCN, which indicates that N3C defects were introduced in N_V-PCN. As shown in Figure S3, N3C has two different positions: site ① and site ②. The out-of-plane heptazine bending modes in PCN and N_V-PCN remain unchanged (Figure 2c), which excludes the generation of N3C defects at site ①. Based on these results, the structure of N_V-PCN catalyst was deduced (Figure 2e).

Since N_V-PCN was used as a PDT agent for cancer therapy, its optical properties and band structure were examined by UV-vis diffuse reflectance spectra, Tauc plots, and Mott–Schottky plots. As shown in the inset in Figure 2f, the bandgap (E_g) of PCN is 2.78 eV, which caused by the n–π* electronic transitions of the conjugated PCN framework and is close to that reported for graphitic carbon nitride.¹⁷ Compared with PCN, N_V-PCN shows larger E_g of 2.85 eV, which is attributable to the quantum confinement effect induced by the smaller nanosheet of N_V-PCN than PCN.¹⁸ The slightly stronger light-absorption capacity for visible light of N_V-PCN than PCN (Figure 2f) might attributable to the midgap state introduced by N_V.¹⁹ In addition, a Mott–Schottky plot was used to obtain the semiconductor type and flat-band position of the PCN and N_V-PCN samples. As illustrated in Figure S4, the slopes of the linear parts in the two curves are positive, indicating the typical n-type semiconductor characteristics of PCN and N_V-PCN.²⁰ The flat-band potentials (E_fb) of PCN and N_V-PCN were determined to be −1.07 and −1.16 eV (vs Ag/AgCl at pH 6.6), corresponding to the conduction-band potential at −0.48 and −0.57 eV (vs RHE) (relevant equations: E(NHE) = E(Ag/AgCl) + 0.197, E(RHE) = E(NHE) + 0.0591 × pH).²¹ Then, the valence-band position was calculated by adding the conduction-band potential to the bandgap, which was 2.30 eV (PCN) and 2.28 eV (N_V-PCN). Based on these results, the band structures of PCN and N_V-PCN were deduced, and these are shown in Figure 2g. Therefore, PCN and N_V-PCN have suitable band structures to catalyze the reaction of H₂O₂/•OH+ OH⁻.

In situ DRIFT spectroscopy measurements (conducted in a confined space and purged for 10 min after adding H₂O₂) were performed to reveal the mechanism of photocatalytic •OH generation over PCN and N_V-PCN. As shown in Figure 2h, compared with the pristine PCN, the peak intensity is significantly enhanced after introducing N_V, indicating that N_V facilitates the adsorption of H₂O₂ on carbon nitride. The peaks at 1602, 1405, and 854 cm⁻¹ can correspond to the stretching vibrations of the N–C=N bond of N_V-PCN, CN–O bond between N_V-PCN and H₂O₂, and HO–OH bonds of H₂O₂, respectively.²² For N_V-PCN, all peaks become weaker as light-irradiation time increases and the HO–OH stretching vibration peak moves to the direction of the high wave from 850 to 856 cm⁻¹, which is attributable to the reduction in H₂O₂ by photoelectrons on N_V-PCN into •OH and OH⁻. Therefore, we speculate that H₂O₂ is adsorbed on the N site of the heptazine ring attached to N_V on N_V-PCN.

To evaluate the ability of PCN and N_V-PCN to generate •OH, we examined the in situ EPR spectrum to measure •OH. Figure 2i exhibits the characteristic 1:2:2:1 •OH radical signal at different time points,²³ indicating that both PCN and N_V-PCN catalysts can reduce H₂O₂ to •OH. For PCN, •OH increases within 1–3 min, but stabilizes at 4 min, which means that the production of •OH equals annihilation.²⁴ For N_V-PCN, •OH continues to rise within 1–4 min, and the overall signal intensity on N_V-PCN is much more prominent than that on PCN, indicating the stronger ability of of N_V-PCN to produce •OH than PCN with equivalent PCN (×1) and even higher than threefold PCN (×3) at 4 min (Figure S5), which is attributable to the more active sites for reduction of H₂O₂ on N_V-PCN than that on PCN.

N_V-PCN Upregulated •OH Expression in Cal-27 Cells under LED Irradiation

PDT is an innovative approach in cancer treatment, offering distinct advantages over conventional therapeutic methods, such as low toxicity, precision, spatiotemporal control, and minimal invasiveness.²⁵ To circumvent the resistance to O₂-dependent PDT in hypoxic tumors, we designed a novel non-O₂-dependent type I photosensitizer—N_V-PCN. It is well established that the level of H₂O₂ in cancer cells is significantly higher than that in normal cells.²⁶ The aim of this research was to utilize N_V-PCN to convert the overexpressed H₂O₂ within tumors into highly toxic •OH, thereby destroying cancer cells. Although the generation of •OH has been investigated in material characterization, further investigation is warranted to explore its potential in biological applications.

Therefore, to further validate N_V-PCN–promoted tumoral •OH expression by PDT, Cal-27 cells were cocultured with N_V-PCN and intracellular •OH levels determined using an •OH probe, which can react with •OH generated from the reduction of H₂O₂ to emit green fluorescence. In Figure 3a, the intracellular •OH was detected via flow-cytometry analysis and CLSM imaging, respectively. As shown in Figure 3b, the ratio of •OH-producing cells is significantly increased in the N_V-PCN + light group, indicating that more •OH was generated. In Figure 3c, we can clearly see that the N_V-PCN + light group exhibits stronger green fluorescence intensity, indicating N_V-PCN possesses similar photocatalytic activity in biological applications. In that case, we propose that N_V-PCN, a novel light photocatalyst, can act as a Fenton-like agent to augment efficient intracellular expression of •OH and induce cell apoptosis, thus achieving PDT.

Figure 3 PDT definitely promoted tumoral •OH expression in vitro. (a)Depicts a schematic diagram of detecting intracellular •OH in Cal-27 cells that have been pre-exposed to light, utilizing a •OH fluorescent probe. (b) Flow-cytometry analysis of •OH production in Cal-27 cells. (c) CLSM images of Cal-27 cells exposed to various conditions, where green fluorescence reflects •OH expression.

In Vitro Anticancer Effects of N_V-PCN Irradiated with LED Light

Prior to assessing the therapeutic effects of N_V-PCN upon white LED-light illumination, it was necessary to use the standard CCK-8 assay to evaluate the cytotoxic effects, which is a very important issue in the medical application of nanomaterials. After incubation with PCN and N_V-PCN for 24 h, no obvious cytotoxicity was observed in Cal-27 cells, even at concentrations up to 1 mg·mL⁻¹, indicating the negligible cytotoxicity of PCN and N_V-PCN (Figure 4a and b). By contrast, when exposed to white LED light, cell viability gradually declined with increasing concentrations of N_V-PCN, and the cell viability of the group incubated with 2 mg·mL⁻¹ N_V-PCN was the lowest among all groups. Nevertheless, even under nonirradiated conditions, 2 mg·mL⁻¹ N_V-PCN still affected cell viability. Therefore, we selected 1 mg·mL⁻¹ as the optimal concentration for subsequent cell experiments. When adding isopropanol (scavenger of •OH)²⁷ into the system (N_V-PCN + light group), cell viability was significantly enhanced (Figure 5a), which showed that •OH played an key role in the N_V-PCN photocatalytic inactivation of cancer cells. In addition, the larger red fluorescence area in the live and dead images for the N_V-PCN + light group corroborates the results of the CCK-8 assay (Figure S6). The results suggest that N_V-PCN not only possesses great biocompatibility but also exhibits more efficient photodynamic killing ability of cancer cells than PCN under illumination. Therefore, this study deeply investigated the biological properties of N_V-PCN.

Figure 4 (a) Optimal concentrations of N_V-PCN acting on cells under light and dark conditions. **P<0.01, ****P<0.0001 compared to 0 μg/mL N_V-PCN + light. (b) Hemolysis values of various samples collected from the supernatants. ****P<0.0001 compared to the other groups.

Figure 5 N_V-PCN as a photosensitizer induces dual damage to cancer nuclear DNA and mitochondria. (a) Relative viability of Cal-27 cells incubated with PCN and N_V-PCN at a concentration of 1 mg·cm⁻¹ for 24 h with white LED light illumination for 30 min. (b) Cellular uptake evaluation of Cal-27 cells treated with N_V-PCN for 0.5–6 h using CLSM images. (c) Immunofluorescence images of γH₂AX foci (green) in Cal-27 cells treated withcontrol, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. Cell nuclei were stained with DAPI (blue). (d) Confocal microscopy images of the JC-1 probe in Cal-27 cells. (e) Flow cytometry of total ROS generation in Cal-27 cells under different treatments using DCFH-DA as intracellular total ROS indicator. Significance calculated by one-way ANOVA: *P<0.05, ****P<0.0001 compared to the control group.

Next, assessment of the capacity of internalizing sufficient N_V-PCN in cancer cells was conducted, because this is critical for further therapeutic effects. Dark-field scattering microscopy was used to visualize the intracellular distribution of N_V-PCN. There was obvious signal enhancement in the N_V-PCN group compared with the control group, showing the efficient uptake of N_V-PCN by Cal-27 cells (Figure S7). As shown in Figure 5b, green fluorescence derived from N_V-PCN is mainly concentrated in the cytoplasm and nucleus and intensifies with prolonged incubation time, indicating that the effective cellular uptake of N_V-PCN is dependent on incubation duration.

N_V-PCN, a potent photosensitizer, converts intracellular H₂O₂ to •OH under white LED-light irradiation. Among ROS, •OH exerts greater damage to cancer cells due to its more aggressive effect on nuclei, inducing DNA double-strand breaks.²⁸ Here, we evaluated DNA double-strand breaks by γH₂AX staining using immunofluorescence labeling (green) and Western blot. The results showed that a prominently higher density of γH₂AX foci occurred in the N_V-PCN + light and Ce6 + light group than in the control, light, and N_V-PCN-alone groups (Figure 5c and S8), indicating that N_V-PCN and Ce6 can greatly increase light-induced DNA damage. However, it has been observed that the occurrence of DNA damage in cancer cells promotes the initiation of DNA damage–repair mechanisms, which results in tumor resistance.²⁹

Moreover, •OH has the potential to induce mitochondrial damage due to the superior reactivity of •OH.³⁰ Considering this, the mitochondrial functions of the different groups were evaluated by analyzing the MMP of cells. As depicted in Figure 5d, when compared with the single-treatment groups (control, N_V-PCN, and light), Cal-27 cells treated with Ce6 and N_V-PCN irradiated with light transformed more JC-1 polymer to JC-1 monomer on the mitochondria, suggesting declining MMP in Cal-27 cells, especially in the N_V-PCN + light group, revealing that more •OH produced by N_V-PCN during PDT eventuates in mitochondrial dysfunction. Ce6, an FDA-approved type II photosensitizer, predominantly functions by leveraging the available oxygen in tumors to generate singlet oxygen, a key mechanism behind its therapeutic effectiveness. Nonetheless, the tumor microenvironment is inherently hypoxic, implying that it lacks the sufficient substrate necessary for the generation of reactive oxygen species (ROS) that are crucial for inflicting mitochondrial damage.³¹ Mitochondria regulate ROS and maintain cellular redox balance. Dysfunction in mitochondria boosts ROS, aggravating DNA damage and possibly leading to cellular dysfunction and disease progression.³² To further verify the total intracellular ROS generation of N_V-PCN under LED illumination, DCFH-DA was used as a fluorescent probe to monitor the intracellular production of ROS. DCFH-DA is hydrolyzed by esterase after entering the cell to form DCFH, which can react with ROS in cells and exhibit green fluorescence. Notably, intense green fluorescence was observed in the N_V-PCN + light (Figure S9). In contrast, negligible fluorescence was observed in the control, N_V-PCN, and light groups. We also quantitatively detected the generation levels of total intracellular ROS in Cal-27 cells using flow cytometry (Figure 5e), and obtained similar results to the aforementioned.

In eukaryotic cells, mitochondria are critical for regulating intracellular energy.³³ After mitochondrial damage, intracellular ATP content decreases.³⁴ As shown in Figure 6a, compared with the single-treatment groups, the ATP content of the N_V-PCN + light group was significantly reduced, leading to the downregulation of DNA-damage repair. However, the Ce6 + light group exhibited enhanced damage repair compared to the N_V-PCN + light group. 53BP1 and GADD45A, key factors in the DNA damage–repair process,^35,36 were further evaluated. N_V-PCN + light treatment markedly decreased the protein expression of 53BP1 and GADD45A (Figure 6b). Additionally, the downregulation of 53BP1 and GADD45A was further confirmed by Western blot analysis (Figure 6c), and cancer cells treated with N_V-PCN + light exhibited failed DNA-damage repair.

Figure 6 N_V-PCN-mediated PDT promoted Cal-27 cell apoptosis. (a) Intracellular ATP levels of Cal-27 cells after various treatments. (b) Expression of 53BP1 and GADD45A in Cal-27 cells examined by fluorescence microscopy. (c) Expression levels of γH2AX, GADD45A, 53BP1, pro-caspase 3, cleaved caspase 3, and β-actin proteins in cells treated for 24 h in different groups detected by Western blot experiments. β-actin protein was used as the internal control. (d) EDU assay of Cal-27 cells with control, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. (e) Transwell assay of Cal-27 cells with control, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. The concentration of N_V-PCN was 1 mg·mL⁻¹. Significance calculated by one-way ANOVA: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Then, we evaluated their therapeutic effects upon light irradiation by EDU assays. As illustrated in Figure S10, there was 80%, 76%, and 60% cell proliferation of Cal-27 cells after treatment by N_V-PCN alone, light alone, and Ce6 + light, respectively, while only 26% survived under N_V-PCN + light treatment (smaller red fluorescence area, Figure 6d). Similarly, the Transwell assays revealed less cell migration in the N_V-PCN + light group than in the other three groups (Figure 6e). These results, along with previous findings, raise the possibility that compared to Ce6, N_V-PCN would induce a more powerful anticancer effect due to enhancing ROS generation and inhibiting cancer-cell proliferation under light irradiation.³⁷

In addition, flow cytometry was utilized to quantitatively detect cell apoptosis under various treatments by annexin V–FITC/propidium iodide double staining. As shown in Figure 7a, no obvious apoptosis (early apoptosis or late apoptosis) was detected in the control, N_V-PCN-, or light-alone groups. On the contrary, when the cells were treated with Ce6 and N_V-PCN under light irradiation, early-apoptosis cells and late-apoptosis cells increased to 3.55% and 3.66% and to 14.51% and 33.42%, respectively. The results of Western blot and immunofluorescence analysis further confirmed that the apoptosis-related protein cleaved caspase 3 was activated and its expression was significantly greater in the N_V-PCN+ light group than in the other groups (Figure 6c, 7b), indicating that N_V-PCN + light cotreatment induces a powerful PDT effect and effectively triggers tumor-cell apoptosis. Taken together, these results indicate that the as-prepared N_V-PCN under LED irradiation enhanced anticancer activity by inducing •OH-mediated DNA damage and mitochondrial dysfunction, downregulating ATP, amplifying ROS storms, inhibiting DNA-damage repair, and upregulating apoptosis-related proteins.

Figure 7 (a) Qualitative flow-cytometry data plot indicating the increase in apoptosis of Cal-27 cells after different treatments for 24 h. (b) Immunofluorescence images of cleaved caspase 3 (green) in Cal-27 cells treated with control, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. Cell nuclei were stained with DAPI (blue).

In Vivo Antitumor Therapeutic Effect and Biosafety of N_V-PCN Irradiated with LED Light

The excellent performance of N_V-PCN at the cellular level prompted us to evaluate the PDT efficacy of N_V-PCN on solid cancers in BALB/c nude mice bearing Cal-27 tumors. Animal experiments were performed according to the protocols approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (DW2023049). The treatment protocol is shown in Figure 8a. Prior to conducting the antitumor study, the biodistribution of Cy5.5–N_V-PCN after intratumoral injection was detected by tracking Cy5.5 fluorescence using an in vivo imaging system.³⁸ As shown in Figure 8b, the long-term distribution of Cy5.5–N_V-PCN fluorescence over 72 h indicates the excellent tumor-accumulation capacity of N_V-PCN.³⁹ It was observed that in addition to tumor tissue, kidney, one of the main metabolic organs, also exhibited much fluorescence aggregation compared with the control group within 6 h, indicating that some N_V-PCN can be excreted mainly through the kidneys without apparent impacts on the remaining organs.

Figure 8 In vivo antitumor effect of N_V-PCN-mediated PDT. (a) Schematic of the therapeutic process for cancer-bearing nude mice. (b) Fluorescence images of Cal-27 cancer-bearing mice and ex vitro fluorescence images of major organs and tumor tissue after intratumoral injection of Cy5.5–N_V-PCN at different time points. (c) Time-dependent surveillance of body weight for mice with different treatments over 22 days (n=4). (d) Time-dependent surveillance of tumor volume for mice with different treatments over 22 days (n=4). (e) Cancer images of each group derived from BALB/c mice at day 22 posttreatment. (f) H&E and immunohistochemical staining of tumor tissue of mice after various treatments. Significance calculated by one-way ANOVA: ****P<0.0001.

Thereafter, the in vivo anticancer effects of N_V-PCN–mediated PDT were investigated. During the experiments, the weight of mice in all groups changed slightly within 22 days (Figure 8c), suggesting that N_V-PCN has no obvious systemic side effects in vivo. Compared with the control group and the single-treatment groups (N_V-PCN, light), Ce6 + light and N_V-PCN + light effectively suppressed tumor growth, with the N_V-PCN + light group showing a more pronounced effect (Figure 8d). The satisfactory photosensitizing effect is attributed to the enhanced generation of ROS, leading to the death of cancer cells in an apoptosis pathway. After 22 days, all mice were euthanized to harvest tumor tissue (Figure 8e) and weigh them to directly explore the efficacy of different treatments. The tumor weights are shown in Figure 8e. Mice that received Ce6 + light and N_V-PCN + light treatment had significantly smaller tumor volume, which matches well with the tumor-growth curves in Figure 8d, further indicating that N_V-PCN presents significant advantages in anticancer therapy.⁴⁰ The therapeutic effect was also confirmed by pathological results (Figure 8f). All groups showed varying degrees of tumor necrosis on H&E staining. Remarkably, the N_V-PCN + light group showed tumor-tissue damage (approximately 75% and 80%). Moreover, cancer cells in the N_V-PCN + light group exhibited the weakest Ki67 signal and the strongest TUNEL signal, showing that this treatment can maximally inhibit cell proliferation and promote cell apoptosis. It is particularly noteworthy that N_V-PCN PDT was confirmed to effectively increase the expression of γH₂AX and cleaved caspase 3, implying serious DNA damage and apoptosis.

Nanomedicine safety is a critical concern in its application in biomedicine. Consequently, experiments including H&E staining of main organs and blood hematology were performed to ensure the safe application of N_V-PCN and Ce6 in vivo. We collected the major organs (heart, liver, spleen, lung, and kidney) after different treatments for H&E staining, and the histological morphology of the major organs appeared unaffected in all the groups (Figure 9a). The blood biochemistry and hematology tests also showed negligible effects in the significant parameters (Figure 9b and c). All of these results demonstrate that N_V-PCN, similar to Ce6, is a safe nanoplatform for cancer therapy.

Figure 9 In vivo toxicity and safety assessment of N_V-PCN. (a) Hematoxylin and eosin–stained tissue sections from the mice to monitor histological changes in heart, liver, spleen, lung, and kidney 22 days after intratumoral injection of the N_V-PCN solution. (b) Blood biochemistry analysis of the mice treated with Ce6 and N_V-PCN. (c) Blood hematology analyses of mice on the last day.

Methods

Catalyst Preparation

Pristine PCN was obtained by annealing melamine powder (10 g) at 550°C for 240 min (heating rate 3°C min⁻¹) and grinding it homogeneously under an air atmosphere. Subsequently, PCN (0.3 g) was subjected to annealing at 520°C for 60 min (heating rate of 10°C min⁻¹) under an argon atmosphere to synthesize N_V-PCN.

Characterization

Transmission electron microscopy (TEM) images were obtained using a Tecnai G2 F20 STwin. Before measurement, a dispersion solution of PCN and N_V-PCN NSs was deposited on a carbon film supported by copper grids. X-ray diffraction (XRD) patterns were attained with a Bruker D8 Advance equipped with Cu Ka radiation (40 kV). Fourier-transform infrared (FTIR) spectra were recorded by a Bruker Tensor II spectrometer using a KBr pellet. X-ray photoelectron spectroscopy (XPS) was performed with a VG Scientific Escalab Mark II spectrometer. UV-vis diffuse reflectance spectra were obtained with a Shimadzu UV-3600 spectrometer. Electrochemical measurement was performed on a CHI 760E workstation using a conventional three-electrode configuration, where Ag/AgCl and platinum plates were used as reference and counter electrode, respectively. The working electrode was prepared by mixing catalyst (2.5 mg), water (300 μL), ethanol (200 μL), and Nafion (5 wt%, 25 μL) evenly. The slurry (20 μL) was then spread to 0.5 cm² on a fluorine-doped tin oxide glass electrode. After the electrode had dried, the edge portion of the electrode was sealed with epoxy adhesive. Motto–Schottky plots and photocurrent signals were collected using 0.1 M of Na₂SO₄ solution as electrolyte. In situ DRIFT spectra were recorded in a sealed in-site reaction cell (equipped with Praying Mantis diffuse reflectance accessory and MCT detecor).

The catalyst was added to the reaction cell and processed at 100°C under argon for 1 h. Then, 10 μL of H₂O₂ solution was added, purged for 10 min under argon conditions, and the test data obtained after the catalyst had started to illuminate. In situ electron paramagnetic resonance (EPR) measurements were conducted on a Bruker EMXplus 10/12 spectrometer. For preparation of the test samples, 5 mg of catalyst was ultrasonically dispersed in 1 mL of acetonitrile, and 45 μL of the above mixture and 5 µL of H₂O₂ were mixed with 20 μL of DMPO acetonitrile solution (1 mg·μL⁻¹). The EPR spectra were measured at an interval of 30 seconds and swept for 30 seconds with no superposition of signals. A BD FACSCanto II flow cytometer was used to perform flow-cytometry analyses. A multifunctional microplate reader was used to perform cell-viability and hemolysis experiments (Infinite M1000 Pro, Tecan). A white light–emitting diode (LED) with emission centered at 400–600 nm purchased from Shenzhen Zhongyu Technology (China) was used as the white-light source. The intensity of the white LED for photocatalysis and PDT was 50 mW·cm⁻² for 30 min.

Cell Culture

Human oral squamous carcinoma cells (Cal-27) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Cal-27 cells were kept in DMEM (Gibco) supplemented with 10% FBS, 1% penicillin–streptomycin and 1% L-glutamine (Meilunbio) at 37°C in a cell incubator (Thermo Scientific) containing 5% CO₂. For cell passage, cells were digested with 0.25% trypsin–EDTA (Gibco) and then resuspended in fresh culture medium.

Measurement of Endogenous •OH

After coculturing N_V-PCN or Ce6 and Cal-27 cells in a 24-well plate (1×10⁵ cells/well) for 6 h, cells were exposed or received no exposure to LED irradiation (50 mW·cm⁻², 30 min). The culture medium was removed and each well washed repeatedly with PBS. Subsequently, the cells were incubated with HKOH-1r (MCE, HY-D1159) in a cell incubator for 30 min. •OH level was detected and quantified using confocal laser scanning microscopy (CLSM, Olympus, FV3000) and flow cytometry (Agilent Corporation, NovoCyte).

Cellular Uptake

The Cal-27 cells were seeded in 24-well plates (1×10⁵ cells/well) and cultured for 24 h. After incubation with N_V-PCN, Cal-27 cells were fixed with 4% paraformaldehyde (PFA, Leagene), stained with DAPI (Beyotime), and finally the uptake of Cal-27 cells was observed with CLSM.

Cytotoxicity Assay

Cell viability was assessed using Cell Counting Kit 8 (CCK-8). The Cal-27 cells were cultured in 96-well plates (1.2×10⁴ cells/well). After 24 h of incubation, the cells were treated with N_V-PCN (1 mg·mL⁻¹, 100 µL) for 6 h and then irradiated with the white LED (50 mW·cm⁻², 30 min). After 24 h, 100 μL of fresh DMEM containing CCK-8 (10%) was added and the treatment continued at 37°C for 30 min. Absorbance was then measured at 450 nm using a full-wavelength enzymograph (SpecteaMax plus 384, USA) to assess cell viability.

A fluorescent live/dead cell assay was applied to visualize the cell viability of N_V-PCN combined with visible light irradiation in Cal-27 cells. Typically, the cells were treated using the same method as described above and then stained with a Live/Dead Cell Staining Kit (BestBio, China) in accordance with the manufacturer’s instruction. Afterwards, live and dead cells, emitting green and red fluorescence, respectively, were observed using CLSM.

In addition, hemocompatibility assays were used to measure the cytotoxicity of N_V-PCN. Fresh blood was obtained from BALB/c mice, red blood cells acquired via centrifugation (3000 rpm, 15 min), and then these were mixed with N_V-PCN concentrations of 200, 400, 600, 800, and 1000 ug·mL⁻¹. PBS and ddH₂O were set as the negative and positive control group, respectively. After incubation for 4 h, the solution was centrifuged (3000 rpm, 15 min) and the absorbance spectra of the supernatant were measured at 540 nm.

DNA Damage by N_V-PCN

Cal-27 cells (1×10⁵/well) were seeded into 24-well plates for 24 h and then incubated with N_V-PCN or Ce6 (1 mg·mL⁻¹, 500 µL) for 6 h. Next, those cells were exposed or received no exposure to LED irradiation. PFA (4%) and Triton X-100 (0.5%) were used to fix and permeate the cells, respectively. Then, the cells were treated with blocking buffer (1% BSA, 30 min) at room temperature and further incubated with anti-phospho-histone γH₂AX rabbit monoclonal antibody (UpingBio, YP-Ab-01510, dilution 1:1000) at 4°C overnight. Then, fluorescein isothiocyanate (FITC; Beyotime Biotechnology, dilution 1:800) was added and incubated at room temperature for 1 h after being washed with PBS three times to remove excess antibody. Cell nuclei were stained by DAPI for 5 min. Finally, fluorescence images were acquired with CLSM.

Mitochondrial Membrane Potential Analysis

Cal-27 cells were inoculated in 24-well plates (1×10⁵ cells/well) for 24 h. The cells were then treated with control, N_V-PCN, and Ce6 in the dark for 6 h with or without LED irradiation. JC-1 dyeing solution (configured according to manufacturer’s instructions) was then added and incubated at 37°C for 20 min. Finally, the cells were washed three times with JC-1 dye buffer and images taken by CLSM.

Total Intracellular ROS Generation

Cal-27 cells (2 × 10⁶/well) were seeded into six-well plates. Five groups were set: (a) control, (b) N_V-PCN, (c) light only, (d) Ce6 + light, and (e) N_V-PCN + light. Then, cells in the corresponding groups were incubated with control, N_V-PCN, and Ce6 (1 mg·mL⁻¹, 500 µL). After 6 h, the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Meilunbio, MA0219) was added as a fluorescence probe and the mixture incubated in the dark for 30 min. Afterwards, groups (c), (d), and (e) were disposed of” modified to “Afterwards, groups (c), (d), and (e) were exposed to illumination. Finally, flow cytometry and fluorescence microscopy (Leica, Germany) were used to study the generation of intracellular ROS.

Detection of Intracellular ATP

The Cal-27 cells were cultured in 96-well plates (1.2×10⁴ cells/well). After 24 h of incubation, the cells were treated with N_V-PCN and Ce6 (1 mg·mL⁻¹, 100 µL) for 6 h, followed by light irradiation, then after incubation for another 24 h, the cells were collected. In conjunction with an ATP chemiluminescence assay kit (Elabscience, E-BC-F002), the fluorescence value of each well was detected by a Varioskan multifunctional enzyme labeler (Thermo Scientific, Varioskan LuX).

Immunofluorescence Staining

The Cal-27 cells were seeded into 24-well plates (1×10⁵ cells/well) for 24 h and then incubated with N_V-PCN and Ce6 (1 mg·mL⁻¹, 500 µL) for 6 h. Next, they were exposed or received no exposure to LED irradiation. PFA (%) and Triton X-100 (0.5% were used to fix and permeate the cells, respectively. Then, the cells were treated with blocking buffer (1% BSA, 30 min) at room temperature and further incubated with 53BP1 (Beyotime), GADD45A (Bioss), and cleaved caspase 3 (Uping Bio) primary antibody at 4°C overnight. Subsequently, Cy5-labeled or FITC-labeled (Beyotime Biotechnology, dilution 1:800) goat anti-rabbit IgG was added and incubated at room temperature for 1 h after being washed with PBS three times to remove excess antibody. Cell nuclei were stained with DAPI for 5 min. Finally, fluorescence images were acquired with CLSM.

In Vitro Anticancer Effect of N_V-PCN

The Cal-27 cells were seeded in 24-well plates (1×10⁵ cells/well) for 24 h and treated with N_V-PCN or Ce6 for 6 h. To evaluate the PDT effect, the cells were exposed or received no exposure to LED irradiation. The cells were then analyzed using 5-ethinyl-2’-deoxyuridine (EdU; KeyGen), Transwell assays, and annexin V–FITC/propidium iodide (KeyGen, Nanjing, China) in accordance with the manufacturer’s guidelines.

Western Blotting Assay

The Cal-27 cells were seeded in a six-well plate (2 × 10⁶ cells/well) and cultured for 24 h. Then, N_V-PCN (1 mg⋅mL⁻¹) was added to two groups (N_V-PCN and N_V-PCN + light) for 6 h. The cells of the light or N_V-PCN + light group were irradiated with white LED light (50 mW·cm⁻², 30 min). After 24 h of incubation, cells were collected and lysed by precooled RIPA buffer for 30 min. After centrifugation (12,000 rpm) for 20 min at 4°C, the supernatant was mixed with the loading buffer and protein concentrations of the four groups determined using a BCA protein assay kit (KeyGen, BioTECH). Proteins were then separated and transferred. The membranes were blocked at room temperature (protein-free rapid blocking solution, Boster) for 20 min. After that, the membranes were incubated with primary antibodies overnight at 4°C. These antibodies were pro-caspase 3 (UpingBio, YP-Ab-00345, dilution 1:1500), cleaved caspase 3 (UpingBio, YP-Ab-00003, dilution 1:1500), γH2AX (UpingBio, dilution 1:1000), GADD45A (Bioss, bs-1360R, dilution: 1:200), 53BP1 (Abcam, ab243868, dion:luti 1:1000), and β-actin (ABclonal, AC038, dilution 1:10,000). Then, the membranes were washed and incubated with HRP-conjugated secondary antibody (UpingBio, YP848537-H, dilution 1:10000) for 1 h. Finally, stained with the ECL detection kit (Meilunbio, MA0186), the protein bands were observed using Compass software (Bio-Rad chemidoc XRS+, Universal Hood II) and the appropriate protein gray values calculated.

Transwell Assay

The migration ability of Cal-27 cells was detected by a Transwell assay. Cal-27 cells were seeded in 6-well plates at a density of 2×10⁶ cells per well. After 24 h, the cells were treated with N_V-PCN (1 mg·mL⁻¹, 100 µL) for 6 h and then irradiated with white LED light (50 mW·cm⁻², 30 min) for another 24 h. The upper chambers of Transwell plates (Corning Inc.; Corning, NY, USA) use 8.0 μm-pore filters. Cal-27 cells in different groups were collected and seeded on the upper chambers (1.0 × 10⁵ cells per chamber) in 200 μL serum-free medium. In contrast, the lower chambers were filled with 600 μL of DMEM medium containing 20% (V/V) FBS to induce cell invasion. After 24 h, the non-invading cells on the upper side of the filters were removed, and the invading cells on the lower side were fixed with 4% (W/V) PBS-buffered PFA for 30 min and stained with 0.2% (W/V) crystal violet for 10 min. The stained cells were visualized and counted in three random fields using an inverted microscope.

In Vivo Biodistribution and Cancer Accumulation of N_V-PCN

In order to evaluate the in vivo biodistribution of N_V-PCN, the healthy mice were intratumorally injected with Cy5.5-labeled N_V-PCN (5 mg·mL⁻¹, 50 μL). At time points of 1 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h), the mice were euthanized to collect the main organs (heart, liver, spleen, lung, and kidney) and tumor tissue. Fluorescence was visualized with a small-animal live optical 3D imaging system (PerkinElmer, IVIS Spectrum). Based on in vivo imaging-system observations, mice were euthanized after injection of Cy5.5–N_V-PCN at different time points, and then the harvested cancer tissue and major organs were analyzed by ex vivo fluorescence imaging.

In Vivo Anticancer Effects of N_V-PCN

To construct a tumor-bearing BALB/c mouse model, harvested Cal-27 cells were suspended in a suitable amount of PBS. Cancer cells were injected into the proximal axilla of the right hind limbs of mice to construct an OSCC mouse model. The mice were then injected with 50 µL (2 × 10⁷ cells) of the suspension into their right hind-limb axilla. When tumors had grown to 100 mm³, the mice were divided into four groups——–—(a) PBS, (b) N_V-PCN, (c) light only, (d) Ce6 + light, and (e) N_V-PCN + light—that were treated with PBS, N_V-PCN, or Ce6 solution via intratumoral injection every 2 days for a total of four times with or without light irradiation. The tumor volume was measured with a vernier caliper and calculated as (length × width²)/2 for 22 days. After euthanasia, the cancer tissue and main organs (heart, liver, spleen, lung, kidney) were weighed, fixed in 4% PFA solution, and tissue slices embedded in paraffin. For further evidence of cancer apoptosis, tumor slices were stained with HE, Ki67 (Abcam, ab15580), Tunel (Beyotime), γH₂AX, and cleaved caspase 3 antibody, and immunofluorescence images were captured by CLSM.

In Vivo Safety Assessment

Blood samples were collected from each group. About 100 μL of the samples were treated with anticoagulant (potassium EDTA) for hematology analysis. The residual blood was precipitated at room temperature for 2 h, and the plasma was collected by centrifugation at 3500 g for 10 min to assess liver (AST, ALT) and kidney (CRE, urea) function indices. Additionally, main organs (heart, liver, spleen, lung, kidney) were stained with H&E to observe changes in tissue structure using light microscopy. All analyses were conducted at Wuhan Xavier Biotechnology.

Statistical Analysis

All data are presented as means ± standard deviation (SD) of at least three independent replicates for each experiment. Statistical analysis was performed using GraphPad Prism 9. Data were compared using Student’s t test and one-way ANOVA. Statistical differences are indicated by asterisks in the figures: *P<0.05, **P<0.01, and ***P<0.001.

Conclusion

In summary, we designed nitrogen vacancy (N_V)–modified PCN (N_V-PCN) for PDT of Cal-27 cell-induced OSCC that effectively induced Cal-27-cell apoptosis by triggering DNA damage and inhibiting DNA-damage repair. The introduction of N_V not only further improved the cell accessibility of PCN by increasing the content of –NH₂ but also provided reactive sites for H₂O₂ reduction and facilitated carrier separation, which are beneficial for large-scale production of •OH. Moreover, EPR and intracellular •OH assays revealed that N_V-PCN exhibited superior •OH-generation efficiency under visible-light irradiation. Therefore, N_V-PCN leads to the explosion of •OH around the nuclei and mitochondria of Cal-27 cells under illumination, which effectively kills Cal-27 cells via synchronously leading to nucleus DNA damage and mitochondrial dysfunction. Then, mitochondrial dysfunction triggers an ROS storm to intensify DNA damage. It also attenuates the ATP energy chain to inhibit DNA-damage repair. Compared to the O₂-dependent photosensitizer Ce6, N_V-PCN-based PDT has stronger antitumor efficacy in vitro and in vivo. Therefore, this kind of nanoparticle not only represents an efficient photosensitizer for enhanced PDT of cancer but also opens up new avenues for in-depth study on carbon nitride-based cancer PDT.

Ethics Approval and Consent to Participate

The animal experiments were conducted in accordance with the Regulations on the Management of Laboratory Animals of Shanxi Province and Guidelines for the Care and Use of Laboratory Animals following the approval of the Ethics Committee of the Second Hospital of Shanxi Medical University (DW2023049). Every effort was made to reduce the number of mice used and alleviate their suffering.

Acknowledgments

Thank you to all the researchers in our laboratory for providing theoretical and technical support during the research.

Author Contributions

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas, took part in drafting, revising, or critically reviewing the article, gave final approval to the version to be published, have agreed on the journal to which the article has been submitted, and agree to be accountable for all aspects of the work.

Funding

This work was supported by the National Natural Science Foundation of China (82071155, 82271023, and 82301052), Basic Research Project of Shanxi Province (202203021223006 and 202403021212211), Graduate Education Innovation Project of Shanxi Province (2023SJ139), Basic Research Project of Shanxi Province (202303021212131 and 202303021212132), and Health Commission of Shanxi Province (2022XM14).

Disclosure

The authors declare no competing interests.

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Nutrition literacy across adolescence stages in Egypt: a quartile-based analysis for tailored educational strategies | BMC Public Health

Study design and target groups

A cross-sectional study was conducted across Egypt’s distinct geographical and socioeconomic regions from January to September 2022. The study targeted adolescents of both genders, aged 10–19 years, who consented to participate. The participants were categorized according to the World Health Organization (WHO) criteria for the stages of adolescence [40], early adolescence (10–13 years), middle adolescence (14–16 years), and late adolescence (17–19 years). The study aimed to assess the nutritional literacy of adolescents across these age groups.

Sample size calculation and selection

The sample size was calculated based on an estimated proportion of 18.1% for adolescents with inadequate total nutrition literacy (TNL) in each adolescence stage. A sample size of 297 provided a two-sided 97% confidence interval with a margin of error of 0.100. The sample size was calculated as follows [41].

Numeric results for Two-Sided confidence intervals for one proportion

Confidence interval formula: exact (Clopper-Pearson)

Confidence Level	Sample Size (N)	Target Width	Actual Width	Proportion (P)	Lower Limit	Upper Limit	Width if P = 0.5
0.950	950	0.050	0.050	0.181	0.157	0.207	0.065

To ensure representation across the three stages of adolescence and socioeconomic strata, the sample size was increased to 1,050 participants, with 350 adolescents from each age group (early, middle, and late adolescence) [42]. By focusing on adolescents, the study captures a critical period of development during which nutrition literacy can significantly impact both immediate and long-term health outcomes.

Study setting and participant selection

Participants were recruited from households through a multi-stage random sampling approach. Stage one was the selection of governorates to represent the main districts of four Egyptian regions. each governorate represented distinct geographical and dietary regions of Egypt: Cairo (representative of the Greater Cairo region representing Urban/metropolitan region); Fayoum (representative of Upper Egypt representing Agricultural region); Al Dakhlyia (representative of the Delta region) and Marsa Matrouh (representative of a border/frontier governorate). Each region has unique dietary habits and crops, contributing to the study’s focus on the diversity of dietary patterns across Egypt. These governorates were randomly chosen to capture various dietary practices, socioeconomic backgrounds, and cultural influences on nutrition. This selection enhances the study’s relevance to Egypt’s broader context [43, 44]. Cairo is a dense urban setting with high dietary diversity, while Fayoum and Al Dakhlyia represent agricultural areas with traditional dietary practices, and Marsa Matrouh, a frontier region, has limited food access compared to other areas.

For each governorate, both urban and rural areas were targeted for addressing the regional variability in nutrition literacy. By including both urban and rural households in each governorate, this study accounts for these disparities, enhancing the generalizability of the findings. During phase three, participants were stratified by socioeconomic status (SES) using the Economic Research Forum and CAPMAS (Central Agency for Public Mobilization and Statistics) wealth index (low, middle, and high). To minimize selection bias, random sampling was conducted in urban and rural districts within each SES group, with three cities and three local village units chosen per stratum. This structured approach also addresses potential oversampling biases in urbanized areas, ensuring that rural adolescent voices are represented adequately [45]. This selection was designed as part of a broader effort to identify children at high risk for autism, and it adhered to the study’s inclusion and exclusion criteria [46]. Adolescents were randomly selected through a community house-to-house approach.

For each targeted governorate, 45 participants (15 per adolescent stage) were recruited from each social class, resulting in a total of 225 adolescents from each governorate, with the exception of the Cairo governorate, which had 270 participants (90 per each social class, 30 per each adolescent stage).

Inclusion criteria

The study included adolescents aged 10–19 years, classified according to the World Health Organization (WHO) adolescence stages, encompassing early, middle, and late adolescence. Both male and female participants were eligible, provided they had been residing in the selected governorates for at least one year to ensure their dietary habits reflected the local environment. To account for variations in educational background, the study included adolescents actively attending school in public, private, or community-based educational institutions, as well as those who had dropped out of school but had completed at least six years of formal education, ensuring they possessed the necessary literacy skills to engage with the study materials. Additionally, informed parental consent and adolescent assent were mandatory for participation.

Exclusion criteria

Adolescents were excluded from the study if they had diagnosed cognitive impairments or severe learning disabilities that could hinder their ability to complete the nutrition literacy assessment. Those who had not completed at least six years of formal education were also excluded, as they might lack the foundational literacy skills required for the questionnaire To minimize confounding factors, adolescents with chronic medical conditions affecting nutrition intake, such as diagnosed eating disorders or metabolic disorders, were excluded unless their condition was a specific focus of the study. Additionally, non-Egyptian adolescents or those who had moved to Egypt within the past year were not included, as their dietary habits and environmental influences might not align with the local context. To prevent potential clustering biases within families, only one adolescent per household was selected for participation.

Data collection instruments and procedures

Questionnaire administration

A self-administered questionnaire was utilized to gather data from the adolescent participants. This questionnaire was filled out under the guidance of the research team to ensure clarity and accuracy. The questionnaire was adapted from a previously validated tool, originally designed and published by Hoteit and colleagues [47], ensuring its relevance to the adolescent population in the context of nutrition literacy (NL) assessment. A pilot study involving 10% of the participants was conducted before the main study to enhance clarity, minimize ambiguity, and address potential sources of measurement error.

The questionnaire was divided into two major sections:

Demographic and socioeconomic information

This section focused on collecting essential background information on the enrolled adolescents. The demographic variables collected included the participants’ age, gender, educational level, and details on their primary caregiver (i.e., who was primarily responsible for their daily care). Additionally, the study gathered data on the education levels of both parents, as parental education often influences the nutritional habits and literacy of children. Household Crowding Index: Calculated as the number of co-residents (excluding newborns) divided by the number of rooms (excluding kitchens and bathrooms [48,49,50].

Parents provided self-reported anthropometric data (weight and height) to calculate Body Mass Index (BMI), facilitating assessment of nutritional status in line with WHO’s BMI-for-age guidelines. Participants were instructed to provide recent height and weight measurements to reduce potential reporting biases, particularly in anthropometric data. However, the reliance on self-reported anthropometrics remains a limitation, as it introduces the possibility of data inaccuracy, an issue common in large-scale, self-administered surveys Participants also reported on their intake of vitamins and minerals, providing insight into their dietary supplementation habits.

Vitamins assessment

Assessing adolescents’ consumption of dietary supplements focused on participants’ report on vitamins D, C, A, B12, and folic acid intake. Assessment of these particular vitamins was crucial due to the essential roles these micronutrients play during this critical developmental stage. Adolescence is marked by rapid growth and physiological changes, increasing the demand for nutrients that support bone development, immune function, cognitive maturation, and overall health. Monitoring supplement intake in this demographic helps identify nutritional gaps and informs interventions aimed at promoting balanced diets rich in essential vitamins.

Vitamin D

is vital for calcium absorption and bone mineralization, processes that are foundational during the adolescent growth spurt. Adequate vitamin D levels are necessary to achieve optimal bone density, reducing the risk of osteoporosis and fractures later in life [51]..

Vitamin C

serves as a potent antioxidant and is essential for collagen synthesis, which is integral to the structural integrity of skin, blood vessels, and connective tissues. It also enhances immune defense mechanisms, aiding in the prevention and recovery from infections [52, 53]..

Vitamin A

is crucial for vision, immune competence, and cellular differentiation. During adolescence, sufficient vitamin A intake supports the development of epithelial tissues and bolsters the body’s ability to combat pathogens [54].

Vitamin B12

is indispensable for neurological function and the formation of red blood cells. Its role in DNA synthesis and myelination of nerve fibers is particularly pertinent during adolescence, a period characterized by significant cognitive and physical development [55]..

Folic acid (vitamin B9)

is essential for DNA synthesis and repair, supporting rapid cell division and growth. Adequate folic acid intake is vital during adolescence to prevent megaloblastic anemia and to support neural development [56].

Minerals assessment

Understanding which supplements adolescents consume provides insights into existing nutrient gaps and overall dietary patterns. Dietary supplements, such as multivitamin/mineral products, have been shown to help fill nutrient gaps and improve micronutrient sufficiency among children and adolescents. However, there is a concern about the over-reliance on supplements as substitutes for whole foods, which can lead to lower overall energy intake and lack of consumption of other critical nutrients found in whole foods [57, 58]. The current study focused on participants’ report on consumption of dietary supplements, specifically calcium, magnesium, iron, and zinc, that is grounded in their critical role in growth, development, and overall health during this life stage [59]. Adolescence is a period of rapid skeletal growth and bone mineralization, making calcium and magnesium essential for maintaining strong bones and preventing future conditions like osteoporosis and fractures. Since bone mass peaks during adolescence, ensuring adequate intake of these minerals is crucial for long-term musculoskeletal health [60, 61].

Beyond skeletal development, iron and zinc are fundamental for cognitive function, immune health, and metabolic processes. Iron deficiency is a leading cause of anemia among adolescents, particularly in females due to increased iron loss from menstruation, which can lead to fatigue, decreased concentration, and poor academic performance [62]. Similarly, zinc plays a key role in immune function, wound healing, and enzymatic reactions, helping adolescents maintain overall health and fight infections during a stage of high physiological demand [63].

In this study, data on vitamin and mineral intake reflect self-reported use of dietary supplements only, specifically including calcium, magnesium, iron, zinc, and multivitamin preparations. These data do not include intake from food sources. Dietary intake of vitamins and minerals from regular meals was assessed as part of a broader project; however, those findings are presented in a separate manuscript.

Nutrition literacy and food literacy assessment

The second part of the questionnaire measured the nutrition literacy (NL) of the adolescents and the food literacy of their parents. Nutrition literacy refers to the ability to obtain, process, and understand basic nutrition information needed to make appropriate health decisions.

To assess NL, the Adolescent Nutrition Literacy Scale (ANLS) developed by Bari [64], was utilized. This comprehensive tool consists of 22 questions, categorized into three distinct components:

Functional Nutrition Literacy (FNL): This component, composed of 7 questionsassessing basic nutritional information comprehension. It evaluated the adolescents’ ability to comprehend and use basic nutritional information, such as their understanding of nutrition-related scientific terms, dietary guidelines, and the recommendations provided by public health professionals. For instance, it included questions assessing participants’ familiarity with international dietary guidelines, such as those from the World Health Organization (WHO) regarding fruit and vegetable intake. The scoring range for FNL is 7–35, with a cut off score of ≥ 21 indicating adequate functional literacy.
Interactive Nutrition Literacy (INL): The 6 questionsin this component measured the adolescents’ skills in seeking out, discussing, and applying nutrition-related informationwithin social contexts, including communication with peers, family members, and health professionals. The ability to engage with nutrition topics and translate this knowledge into practical actions, such as modifying dietary habits based on newly acquired information, was a key aspect of this component. The score for INL ranges from 6 to 30, with a cut off score of ≥ 18 considered adequate.
Critical Nutrition Literacy (CNL): The9 questionsin this section focused on the adolescents’ ability to critically assess nutrition information and influenceothers’ dietary practices. It assessed participants’ engagement in activities that promote healthy eating, support for policies that improve dietary habits, and their ability to evaluate the credibility of nutrition-related information, particularly from social media and other sources. The score range for CNL is 9–45, with a cut off score of ≥ 27 indicative of sufficient critical literacy.

Total nutrition literacy (TNL)

was calculated as the sum of the three components (FNL, INL, CNL), yielding a total possible score between 22 and 110. A cut off score of ≥ 66 reflected adequate overall nutrition literacy. This metric provided a comprehensive view of the adolescents’ ability to understand, interact with, and critically evaluate nutrition information.

To assess parental food literacy, the validated Short Food Literacy Questionnaire (SFLQ) developed by Gréa Krause et al. [65] was used. The parental food literacy questionnaire was composed of 12 questions, divided across three dimensions similar to those assessed in the adolescent scale but with fewer questions per category: Functional food literacy (6 questions); Interactive food literacy (2 questions); Critical food literacy (4 questions).

The parental food literacy score ranged from 7 to 52, with a cut off score of ≥ 36 indicating adequate food literacy. This allowed for comparison between the literacy levels of parents and their children, providing a deeper understanding of family dynamics regarding nutrition knowledge and behaviors.

Nutritional and growth status assessment

In addition to the self-reported data of the parents, a physical assessment of nutritional status was conducted. By conducting in-person measurements, this study enhances data reliability and consistency across rural and urban participants. Additionally, anthropometric data allows for exploring relationships between growth status and nutrition literacy, which could reveal developmental implications of inadequate nutrition literacy during adolescence. Anthropometric measurements of weight and height were taken using standardized equipment and techniques. Weight was measured with a Seca Scale Balance, while height was recorded using a Holtain portable anthropometer. These measurements were critical for evaluating the growth status of the adolescents, as weight and height are primary indicators of nutritional health. The BMI was calculated as weight (in kilograms) divided by height (in meters) squared based on the WHO growth standards with the help of the Anthro-Program of PC [66]. The body mass index (BMI) was evaluated as follows: underweight if BMI is less than 18.5, normal/healthy weight if BMI is 18.5 to 24.9, overweight is BMI is 25.0 to 29.9, and obese if BMI is 30.0 or higher [67]. The BMI classification provided an additional layer of insight into the participants’ nutritional health, correlating with their dietary habits and nutrition literacy levels.

Measures to ensure validity and reliability of tools used for NL and FL assessment

Both ANLS that is developed by Bari [64] and SFLQ that is developed by Gréa Krause et al. [65] have been translated, culturally adapted and utilized in Arabic-speaking contexts. They have been adapted in a study to assess the nutrition literacy of adolescents across countries including Lebanon, Bahrain, Egypt, Jordan, Kuwait, Morocco, Palestine, Qatar, Saudi Arabia, and the United Arab Emirates [68]. The study involved 5,401 adolescent-parent dyads and found that 28% of adolescents had poor nutrition literacy. However, the study did not detail the process of translating or validating the ANLS and SFLQ for each specific Arabic-speaking context and it did not provide specific psychometric properties of the Arabic-translated tools.

We have conducted a multistep process to mitigate this for ensuring the tools appropriateness for our target population. Initially, a pilot test was conducted before large-scale implementation to ensure the clarity and appropriateness of the translated Arabic ANLS and SFLQ tools. This step involved administer of the Arabic versions of the tools for 10% of different participants as a pilot sample (n = 105) to assess their usability and ensure that participants could complete the questionnaire without difficulty. Subsequently, to assess internal consistency, the study employed Cronbach’s alpha [69]. with a larger sample of 330 participants, achieving high values of Cronbach’s alpha (0.89 for ANLS and 0.86 for SFLQ, ≥ 0.8) that indicated strong reliability [70]. To assess the stability of responses over time, a subset of 105 participants completes the Arabic SFLQ twice, with a two-week interval (test-retest reliability). The Intraclass Correlation Coefficient (ICC) was calculated to measure consistency, achieving ICC of 94% and 92% respectively indicating excellent reliability (ICC ≥ 0.75) [71]. This comprehensive approach ensured that the Arabic ANLS and SFLQ are scientifically sound and culturally relevant tool for assessing nutrition literacy among Arabic-speaking adolescents.

Statistical analysis

Data were analyzed using the Statistical Package for Social Sciences (SPSS), version 26. Various statistical techniques were employed to summarize and analyze the collected data: Categorical variables (e.g., gender, social class, nutritional literacy categories) were summarized as numbers and percentages. Continuous variables (e.g., BMI, literacy scores) were presented as means and standard deviations.

Statistical significance was determined using: Pearson’s Chi-square test (χ²) and Fisher’s exact test to assess associations between categorical variables. Z-tests were applied for comparisons of proportions.For comparisons of means between groups, the t-test and ANOVA were utilized. Crude Odds Ratio (COR) with 95% confidence intervals (CI) were calculated to examine associations between adolescence stages and nutritional literacy. Logistic regression analysis was conducted to identify significant predictors of adequate TNL among the adolescents.A p-value < 0.05 was considered statistically significant, indicating a meaningful association or difference, while a p–value < 0.01 was considered highly important, highlighting particularly strong associations or differences.

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July 5, 2025

Search continues after Pakistan building collapse kills 14

WASHINGTON: US President Donald Trump said on Friday that he has discussed sanctions with Russian President Vladimir Putin, who is worried about them and understands they might be forthcoming.

Trump, who spoke on Friday with Ukrainian President Volodymyr Zelensky, also told reporters aboard Air Force One that he had discussed the possibility of sending Patriot air defense missiles to Kyiv.

Trump spoke with Zelensky a day Russia stepped up its attack on Ukraine, sending waves of drones and missiles on Kyiv overnight in what Ukrainian officials described as the largest aerial assault since Russia’s invasion began more than three years ago. One person was killed and at least 26 others, including a child, were wounded.

Asked Friday night by reporters about the call, Trump said, “We had a very good call, I think.”

The two leaders how Ukrainian air defenses might be strengthened, possible joint weapons production between the US and Ukraine, and broader US-led efforts to end the war with Russia, according to a statement by Zelenksy.

When asked about finding a way to end the fighting, Trump said: “I don’t know. I can’t tell you whether or not that’s going to happen.”

The US has paused some shipments of military aid to Ukraine, including crucial air defense missiles. Ukraine’s main European backers are considering how they can help pick up the slack. Zelensky says plans are afoot to build up Ukraine’s domestic arms industry, but scaling up will take time.

‘I’m very disappointed’

The attack on Kyiv began the same day a phone call took place between Trump and Russian President Vladimir Putin.

Asked if he made any progress during his call with Putin on a deal to end the fighting in Ukraine, Trump said: “No, I didn’t make any progress with him today at all.”

“I’m very disappointed with the conversation I had today with President Putin because I don’t think he’s there. I don’t think he’s looking to stop (the fighting), and that’s too bad,” Trump said.

According to Yuri Ushakov, Putin’s foreign affairs adviser, the Russian leader emphasized that Moscow will seek to achieve its goals in Ukraine and remove the “root causes” of the conflict.

“Russia will not back down from these goals,” Ushakov told reporters after the call.

Russia’s army crossed the border on Feb. 24, 2022, in an all-out invasion that Putin sought to justify by falsely saying it was needed to protect Russian-speaking civilians in eastern Ukraine and prevent the country from joining NATO.

Zelensky has repeatedly called out Russian disinformation efforts.

Russia has been stepping up its long-range attacks on Ukrainian cities. Less than a week ago, Russia launched what was then the largest aerial assault of the war. That strategy has coincided with a concerted Russian effort to break through parts of the roughly 1,000-kilometer (620-mile) front line, where Ukrainian troops are under severe pressure.

Russia launched 550 drones and missiles across Ukraine during the night, the country’s air force said. The majority were Shahed drones, but Russia also launched 11 missiles in the attack.

Alya Shahlai, a 23-year-old Kyiv wedding photographer, said that her home was destroyed in the attack.

“We were all in the (basement) shelter because it was so loud, staying home would have been suicidal,” she told The Associated Press. “We went down 10 minutes before and then there was a loud explosion and the lights went out in the shelter, people were panicking.”

Five ambulances were damaged while responding to calls, officials said, and emergency services removed more than 300 tons of rubble.

Trump, Zelensky talks

In Friday’s call, Zelensky said he congratulated Trump and the American people on Independence Day and thanked the United States for its continued support.

They discussed a possible future meeting between their teams to explore ways of enhancing Ukraine’s protection against air attacks, Zelensky said.

He added that they talked in detail about defense industry capabilities and direct joint projects with the US, particularly in drone technology. They also exchanged views on mutual procurement, investment, and diplomatic cooperation with international partners, Zelensky said.

Peace efforts have been fruitless so far. Recent direct peace talks have led only to sporadic exchanges of prisoners of war, wounded troops and the bodies of fallen soldiers. No date has been set for further negotiations.

Ukrainian officials and the Russian Defense Ministry said another prisoner swap took place Friday, though neither side said how many soldiers were involved. Zelensky said most of the Ukrainians had been in Russian captivity since 2022. The Ukrainian soldiers were classified as “wounded and seriously ill.”

Constant buzzing of drones

The Ukrainian response needs to be speedy as Russia escalates its aerial attacks. Russia launched 5,438 drones at Ukraine in June, a new monthly record, according to official data collated by The Associated Press. Ukrainian Foreign Minister Andrii Sybiha said earlier this week that Russia also launched more than 330 missiles, including nearly 80 ballistic missiles, at Ukrainian towns and cities that month.

Throughout the night, AP journalists in Kyiv heard the constant buzzing of drones overhead and the sound of explosions and intense machine gun fire as Ukrainian forces tried to intercept the aerial assault.

“Absolutely horrible and sleepless night in Kyiv,” Ukrainian Foreign Minister Andrii Sybiha wrote on social media platform X. “One of the worst so far.”

Ukraine’s Economy Minister Yuliia Svyrydenko described “families running into metro stations, basements, underground parking garages, mass destruction in the heart of our capital.”

“What Kyiv endured last night, cannot be called anything but a deliberate act of terror,” she wrote on X.

Kyiv was the primary target of the countrywide attack. At least 14 people were hospitalized, according to Kyiv Mayor Vitali Klitschko.

Zelensky called the Kyiv attack “cynical.” In Moscow, the Defense Ministry claimed its forces targeted factories producing drones and other military equipment in Kyiv.

Russia strikes 5 Ukrainian regions

Ukrainian air defenses shot down 270 targets, including two cruise missiles. Another 208 targets were lost from radar and presumed jammed.

Russia successfully hit eight locations with nine missiles and 63 drones. Debris from intercepted drones fell across at least 33 sites.

In addition to the capital, the Dnipropetrovsk, Sumy, Kharkiv, Chernihiv and Kyiv regions also sustained damage, Zelensky said.

Emergency services reported damage in at least five of Kyiv’s 10 districts.

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July 5, 2025
Advances in perioperative nutritional management in Metabolic and Bari

Introduction

Obesity has emerged as a global epidemic and has a significant impact on human health and socio-economic outcomes. According to the latest data, the total number of children with obesity, adolescents with obesity and adults with obesity worldwide has exceeded 1 billion. In 2022, 159 million children with obese and 879 million adults with obese worldwide. Obesity is prevalent not only in developed countries, but also rapidly spreading in low- and middle-income countries. More than 750 million adolescents (5–19 years) worldwide are expected to be overweight or obese by 2035.^1,2 The negative health impacts of obesity are multifaceted. Obesity is an important risk factor for non-communicable diseases such as cardiovascular disease, type 2 diabetes, and certain cancers.³ In addition, obesity is associated with multiple psychosocial problems, such as impaired self-esteem, social discrimination, and decreased quality of life. Obesity in childhood and adolescence not only impacts their immediate health, but also increases the risk of chronic diseases in adulthood.⁴ Obesity also places a huge burden on the global economy, and the global cost of overweight and obesity is expected to reach $3 trillion per year by 2030.⁵

Metabolic and bariatric surgery (MBS) has a crucial role in the treatment of severe obesity. MBS is one of the most effective methods to achieve sustained long-term weight loss, especially for those patients who fail to achieve successful weight loss with diet, exercise, and medical therapy. Surgery promotes a significant decrease in body weight by altering the anatomy and function of the gastrointestinal tract, reducing the intake and absorption of food, while affecting the appetite regulation mechanisms of patients.⁶ MBS is not only effective in reducing body weight, but also significantly improves or resolves a variety of metabolic diseases associated with obesity, such as type 2 diabetes, hypertension, hyperlipidemia and obstructive sleep apnea.⁷ The improvement of these diseases not only improves the quality of life of patients, but also reduces long-term medical costs and the risk of death.⁷ MBS addresses not only the physical aspects of obesity but also significantly impacts mental health and overall patient experience. Obesity is often associated with psychological challenges such as depression, anxiety, and low self-esteem, which can be exacerbated by social stigma and discrimination. These psychological factors play a crucial role in the success of MBS and the long-term outcome of patients. A comprehensive patient-centered approach that integrates psychological support and addresses patient experience is essential to optimize surgical outcomes and improve quality of life. Preoperative psychological assessment is essential to identify patients who may be at risk for adverse mental health outcomes. This includes screening obese people for prevalent depression, anxiety, and eating disorders. Providing psychological support and counseling before surgery can help patients develop coping strategies and improve mental health, thereby enhancing the preparation of surgery and postoperative recovery. Following surgery, patients’ mental health should continue to be prioritized. Many patients experience significant lifestyle changes following MBS, which can lead to emotional and psychological challenges.¹ Regular follow-up with mental health professionals can help address these issues and provide ongoing support. In addition, support groups and peer coaching programs have been shown to be beneficial in improving patient experience and long-term adherence to lifestyle changes. Patient education and participation in decision-making processes are critical components of a patient-centered approach. Patients should be fully informed about the surgical procedure, potential risks, and expected outcomes. Involving patients in the development of their nutrition and lifestyle programs can enhance their sense of control and improve compliance with postoperative recommendations. However, it is important to note that MBS, like any surgical intervention, carries certain risks and potential complications. These may include surgical site infections, bleeding, thromboembolic events, and anesthesia-related risks. Additionally, some patients may experience long-term complications such as nutritional deficiencies, dumping syndrome, or gastrointestinal reflux.⁸ Although surgery carries certain risks and complications, its combined benefits in the treatment of severe obesity far outweigh these potential risks, providing patients with a comprehensive and lasting solution.⁸ However, it is important to note that MBS, like any surgical intervention, carries certain risks and potential complications.

Perioperative nutritional management plays a crucial role in MBS. First, in the preoperative period, the main goal of nutritional management is to improve the nutritional status of patients and reduce the degree of malnutrition, thereby improving the patient tolerance to surgical trauma and reducing the incidence of postoperative complications.⁹ To achieve this, specific interventions are essential. Preoperative dietary modifications should prioritize low-fat and low-energy diets, which can help reduce overall caloric intake while ensuring adequate nutrient intake. Additionally, multivitamin and mineral supplements, including vitamin D, iron, and folic acid, should be administered to address common micronutrient deficiencies observed in obese patients. These supplements are crucial for optimizing nutritional status and preventing postoperative complications such as anemia and metabolic bone disease. Reasonable nutritional therapy, such as low-fat, low-energy diet and multivitamin and mineral supplementation, can effectively reduce the patient’s weight, reduce liver volume, improve surgical field exposure, and increase the success rate of surgery.^10,11 In addition, preoperative nutritional management can also help patients adapt to the feeding pattern in the postoperative volume-restricted state and reduce the loss of lean body tissue and bone mass after surgery. During the intraoperative period, perioperative nutritional management helps to maintain blood glucose levels and fluid balance in patients and reduce the damage of surgical stress to the body. After surgery, nutritional management focuses on promoting rapid recovery of patients and preventing the occurrence of malnutrition.¹² A low-energy, high-protein diet should be given early after surgery to maintain muscle mass and promote wound healing. A high-protein diet typically refers to a diet that provides at least 1.2 to 1.5 grams of protein per kilogram of body weight per day, which is higher than the general dietary recommendation for the average adult. For example, a patient weighing 70 kg should aim to consume between 84 and 105 grams of protein daily. This recommendation is based on evidence that higher protein intake supports muscle preservation and overall metabolic health during the recovery phase.¹² Diverse Protein Sources To achieve the recommended protein intake, patients should be encouraged to consume a variety of protein-rich foods. These can include: Animal Proteins: Lean meats such as chicken breast (31 grams of protein per 100 grams), turkey (28 grams of protein per 100 grams), and fish-like salmon (20 grams of protein per 100 grams) and cod (17 grams of protein per 100 grams). Dairy products such as Greek yogurt (10 grams of protein per 100 grams) and cottage cheese (11 grams of protein per 100 grams) are also excellent sources. Plant-Based Proteins: Legumes like lentils (9 grams of protein per 100 grams) and chickpeas (8 grams of protein per 100 grams), as well as tofu (8 grams of protein per 100 grams), provide essential amino acids and support a balanced diet for patients who prefer or require vegetarian options. These foods not only provide essential amino acids but also help in maintaining satiety and supporting overall health. It is important to note that protein sources should be easily digestible, especially in the early postoperative period when patients may experience gastrointestinal sensitivity. In addition to macronutrient management, postoperative micronutrient supplementation is crucial to prevent deficiencies. Patients should receive regular supplementation with key vitamins and minerals, including vitamin D, vitamin B12, folic acid, and iron. Supplementation should be tailored based on individual nutritional assessments and laboratory tests to ensure adequate levels of these micronutrients. Nutritional therapy should be started as early as possible in patients with malnutrition or nutritional risk, and enteral or parenteral nutrition support should be used if necessary.¹³ In addition, long-term nutritional monitoring and supplementation are required after surgery to prevent and correct micronutrient deficiencies and maintain the long-term nutritional status of patients. Specifically, it is recommended to perform comprehensive nutritional assessments every 3 months in the first year after surgery, including blood tests for micronutrients such as iron, zinc, copper, vitamin B1, B9, B12, D, A, and E, as well as bone mineral density testing and liver and kidney function indicators. In the second year, monitoring can be adjusted to biannual, and then at least once a year focusing on key indicators. For patients at high risk of nutritional deficiencies, such as those with severe preoperative malnutrition or postoperative complications, more frequent monitoring and personalized supplementation plans are essential. In conclusion, perioperative nutritional management runs through the whole process of MBS and is of great significance to improve the surgical effect and promote the postoperative recovery of patients. To maintain good weight loss, scientific nutritional management is still required during the perioperative period.¹⁴ However, perioperative nutritional management also faces several challenges. These include patient compliance with dietary and supplement regimens, variability in nutritional needs based on individual factors such as surgical type and pre-existing conditions, and the necessity for long-term monitoring to prevent and address nutritional deficiencies. Addressing these challenges is essential to optimize patient outcomes. The aim of this literature review is to present the latest advancements in perioperative nutritional management in MBS and provide insights for optimizing the nutrition of these patients.

Prevalence and Influencing Factors of Preoperative Malnutrition

Preoperative malnutrition is prevalent in patients with MBS and has a high incidence. Studies have shown that obese patients have prevalent deficiencies of multiple micronutrients before surgery, including vitamin D, iron, folic acid, vitamin B12, vitamin A, thiamine, and zinc. Preoperative vitamin D deficiency may be present in up to 76%, iron deficiency in 6% to 50.5%, folic acid deficiency in 0% to 56%, low MCV in 19% to 47.9%, and anemia in 15.8% to 19.6%.^15–19 This malnourished condition not only impacts the quality of life of patients, but may also increase the risk of postoperative complications, such as anemia, neurological disorders, and metabolic bone disease.^20–22 To address these deficiencies before surgery, specific interventions are recommended based on the type and severity of the deficiency. For patients with vitamin D deficiency, oral vitamin D supplements are typically prescribed, with the daily dose adjusted according to serum 25(OH)D levels. The goal is to maintain serum 25(OH)D levels above 30 ng/mL. For iron deficiency, oral iron supplements are usually the first-line treatment, although intravenous iron may be considered in cases of severe deficiency or poor gastrointestinal absorption. Regular monitoring of serum ferritin and hemoglobin levels is essential to assess the effectiveness of the supplementation. In cases of severe anemia, additional interventions such as intravenous iron or even transfusions may be necessary (Table 1).

Table 1 Common Micronutrient Deficiencies and Recommended Supplementation Strategies in Metabolic and Bariatric Surgery

The occurrence of preoperative malnutrition is influenced by multiple factors. Obese patients usually have long-term imbalance in dietary intake, and their diet is often dominated by foods with high calorie and low nutritional quality, resulting in insufficient intake of micronutrients.²³ Second, obesity itself decreases the bioavailability of certain nutrients, for example, vitamin D is easily taken up by adipose tissue due to its lipid solubility, thereby reducing its concentration in blood.²⁴ In addition, obesity-related chronic inflammation can also affect nutrient absorption and utilization, such as iron absorption and utilization may be negatively affected by chronic inflammation. Female patients have a higher risk of preoperative malnutrition due to menstrual blood loss and other reasons, particularly in terms of iron and vitamin D deficiency.²⁵ Ethnic differences may also have an impact on the development of preoperative malnutrition, and dietary habits and lifestyles vary among ethnic groups, resulting in differences in the type and degree of their nutritional deficiencies.²⁶

In summary, preoperative malnutrition is highly prevalent in patients those being considered for MBS, and its occurrence is influenced by multiple factors such as dietary habits, obesity itself, chronic inflammation, gender, and ethnicity. Tailoring nutritional interventions based on the severity of deficiencies is crucial. For mild deficiencies, oral supplements and dietary adjustments may suffice, while more severe cases may require higher doses, intravenous administration, or additional medical interventions. Correction of preoperative malnutrition is important to improve the preoperative status of patients and prevent postoperative complications.

Key Indicators and Methods of Preoperative Nutritional Assessment

Preoperative nutritional assessment for weight loss is an important link to ensure the safety of surgery and postoperative recovery. Key indicators and methods include comprehensive body composition analysis of patients, measurement of height and weight to calculate body mass index (BMI), assessment of body fat percentage, waist circumference, hip circumference and waist-to-hip ratio, and understanding of visceral fat area content, which are important indicators for judging the degree of obesity and health risks.²⁷ At the same time, micronutrient levels in blood, such as vitamin B1, vitamin B12, vitamin A, vitamin D, zinc, and copper, as well as mineral contents such as calcium, phosphorus, iron, potassium, sodium, and chloride, are measured to identify potential nutritional deficiencies.^28,29 In addition, the nutritional status and metabolic function of patients were assessed by blood tests to understand the content of macronutrients such as protein, fat, and carbohydrates in patients.³⁰ The evaluation methods mainly include detailed history inquiry, understanding the dietary habits, past disease history and drug use of patients; physical examination, observing the body size, skin condition and hair distribution of patients; laboratory tests, such as blood routine, blood biochemistry, liver and kidney function, blood glucose, blood lipid and endocrine hormone levels, such as insulin and thyroid hormone, to evaluate the metabolic status and endocrine function of patients.¹¹ Through comprehensive analysis of these indicators and methods obtained information, can comprehensively understand the nutritional status of patients, for the development of personalized preoperative nutritional intervention program to provide the basis, reduce the risk of postoperative complications, and promote postoperative recovery of patients.

It is also important to consider the educational and socioeconomic status of each individual with obesity during preoperative nutritional assessment. Not all individuals have the same access to preoperative weight loss programs, and some may not be able to afford these diets, which can sometimes be expensive. Social discrimination should also be taken into account when making these decisions, and certain social groups may need further support to ensure equitable access to care. At present, the commonly used nutritional risk screening tools are Nutritional Risk Screening Scale (NRS2002), Malnutrition Universal Screening Tools (MUST) and Mini-nutritional Assessment Short Form (MNA- SF). NRS2002 is recommended as the preferred tool for nutritional risk screening in inpatients by multiple nutrition societies internationally based on strong evidence-based evidence.³¹ However, obese patients, especially those with moderate to severe obesity and diabetes, often have micronutrient deficiencies before surgery,³² and nutritional screening using NRS-2002 is not accurately assessed at this time. Therefore, it is equally important for such patients to use nutritional assessment methods for nutritional screening. The nutritional status of the body was determined by subjective and objective methods such as clinical examination, anthropometry, biochemical examination, body composition measurement, and multiple comprehensive nutritional evaluations of the patients, so as to provide all-round nutritional guidance for the patients.³³

Type and Effect of Preoperative Dietary Management

Preoperative dietary management for MBS is an essential component of surgical success and aims to achieve moderate weight loss and improve surgical conditions. Common preoperative dietary patterns include energy restricted diets, low-carbohydrate ketogenic diets (LCKD), and dietary regimens incorporating ready-to-eat low-carbohydrate ketogenic products (RLCKPs).^34,35

Energy restricted diets are the most commonly recommended type of preoperative diet, which promotes weight loss by reducing caloric intake. However, this diet is associated with poorer long-term weight management outcomes and may lead to problems such as weight rebound, increased food craving, binge eating, emotional eating, malnutrition, and eating disorders, thereby reducing future success in changing eating behaviors. In addition, energy restricted diets may increase the likelihood of eating disorders, food consumption anxiety, and internalization of weight stigma, adversely affecting pre- and postoperative outcomes.³⁴

In contrast, LCKD showed better results in preoperative dietary management. Studies have shown that weight loss and left lateral liver segment (LLLS) volume reduction can be safely and effectively achieved with LCKD 4 weeks before surgery, thereby reducing the difficulty of surgery and the risk of complications. Most programs require people to follow a low calorie low carbohydrate diet prior to surgery for between 2 to 6 weeks to reduce the size of the liver and make the surgery safer. LCKD promotes lipolysis and energy expenditure by limiting carbohydrate intake and putting the body into a ketogenic state. However, long-term adherence to LCKD can be challenging because it has limited sweetness options and easily triggers a desire for traditional carbohydrate-rich foods.³⁵ In addition, it is important that patients have access to a dietitian to prepare for surgery. For instance, to help improve the quality of diet and eating patterns.

To address this issue, RLCKP was introduced into preoperative dietary management. RLCKP helps patients adhere more easily to LCKD by replicating the texture and flavor of traditional foods while maintaining low carbohydrate content. The study showed that a 4-week preoperative dietary regimen with RLCKP significantly reduced body weight and LLLS volume, with high patient compliance and satisfaction. The use of RLCKP improves adherence to ketogenic diet regimens and helps to improve the effect of preoperative diet management.³⁶ Patients following an LCKD may experience significant changes in hunger and mood. Studies have shown that while LCKD can effectively promote weight loss, some patients may report increased feelings of hunger or irritability during the initial adaptation phase. However, with proper support and counseling, these symptoms can be managed, and patient satisfaction can be improved. The use of RLCKP can further enhance patient compliance by providing more palatable and familiar food options, thereby reducing the psychological burden associated with dietary changes.³⁶

Overall, the types of preoperative dietary management for MBS are diverse, and different dietary regimens have their own advantages and disadvantages. Although energy-restricted diets are widely used, their long-term effects and effects on eating behavior cannot be ignored. LCKD and RLCKP dietary regimens have shown good results in promoting weight loss and improving surgical conditions, but further studies are still needed to optimize dietary regimens and improve patient compliance, so as to provide more scientific and effective preoperative dietary management strategies for MBS patients.

Necessity and Strategy of Preoperative Micronutrient Supplementation

MBS is an effective treatment for severe obesity and its related complications, however, preoperative and postoperative micronutrient deficiencies are prevalent in patients with MBS and may lead to a variety of complications, such as anemia, neurological diseases and metabolic bone diseases, which seriously affect the quality of life of patients and surgical outcomes. Therefore, preoperative micronutrient supplementation appears particularly necessary.^23,37 Preoperative micronutrient supplementation can not only correct the existing nutritional deficiency status of patients, optimize their nutritional status, create a good physiological basis for surgery, but also prevent the further deterioration of postoperative nutritional deficiency to a certain extent. Studies have shown that preoperative micronutrient deficiency is an important predictor of postoperative deficiency, and preoperative identification and treatment of these nutritional deficiencies can effectively prevent the deterioration of postoperative nutritional status, reduce the incidence of postoperative complications, and promote postoperative recovery of patients.²³

When developing preoperative micronutrient supplementation strategies, patients first need to undergo a comprehensive nutritional assessment, including a detailed history, physical examination, and relevant laboratory tests, such as blood routine, serum ferritin, vitamin D, folic acid, and vitamin B12 measurements, to accurately understand the specific nutritional deficiency of patients. On this basis, a personalized supplementation program is developed according to the type and degree of micronutrients deficient in the patient. For patients with vitamin D deficiency, oral vitamin D supplements can be used, and the daily dose of supplementation depends on serum 25 (OH) vitamin D levels, and it is generally recommended to maintain serum 25 (OH) vitamin D levels above 30 ng/mL.³⁸ For patients with iron deficiency, oral iron or intravenous iron supplementation can be given, and changes in serum ferritin, hemoglobin and other indicators should be monitored to assess the effect of supplementation; patients with folic acid and vitamin B12 deficiency can be corrected by oral or injection of the corresponding supplement.³⁹

In the selection of supplementary methods, oral supplements are the most commonly used modality and have the advantages of convenience and economy, but their absorption may be affected by factors such as gastrointestinal function and drug interactions of patients, so patients’ compliance and supplementary effects need to be closely monitored during supplementation, and other routes of administration such as intramuscular injection or intravenous infusion can be considered when necessary to improve the supplementary effect.⁴⁰ In addition, the timing of micronutrient supplementation before surgery also needs to be reasonably scheduled, and it is generally recommended that supplementation be started several weeks before surgery in order to give the patient sufficient time to correct the nutritional deficiency state while avoiding the potential risks resulting from supplementation near the time of surgery.⁴¹ It is worth noting that preoperative micronutrient supplementation is not a once and for all measure, and it is still necessary to continuously pay attention to the nutritional status of patients after surgery, and timely adjust the supplementation regimen according to the postoperative recovery and changes in nutritional requirements to ensure that patients can maintain a good nutritional status and promote health throughout the perioperative period and long-term follow-up after surgery.

Nutritional Management After MBS

Following MBS, patients often face a variety of nutritional deficiencies, and the types, mechanisms, and risk factors of these deficiencies are complex. These deficiencies not only affect the quality of life of patients, but may also lead to complications such as anemia, neurological diseases, and metabolic bone diseases in severe cases.⁴² The mechanism of nutritional deficiency is mainly related to physiological changes after surgery. On the one hand, surgery will change the anatomy of the digestive tract, such as reduced gastric capacity, reduced intestinal absorption area, etc., thus affecting the intake of food and nutrient absorption. For example, after gastric bypass surgery, food bypasses parts of the stomach and small intestine, resulting in decreased absorption of vitamin B12 and iron. On the other hand, patients may experience dyspeptic symptoms such as nausea and vomiting after surgery, further limiting the intake of food.⁴³ In addition, decreased gastric acid secretion after surgery can also affect the absorption of nutrients, such as vitamin B12 requires intrinsic factors in gastric acid to be absorbed. Finally, risk factors for postoperative nutritional deficiencies include patient gender, BMI, ethnicity, etc.⁴⁴ Female patients are more likely to present with iron deficiency and anemia due to physiological characteristics, such as menstrual blood loss. Patients with a higher degree of obesity may have nutritional deficiencies before surgery due to long-term unbalanced diet, and the risk is further increased after surgery.⁴⁵ People of different ethnic groups may also be at different risk of nutritional deficiencies due to differences in dietary habits and genetic factors. For example, people of certain ethnic groups may be more vulnerable to vitamin D deficiency.²⁶ Therefore, for patients with MBS, nutritional assessment and management before and after surgery are essential to prevent and correct nutritional deficiencies and ensure patient health and surgical outcomes. Additionally, post-bariatric hyperinsulinemic hypoglycemia (PBHH) is an increasingly recognized complication, especially after Roux-en-Y gastric bypass (RYGB). This condition can significantly affect the quality of life of patients and requires strict dietary instructions to avoid its occurrence. According to a recent study by Kehagias et al,⁴⁶ PBHH was observed in a considerable proportion of patients after laparoscopic Roux-en-Y gastric bypass, particularly among those with obesity and type 2 diabetes. The study highlighted the importance of close monitoring and dietary management to prevent and manage this complication. Patients are advised to follow a structured meal plan with frequent small meals and avoid high-carbohydrate foods to minimize the risk of hypoglycemia.

Long-term micronutrient deficiencies after MBS can lead to significant health issues, such as osteoporosis from chronic vitamin D deficiency and persistent anemia from iron deficiency.¹⁵ Regular nutritional monitoring and personalized supplementation are crucial for managing these deficiencies. Patients should undergo periodic screening for key nutrients (eg, iron, vitamin D, B12) and bone mineral density testing to assess osteoporosis risk.⁴⁷ Personalized supplementation plans should be developed based on individual deficiencies and adjusted over time. Additionally, dietary education and lifestyle modifications, such as maintaining a balanced diet and avoiding high-sugar foods, are essential for long-term health. Effective long-term nutritional management requires collaboration among dietitians, surgeons, endocrinologists, and psychologists. Each professional plays a critical role in supporting the patient’s nutritional needs and overall well-being.⁴⁸

The structure and habits of the diet also need to be adjusted after surgery. Patients should avoid foods high in sugar, fat, and salt and choose foods low in calories and fiber to help control weight and prevent the recurrence of obesity. At the same time, patients should be encouraged to develop good eating habits, such as regular quantitative eating, chewing slowly, etc., to promote digestion and absorption. Diversity in diet is also important and should include a variety of vegetables, fruits, whole grains, and high-quality protein sources to ensure that patients have access to comprehensive nutrition.⁴⁹

Postoperative nutritional monitoring is essential for patients receiving MBS, as surgery may lead to problems such as decreased food intake, poor nutritional absorption, etc., causing multiple nutritional deficiencies.⁴⁹ Trace element and vitamin levels, such as iron, zinc, copper, vitamin B1, B9, B12, D, A, E, etc., these nutrients are easily deficient after surgery, and their plasma concentrations need to be measured regularly to assess whether the patient has the corresponding nutritional deficiency; bone mineral density testing, monitoring bone mineral density changes by DEXA and other methods to assess the risk of osteoporosis, because vitamin D deficiency and calcium malabsorption may lead to osteoporosis; liver and kidney function indicators, such as transaminases, bilirubin, urea nitrogen, creatinine, etc., these indicators can reflect the overall metabolic status and organ function of patients and indirectly indicate nutritional status.⁵⁰ In terms of monitoring frequency, it is recommended to perform a comprehensive nutritional monitoring every 3 months in the first year after surgery, including all the above indicators, timely identify nutritional problems and intervene; the second year can be adjusted to biannual monitoring; and then at least once a year, focusing on blood routine, serum protein levels and trace elements, vitamin levels and other key indicators.⁵¹ The frequency of monitoring should be appropriately increased in patients at special nutritional risk, such as those with more postoperative complications, severely inadequate nutritional intake, or specific nutritional deficiency symptoms. Timely intervention for nutritional deficiencies is essential, and once nutritional deficiencies are detected, appropriate supplementation measures should be taken according to the type and degree of nutrients specifically deficient.⁵² For patients with iron deficiency anemia, oral iron or intravenous iron supplementation can be given if necessary, and dietary structure can be adjusted to increase iron-rich food intake; vitamin D deficiency requires vitamin D supplementation, oral or injectable formulations can be selected, and appropriate sun exposure can be encouraged to promote vitamin D synthesis in the body; for patients with protein malnutrition, nutritional status can be improved by increasing high-quality protein food intake or protein supplementation. At the same time, nutrition education should also be strengthened to guide patients to reasonably arrange their diets, avoid bad eating habits such as partial eclipse and picky eating, ensure balanced nutritional intake, and promote postoperative recovery.¹⁸ Perioperative nutritional management in MBS should be tailored to the unique needs of each patient, considering factors such as age, pre-existing comorbidities, and ethnic background. These factors can significantly influence nutritional outcomes and require specific attention.

Collaboration of multidisciplinary teams is essential during postoperative nutritional management. Professionals such as dietitians, surgeons, endocrinologists, and psychologists should participate in the development of nutritional assessment and management plans for patients. Dietitians are responsible for providing personalized dietary advice and nutrition education, surgeons and endocrinologists adjust treatment options according to the specific circumstances of patients, and psychologists help patients cope with psychological problems that may occur after surgery, such as anxiety and depression, which may affect the patient ‘dietary behavior and nutritional status.⁵²

Perioperative nutritional management also varies between specific patient groups in metabolic versus bariatric surgery. Elderly patients may have more complex nutritional problems due to physiological hypofunction. With age, gastrointestinal function decreases, the absorption capacity of nutrients weakens, and deficiencies of nutrients such as protein, vitamin B12, and calcium are more likely to occur. Therefore, more meticulous examination of these nutrient levels is required during preoperative nutritional assessment. At the same time, elderly patients may have sarcopenia, and muscle mass and function should be assessed emphatically preoperatively and judged by measuring grip strength, gait speed, and other indicators.⁵³ Older patients recover more slowly after surgery and may have longer hospital stays. Postoperative nutritional support should pay more attention to maintaining muscle mass and improving physical function, and appropriately increase protein intake, such as by whey protein supplementation. In addition, due to the decreased ability of the elderly to metabolize and excrete drugs, attention should be paid to drug interactions when nutritional preparations are supplemented postoperatively to avoid affecting the efficacy of other drugs or increasing adverse reactions.⁵⁴ For diabetic patients, preoperative glycemic control is essential. Blood glucose management should be optimized before metabolic and bariatric surgery to avoid increased surgical risk due to hyperglycemia. In preoperative dietary management, in addition to conventional low-calorie diets, the proportion and type of carbohydrate intake can be appropriately adjusted, and foods with low glycemic index can be selected to help better control blood glucose. At the same time, blood glucose changes should be closely monitored, hypoglycemic drug doses should be adjusted according to blood glucose levels, and hypoglycemic regimens should be optimized in cooperation with endocrinologists if necessary.⁹ Cardiac function and nutritional status should be assessed preoperatively in patients with cardiovascular disease. In nutritional management, sodium intake should be restricted to reduce edema and cardiac burden. At the same time, adequate protein intake is ensured to maintain myocardial function. For patients with hypertension, preoperative diet should pay attention to blood pressure control, avoid high-salt, high-fat foods, and increase the intake of foods rich in potassium and magnesium, such as green leafy vegetables and fruits, which helps to reduce blood pressure.²⁹ Diet habits vary significantly among ethnic groups, which can influence the development of nutritional management programs. The diet of people in the Mediterranean region is rich in olive oil, fish, vegetables and fruits, and this diet is rich in unsaturated fatty acids, vitamins and minerals. For patients from the Mediterranean region, preoperative dietary management can appropriately adjust the intake ratio of olive oil and fish to meet nutritional needs. However, some people in Asia mainly eat cereals, and vegetable and fruit intake is relatively small, and it is necessary to increase vegetable and fruit intake and improve nutritional structure before surgery.⁴⁴

In conclusion, nutritional management after MBS is a long-term and integrated process that requires the joint efforts of patients, families, and medical teams. Through reasonable dietary modification, nutritional supplementation and multidisciplinary collaboration, the occurrence of postoperative nutritional deficiency and other complications can be effectively prevented, and the health recovery and quality of life of patients can be promoted.

Challenges in Perioperative Nutritional Management

Perioperative nutritional management plays a crucial role in MBS, but it also faces many challenges. Malnutrition and micronutrient deficiencies are prevalent in obese patients preoperatively. Preoperative vitamin D, iron, folic acid, vitamin B12 and other nutrients deficiencies are high due to long-term unbalanced diets and obesity-related physiological changes, such as reduced bioavailability of vitamin D and chronic inflammation affecting iron absorption. Obese patients often have a long history of restricted diets and fluctuations in body weight, resulting in depletion of fat-free mass (FFM), further exacerbating the risk of malnutrition.⁵⁵ The assessment and optimization of preoperative nutritional status is particularly important, but there is no uniform consensus and standard in the definition of preoperative nutritional evaluation, the selection of screening markers, the determination of pathological cut-off values, and the dose of nutritional supplements, which poses a challenge to clinical practice.

Entering the postoperative phase, challenges in nutritional management escalated further. Patients are more prone to nutritional deficiencies after MBS due to factors such as reduced food intake, anatomical changes leading to inadequate nutrient absorption, and decreased gastric acid and endoplasmic reticulum secretion. Especially for some malabsorptive procedures, such as biliopancreatic diversion plus duodenal switch (BPD-DS), the risk of postoperative nutritional deficiencies is higher.⁵⁵ Postoperative nutritional deficiencies not only affect the quality of life of patients, but may also lead to serious complications, such as anemia, neurological diseases and metabolic bone diseases. Therefore, long-term and even life-long monitoring and supplementation of nutrients are required after surgery to prevent and correct nutritional deficiencies. However, the individual differences of patients after surgery are large, different surgical types, basic nutritional status of patients, dietary habits and other factors will affect the effect of nutritional management, how to develop individualized nutritional supplementation program is still a difficult problem.

Future research directions can be developed from the following aspects: First, to strengthen standardized and refined studies of preoperative nutritional assessment. To develop more accurate and comprehensive preoperative nutritional assessment tools and indicators to identify cut-off values for different nutrient deficiencies in order to better identify patients with preoperative malnutrition and provide a basis for preoperative nutritional intervention. Second, to deeply explore the best program of preoperative nutritional intervention. To investigate the effects of different nutritional supplements on preoperative nutritional status and prevention of postoperative nutritional deficiency, and provide more scientific and effective preoperative nutritional intervention strategies for clinical practice. In addition, optimization of postoperative nutritional management is also the focus of future research. Further studies are needed to investigate changes in nutritional requirements at different stages after surgery, explore individualized nutritional supplementation regimens, and how to improve patient compliance with nutritional supplementation. At the same time, attention should also be paid to the impact of postoperative nutritional deficiency on the long-term health of patients, and long-term follow-up studies should be carried out to evaluate the impact of different nutritional management strategies on postoperative complications, quality of life and long-term prognosis of patients, providing a strong evidence-based basis for the continuous improvement of perioperative nutritional management.

Summary

Perioperative nutritional management has a crucial role in MBS. Preoperative nutritional assessment and intervention are essential to improve surgical success. Through preoperative nutritional support, the nutritional status of patients can be improved and the incidence of postoperative complications can be reduced, thereby improving the success rate of surgery. Nutritional management is also essential after surgery. Following MBS, patients may be at risk of deficiencies in nutrients such as protein, vitamin D, calcium, iron, vitamin B12, and folic acid. Deficiencies in these nutrients not only affect the physical health of patients, but may also lead to a decrease in quality of life. Good nutritional management can further enhance this improvement, help patients better adapt to the postoperative lifestyle, and improve their quality of life. In order to further optimize the nutritional management strategy during the perioperative period of MBS, future research and practice need to be explored and improved in the following aspects: First, a more personalized and precise nutritional management program needs to be developed and adjusted according to the specific circumstances and nutritional needs of patients. Second, collaboration among multidisciplinary teams, including dietitians, surgeons, endocrinologists, etc., should be strengthened to jointly develop and implement nutrition management programs. In addition, education and guidance for patients and their families should be strengthened to improve their awareness and compliance with the importance of nutritional management. Through these efforts, the nutritional needs of patients in the perioperative period can be better met, and the success rate of surgery and the long-term quality of life of patients can be improved. Moreover, it is important to address the significant challenges discussed in this review, such as the high prevalence of preoperative malnutrition and the complexity of postoperative nutritional deficiencies. Future research is essential to develop more accurate preoperative nutritional assessment tools and personalized postoperative nutritional supplementation strategies to optimize perioperative nutritional management.

Data Sharing Statement

All data generated or analyzed during this study are included in this published article.

Ethics Approval and Consent to Participate

An ethics statement is not applicable because this study is based exclusively on published literature.

Funding

There is no funding to report.

Disclosure

The authors have no personal, financial, commercial, or academic conflicts of interest in this study.

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Blog

Introduction

Results and Discussion

Material Characterization of NV-PCN

NV-PCN Upregulated •OH Expression in Cal-27 Cells under LED Irradiation

In Vitro Anticancer Effects of NV-PCN Irradiated with LED Light

In Vivo Antitumor Therapeutic Effect and Biosafety of NV-PCN Irradiated with LED Light

Methods

Catalyst Preparation

Characterization

Cell Culture

Measurement of Endogenous •OH

Cellular Uptake

Cytotoxicity Assay

DNA Damage by NV-PCN

Mitochondrial Membrane Potential Analysis

Total Intracellular ROS Generation

Detection of Intracellular ATP

Immunofluorescence Staining

In Vitro Anticancer Effect of NV-PCN

Western Blotting Assay

Transwell Assay

In Vivo Biodistribution and Cancer Accumulation of NV-PCN

In Vivo Anticancer Effects of NV-PCN

In Vivo Safety Assessment

Statistical Analysis

Conclusion

Ethics Approval and Consent to Participate

Acknowledgments

Author Contributions

Funding

Disclosure

References

AED to PKR Rate Today

Study design and target groups

Sample size calculation and selection

Numeric results for Two-Sided confidence intervals for one proportion

Confidence interval formula: exact (Clopper-Pearson)

Study setting and participant selection

Inclusion criteria

Exclusion criteria

Data collection instruments and procedures

Questionnaire administration

Demographic and socioeconomic information

Vitamins assessment

Vitamin D

Vitamin C

Vitamin A

Vitamin B12

Folic acid (vitamin B9)

Minerals assessment

Nutrition literacy and food literacy assessment

Total nutrition literacy (TNL)

Nutritional and growth status assessment

Measures to ensure validity and reliability of tools used for NL and FL assessment

Statistical analysis

Introduction

Prevalence and Influencing Factors of Preoperative Malnutrition

Key Indicators and Methods of Preoperative Nutritional Assessment

Type and Effect of Preoperative Dietary Management

Necessity and Strategy of Preoperative Micronutrient Supplementation

Nutritional Management After MBS

Challenges in Perioperative Nutritional Management

Summary

Data Sharing Statement

Ethics Approval and Consent to Participate

Funding

Disclosure

References

Material Characterization of N_V-PCN

N_V-PCN Upregulated •OH Expression in Cal-27 Cells under LED Irradiation

In Vitro Anticancer Effects of N_V-PCN Irradiated with LED Light

In Vivo Antitumor Therapeutic Effect and Biosafety of N_V-PCN Irradiated with LED Light

DNA Damage by N_V-PCN

In Vitro Anticancer Effect of N_V-PCN

In Vivo Biodistribution and Cancer Accumulation of N_V-PCN

In Vivo Anticancer Effects of N_V-PCN