Use of folic acid nanosensors with excellent photostability for hybrid imaging

Sentinel lymph node (SLN) mapping and tumor-boundary delineation play a key role in cancer surgery, as they have great potential to reduce surgical intervention and increase relapse-free survival rates of patients. The autofluorescence imaging (AFI) method can improve the efficiency of tumor delineation and optimize the scope of surgical intervention, but there are still no fluorescent drugs that can be used with such a method to form a hybrid imaging technique. Another problem is bleaching when fluorescent dyes are conjugated with folic acid. This study reports, for the first time, nanosensors with excellent photostability and compatibility with endoscopes for AFI, which makes simultaneous hybrid imaging possible. After functionalization of the quantum dot (QD) surfaces, we found that they bound effectively to MCF-7 cancer cells. The diagnostic value of simultaneous hybrid imaging using common AFI equipment in delineating tumor boundaries and mapping SLN can reduce the cost of diagnosis and increase its reliability.

Stomach cancer is one of the most common types of cancer in the world, accounting for more than one million cases per year and 5.7% of all cancer diagnoses (van Cutsem et al., 2016; Bray et al., 2018; Sung et al., 2021). Generally, its prognosis is unfavorable, as evidenced by the five-year survival rate and the fact that when diagnosed, most cases already have metastases (Rawla and Barsouk, 2019). The main method of treatment is surgical intervention. The most adequate degree of lymph node dissection during stomach cancer surgery is highly debatable among doctors and researchers because there are still no reliable intraoperative methods to identify affected lymph nodes. D1 gastrectomy or subtotal resection of the stomach with lymph dissection remains the standard operating intervention in most clinics, with D2 and D3 lymphadenectomies being considered extended. To date, there are no clear medical indications as to the extent and adequacy of performing extended intra-abdominal lymph dissection in the case of resectable stomach cancer. Several surgeons have argued in favor of extended (D2) lymphadenectomy, as it would provide better local-regional control of the disease. However, increased morbidity and mortality initially attributed to D2 lymphadenectomy, and the fact that there were no survival benefits registered, made Western surgeons reconsider surgical intervention tactics and turn their attention to a more limited D1 lymphadenectomy (Bonenkamp et al., 1999; Cuschieri et al., 1999; Degiuli et al., 2010).

Nevertheless, a long-term (15-year) follow-up carried out by Dutch researchers changed the overall picture, as it reported a significant decrease in the frequency of relapses after the D2 procedure. D2 lymphadenectomy is currently recommended for all patients suffering resectable advanced stomach cancer (Cummings et al., 2021). Since gastrectomy with D2 lymph dissection involves a high level of surgical intervention, relatively high morbidity and mortality rates are to be expected, even though it is performed in highly specialized medical centers (Songun et al., 2010).

To determine the extent of the malignant process and completely excise the primary tumor on the stomach wall within healthy tissue, it is recommended to use equipment that improves the efficiency of tumor-boundary detection by AFI (Pantalone et al., 2007; Tada et al., 2011). In addition, recent clinical studies have shown that image enhancement using AFI is advantageous for laparoscopic intraoperative diagnosis of serous invasion and peritoneal dissemination in the case of advanced gastric cancer. It also provides a significantly higher frequency of complete resection in early stomach cancer (Ahn et al., 2011; Matsui et al., 2021).

Use of fluorescent dyes for SLN mapping, together with AFI, can increase the effectiveness of diagnostics. Some specialists have been trying to optimize surgical tactics by mapping SLNs with various chemical dyes. One of the first means for mapping was non-specific dyes. Initially, this involved a mucous-membrane injection of methylene blue, after which indocyanine green (ICG), a fluorescent dye, came into use. This agent was more sensitive than chromogenic dyes. Among the available imaging methods, only fluorescence imaging (FI) provides real-time images with millimeter resolution, and this is the reason why it has aroused interest among surgeons considering intraoperative FI (Hokimoto et al., 2018; Kim et al., 2019; Jung et al., 2021). Results of a meta-analysis of 13 clinical trials involving 971 patients have demonstrated that detection of SLNs by near-infrared or FI using FI ICG is technically feasible (He et al., 2018). However, one of the main constraining factors for the introduction of fluorescent mapping of lymph nodes is the high cost of diagnostic equipment and organic dye (ICG), as well as potential low specificity and false-positive results due to the effects of tissue perfusion. A promising way to solve these problems is to develop a nanosensor that will bind specifically to cancer cells and at the same time be compatible with endoscopes having an AFI mode. This combination will provide simultaneous hybrid imaging without the need to develop special equipment.

Folic acid is an important substance in human metabolism and is involved in nucleotide base synthesis; its derivatives have been used for targeted treatment and imaging of tumor tissues since the 1990s. Folic acid has several receptors that ensure its transfer through the cell wall. The most common one is folate receptor-‍‍α (FR-‍α), which is overexpressed in malignant ovary, stomach, colon, and breast tissues (Hashemzadeh et al., 2021; Sakai et al., 2021). Various conjugates related to folic acid and its derivatives have been developed (Liang et al., 2019; Augustine et al., 2021; Shakeri-Zadeh et al., 2021; Skripka et al., 2021), one of which is folate-fluorescein isothiocyanate (FITC) (van Dam et al., 2011). However, FITC emits green fluorescence (about 520 nm) and is not suitable for deep-tissue imaging. For this purpose, dyes emitting in the near-infrared region (650‒900 nm) are most suitable, since their penetration into tissues is high and their autofluorescence is low, which provides a high signal/background ratio (Kelderhouse et al., 2013; Potara et al., 2021). In a study by Huang et al. (2016), a near infrared-light-absorbing carbazole-substituted BODIPY (Car-BDP) conjugate having an emission peak of 755 nm was developed with a quantum yield of 4%, which could increase to 58% when exposed to radiation at a wavelength of 710 nm. There is no data on photostability. Thus, this conjugate requires the use of non-standard and complex equipment. Photostability of the fluorescent agent is an extremely important characteristic because during a long surgery the dye may bleach, which will lead to false-negative results of intraoperative revision using FI. QDs are known to differ from common dyes by high quantum yield and high photostability. However, when they are conjugated with folic acid, the quantum yield decreases and fluorescent dyes bleach quickly as well as in case of conjugation of organic dyes with folic acid (Tung et al., 2002; Moon et al., 2003; Liu et al., 2016; Yan et al., 2016). Only recently has it been possible to achieve an increase in quantum yield and photostability, and only with graphene QDs (Zhang et al., 2019; Saljoughi et al., 2020). It has been found that suppression of nonradiative transitions by amino groups pyrolyzed from pterin plays a key role in the mechanism of high quantum yield and excellent stability. However, these studies report conjugates with a maximum absorption of 230‒300 nm and fluorescence in the high autofluorescence zone of 450‍‒‍550 nm, which makes them unsuitable for mapping sentinel nodes.

For these reasons, we sought to develop a fluor

escent nanosensor with high photostability, low cost, specificity to cancer cells, and suitability for use with common endoscopes for AFI, which would allow for hybrid diagnostics.

This research is the first to synthesize luminescent QD conjugates within the range bordering on infrared radiation. The conjugates revealed photostability sufficient for medical applications. During conjugation with folic acid, we observed an increase in absorbance (Fig. 1a). The spectrofluorimetry data indicate that the fluorescence spectra are not shifted and their proportions do not change, but the fluorescence intensity does (Fig. 1b). The QD samples hardly changed their quantum yields when irradiated over time, which confirms their high photostability (Fig. 1c). Conjugation of QDs with folic acid resulted in a severe loss of photostability (bleaching within 30 min) and a decrease in quantum yield to 15% (Table 1). However, hydroxylation of the shell with diethanolamine led to the unexpected result of increasing the quantum yield to 31% and doubling the photostability (Fig. 1c). We believe that this surface modification led to shielding of the charge in the exciton state, which reduced the probability of its transfer to the folic acid molecule.

Fig. 1. Characterization of the nanoparticles. (a) Comparative spectra of conjugate absorption; (b) Comparative spectra of fluorescence; (c) Kinetics of change of fluorescence quantum yield with irradiation time; (d, e) Transmission electron microscopy (TEM) images of quantum dots (QDs); (f) Size distribution of QD; (g‍‒‍i) Size distribution of hydrodynamic diameters of QD (g), QD-PPG-FA-COOH (h), and QD-PPG-FA-OH (i). a.u.: arbitrary units; PPG-FA: folic acid-conjugated polyethylenimine-modified PEGylated nanographene; COOH: carboxy; OH: oxhydryl.

Table 1.

Optical characteristics of quantum dot samples and conjugates

Sample	Quantum yield (%)	Fluorescence wavelength (nm)	FWHM (nm)
QD	55	680	45
QD-PPG-FA-COOH	15	680	45
QD-PPG-FA-OH	31	680	45

COOH: carboxy; FA: folic acid; FWHM: full width at half maxima; PPG: polyethylenimine-modified PEGylated; QD: quantum dot; OH: oxhydryl.

Microphotographs of the synthesized QDs were obtained, as well as their distribution according to size (Figs. 1d–1f). The samples had a crystalline structure. Analysis of the high-resolution images made it possible to measure the interplanar distances (d) of the samples (Fig. 1e). The obtained d values corres

pond to the reported values of the P6 ₃ mc phase (Razik et al., 1990). Thus, we were able to conclude that the studied samples of synthesized nanocrystals have a hexagonal structure similar to that of wurtzite. Interestingly, most of the QDs are directed along the crystallographic direction.

We also determined the hydrodynamic diameters of the nanoparticles (Figs. 1g‍–‍1i) and found that the addition of folic acid-conjugated polyethylenimine-modified PEGylated nanographene (PPG-FA) increases the hydrodynamic diameters of the particles from 20 to 50 nm. Hydroxylation of nanoparticles has almost no effect on their size.

We evaluated the degree of binding of QD samples and conjugates to the surfaces of CHO, B16-F10, HEK-293, and MCF-7 cells, using the fluorescent signal of nanoparticles. We also found that hydroxylation unexpectedly results in increased efficiency of binding to MCF-7 cells. The highest binding occurred with MCF-7 cells (Fig. 2a). Increased binding to the membrane and accumulation of QD conjugates with folic acid in MCF-7 cells have been reported in several studies (Kumar et al., 2018; Jiao et al., 2019; Yan et al., 2020). The data from this study confirm the specificity of the interaction of folic acid with the folic acid receptor on the surface of cancer cells.

Fig. 2. Binding degrees of QD samples and conjugates to the surfaces of CHO, B16-F10, HEK-293, and MCF-7 cells. (a) Average fluorescence values of nanoparticle samples. The values are expressed as mean±SEM, n=4. (b‍‒‍d) In vivo imaging 24 h after injection of QD-PEG-FA-OH into mice: subcutaneous (b); intravenous (c); and mouse internal organs (d). QD: quantum dot; PPG-FA: folic acid-conjugated polyethylenimine-modified PEGylated nanographene; OH: oxhydryl; RFU: relative fluorescence units; SEM: standard error of the mean.

Substances chosen for concentration testing proved to be non-toxic; the software calculated half maximal inhibitory concentration (IC₅₀) values automatically. The IC₅₀ values of QDs on cell lines HT29 and CHO were measured and were greater than 202.1 nmol/L. Given the low toxicity of the tested samples, IC₅₀ was not determined.

To study the possibility of using the obtained QDs and their conjugates with folic acid under in vivo imaging conditions, mice were given subcutaneous and intravenous injections. We found that at the 24-h time point, when mice were injected subcutaneously, the fluorescence did not change (Fig. 2b). The pronounced fluorescent signal in the imaging of nanoparticles through the skin of the mouse confirms that nanoparticles operate in the low-autofluorescence area of tissues. This indicates the possibility of mapping the affected lymph nodes during submucosal injection of nanoparticles with minimal risk of signal loss.

When mice were administered intravenously, there was no evident fluorescence signal at the 24-h time point (Fig. 2с). Fluorescence was observed mainly in the liver and intestine. The hepatobiliary route of excretion of nanoparticles from the body can be explained by their size (about 50 nm). Particles larger than the pore diameter in the kidney glomeruli cannot be excreted by the renal route (Tang et al., 2016; Madajewski et al., 2018; Yang et al., 2018). Thus, we have shown that the nanosensor is retained by the tissues for a sufficient time for the operation, the fluor

escent signal from the nanosensor passes through the tissues, and when it enters the bloodstream, it is quickly eliminated from the body.

To confirm the possibility of imaging a signal from a nanoparticle using common instruments for minimally invasive surgery, we used endoscopic equipment with an autofluorescence mode, produced by the company K. Storz (Tuttlingen, Germany). It has been shown that equipment in the autofluorescence mode allows effective imaging of the signal from nanoparticles (Fig. 3). Thus, the obtained nanoparticles are promising for use with endoscopes having the autofluorescence mode, which will positively affect the cost of diagnosis. In addition, it will allow the implementation of a hybrid method (tissue autofluorescence+dye fluor

Fig. 3. Ex vivo optical imaging of nanoparticles at 0.6 and 3.0 μmol/L with application (white light), application (autofluorescence), and SI (autofluorescence) modes. SI: submucosal injection.

escence), which can increase the reliability of diagnosis. The submucosal injection pathway can be effective for preoperative mapping of lymph nodes in cancer patients. Detection of tumor dissemination pathways and identification of affected lymph nodes will allow surgeons to significantly optimize treatment tactics.

In summary, we created photostable nanosensors based on folic acid conjugates. These nanosensors have small nuclei and a hexagonal crystal structure, and are fluorescent in boundary to the near-infrared radiation range. Thus, they can be effectively used in hybrid AFI/FI. Due to their optical properties, they are highly luminescent in areas of low tissue autofluor-escence, which ensures their imaging during submucosal injection and even at low dispersion concentrations (0.6 μmol/L), using conventional endoscopes. We expect hybrid imaging to significantly reduce the cost of diagnosis and increase its reliability. Given its excellent characteristics, we believe that the obtained nanosensor is a promising technique for intraoperative mapping of SLNs.

Materials and methods

Detailed methods are provided in the electronic supplementary materials of this paper.

Author contributions

Denis KUZNETSOV: Conceptualization, Methodology, Data curation, Formal analysis, Project administration, Writing original draft, Visualization. Sergey DEZHUROV and Dmitri KRYLSKY: Conceptualization, Data curation, Methodology, Formal analysis, Writing review & editing. Valery NOVIKOV and Valery NESCHISLIAEV: Writing review & editing, Formal analysis. Anastasiia KUZNETSOVA: Validation, Data curation, Visualization.

Compliance with ethics guidelines

Denis KUZNETSOV, Sergey DEZHUROV, Dmitri KRYLSKY, Valery NOVIKOV, Valery NESCHISLIAEV, and Anastasiia KUZNETSOVA declare that they have no conflict of interest.

All institutional and national guidelines for the care and use of laboratory animals were followed.