Preparation of 131I-loaded albumin nanopreparations and uptake in radioiodine-refractory thyroid cancer cells

Preparation of 131I-loaded albumin nanopreparations and uptake in radioiodine-refractory thyroid cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preparation of 131 I-loaded albumin nanopreparations and uptake in radioiodine-refractory thyroid cancer cells Zhenyu Zou, Peng Wen, Wenjie Pan, Haiqing Zhu, Jing Peng, Wei Tan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7371870/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Radioiodine-refractory thyroid cancer is currently a difficult clinical issue and pain point. This may be related to a combination of factors such as cytokines, signaling pathways and the cell microenvironment causing damage to the cell's iodine uptake channels, mainly manifested as the decreased expression of Na/I symporter（NIS） and the loss of the best treatment strategy. This study innovatively combined targeted radionuclide therapy (TRT) with nano delivery system to build a nano targeted radionuclide therapy (NTRT) platform to solve the problem of NIS failure. Results: The nanopreparations were characterized according to their particle size, potential, morphology, encapsulation rate, radiochemical purity and stability, and the uptake of 131 I-cyclodextrin-albumin nanoparticles by SLC5A5 knockout B-CPAP cells was analyzed. the average hydrodynamic diameter, PDI and zeta potential of the prepared 131 I-cyclodextrin-albumin nanoparticles were 132.80±1.60nm, 0.21±0.02 and -29.20±0.30mV. The results of transmission electron microscopy showed that the 131 I-cyclodextrin-albumin nanoparticles were round or elliptical, without an obvious adhesion phenomenon. The distribution was relatively uniform, and the particle size was about 35-55nm. The encapsulation rate of the nanopreparation was 68.70±2.79%, and the radiochemical purity was 91.70±1.67%. SLC5A5 knockout B-CPAP cells can ingest 131 I-cyclodextrin-albumin nanopreparations, and the uptake rate is about 80.60±1.81%. It is concluded that we successfully prepared a 131 I-cyclodextrin-albumin nanoparticle preparation that can be taken up by RAIR-DTC cells with a high uptake rate. Conclusions: We have successfully developed a preliminary NTRT platform, which will be a promising alternative therapy for RAIR-DTC patients who have lost radioiodine therapy due to NIS failure. Radioiodine-refractory thyroid cancer NIS Radionuclide therapy 131I Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Thyroid cancer is one of the most rapidly increasing tumor types [ 1 ] , with the seventh highest incidence rate among our residents and the fourth highest among the female population, and it is expected to be the second most common cancer in women by 2030. Its most common form is differentiated thyroid cancer (DTC), which accounts for about 90% of all thyroid cancers, with a low degree of tumor malignancy and a good prognosis [ 2 ] . However, some patients still develop metastasis or the loss of ability to concentrate iodine and eventually develop radioactive-iodine-refractory differentiated thyroid cancer (RAIR-DTC) [ 3 ] . RAIR-DTC is currently a difficult clinical issue and pain point, and its definition is still disputed. According to the 2015 American Thyroid Association Guidelines for the Diagnosis and Management of Thyroid Nodules and Differentiated Thyroid Cancer, it is characterized by four main conditions [ 2 ] : a) Distant metastases do not take up iodine after initial 131I treatment. b) Distant metastases gradually lose iodine uptake after repeated treatments. c) Distant metastases show only partial iodine uptake after 131I treatment. d) Iodine uptake takes place in distant metastases after 131I treatment, but disease progression occurs within a certain period of time. Currently, most experts believe that the loss of iodine uptake in DTC is mainly related to the expression of Na/I symporter (NIS) [ 4 – 5 ] . With the continuous development of nanotechnology, nanoformulations have received extensive attention from the medical community [ 6 – 7 ], and so far, researchers have developed various types of nanocarriers [ 8 – 11 ] , including polymer nanoparticles, nanoprotein materials, metal-based nanoparticles and liposomes. Among them, human serum albumin has the advantages of good biodegradability, biocompatibility, low immunogenicity and high stability and is widely used in drug delivery systems [ 12 ] . In the present study, we knocked out the NIS protein in DTC via the gene knockdown method, constructed a simulated RAIR-DTC model and innovatively combined radionuclides with a nanodelivery system to develop 131 I–cyclodextrin–albumin nanoparticles. The nanodelivery system incorporates radionuclides and a nanodelivery system that was used to develop a 131 I–cyclodextrin–albumin nanopreparation, which was designed to solve the problem of RAIR-DTC iodine pump failure and the inability to continue to receive 131 I treatment as well as provide new ideas for the treatment of radioiodine-refractory thyroid cancer. Materials and methods Human serum albumin was supplied by Shanghai Yuanye Biotechnology Co., Ltd; Na 131I was supplied by Changsha Atomic High-Tech Pharmaceutical Co., Ltd; iodine and 2-hydroxypropyl-β-cyclodextrin were purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd; fluorescein isothiocyanate was purchased from Hunan HuibaiShi Bio-technology Co., Ltd; anhydrous ethanol was purchased from Hunan Huihong Reagent Co., Ltd; RPMI1640 was purchased from GE Co.; and fetal bovine serum was purchased from Zhejiang Tianhang Bio-technology Co., Ltd. The analytical-grade human papillary thyroid cancer cell line B-CPAP was purchased from Saibaikang (Shanghai) Biotechnology Co. and was used without further purification. A syringe pump was provided by Fresenius Kabi Jianyuan (Changsha) Medical Technology Co., Ltd, and a 0.45um microporous filter membrane was provided by Merck Millipore. The following instruments were also used in this study: a Tecnai G2 F20 S-TWIN transmission electron microscope purchased from FEI, a Carl Zeiss Axio Vert.A1 inverted fluorescence microscope purchased from ZEISS, a PL403 precision electronic balance provided by Mettler Toledo, an Eppendorf 5415 high-speed freezing centrifuge purchased from Eppendorf, a KQ-400/100DB ultrasonic cleaner purchased from Kunshan Ultrasonic Instrumentation Co., a PL403 precision electronic balance supplied by Mettler Toledo and an RM-905 radioactivity meter purchased from Shanghai HeYi Instrumentation Co., Ltd. An MPK5000 Neon™ Transfection Instrument was purchased from Shanghai Hosheng Biochemical Technology Co., Ltd; a pure water system was purchased from ALGA; a PCR instrument and CO2 incubator were purchased from Thermo Fisher Scientific; a DNA electrophoresis instrument was supplied by Beijing Liuyi Biotechnology Co., Ltd.; a biosafety cabinet was purchased from Haier Company; and − 80℃, -20℃ and 4℃ refrigerators were purchased from Haier Company. 2. Preparation of 131I–cyclodextrin–albumin nanoformulations 2.1. Preparation of 131 I–cyclodextrin inclusion complex. A total of 0.6000g of 2-hydroxypropyl-β-cyclodextrin was precisely weighed and placed in a small beaker, and 5mL of distilled water was added to ensure complete dissolution. A precise measurement of 0.0500g of I2 was placed in another small beaker and mixed with 1ml of Na131I solution (about 46MBq), then 4mL of distilled water was added and stirred with a magnetic stirrer until dissolution. The mixture was then reacted to produce Na131I3 solution, which was then poured into the beaker of the above 2-hydroxypropyl-β-cyclodextrin solution before stirring with a magnetic stirrer for 4 hours at 0 ℃ until the 131I3 was fully contained. The resulting 131I–cyclodextrin inclusion solution (hereinafter referred to as the "inclusion") was sealed in an EP tube and stored at 4 ℃. 2.2. Preparation of the 131I–cyclodextrin–albumin nanopreparation. A total of 0.0500 g of human blood albumin (HSA) was precisely weighed and placed in a small beaker containing 10 mL of distilled water. Then, 200 µL of inclusion solution was taken and added to the above solution. The above mixture was sufficiently mixed with a magnetic stirrer at 25°C at 1500 rpm. At the same time, 10 mL of anhydrous ethanol was extracted with a syringe mounted on a syringe pump and dropped into the above mixture at a flow rate of 2 mL/min until the solution became turbid, at which point the injection was stopped. To the above solution, 70 µL of 7% glutaraldehyde solution was added. The beaker was sealed with plastic wrap, and stirring was continued for 3 hours for chemical cross-linking and curing. Finally, the cured 131I–cyclodextrin–albumin nanopreparation was dialyzed with distilled water, which was poured into an ordinary-type dialysis bag (molecular retention of 8000–14000 Da), clamped and dialyzed for 24 hours, and the distilled water was replaced every 6–8 hours to obtain the nanopreparation free of organic solvents and cross-linking agents. The obtained nanopreparation was packed in a centrifuge tube and stored at 4℃. 3. Characterization of 131I–cyclodextrin–albumin nanoformulations 3.1 Dynamic light scattering. A measurement of 2mL of the above nanopreparation was taken, subjected to ultrasonic dispersion for 5 minutes and filtered using a 0.45µm microporous filter membrane to produce a sample. A dynamic light scattering (DLS) particle size analyzer and zeta potential meter were then used to determine the size, potential and polydispersity index of the nanopreparation samples, and the measurements were repeated 3 times. 3.2 Transmission electron microscopy. A total of 1mL of the above nanopreparation was taken with a pipette gun, added dropwise onto a copper mesh and left to stand for 3 minutes to ensure that the nanoparticles were deposited on the mesh. Then, the water was absorbed on the surface with a filter paper and air-dried naturally at room temperature. Next, the morphology of the nanoparticles was observed with a transmission electron microscope, and the particle size distribution of the particles was measured after taking pictures. 3.3 Detection and calculation of encapsulation rate and radiochemical purity. The radioactivity and radiochemical purity of the nanopreparations were measured with a radioactivity meter and Waterman chromatography paper, respectively, and the encapsulation rate and radiochemical purity were calculated. The calculation formula is as follows: Encapsulation rate = activity of 131I loaded in the nanopreparation/activity of total 131I input x 100 percent Radiochemical purity = 131I activity loaded in the nanopreparation/total activity of the nanopreparation x 100%. The radiochemical purity of 131I–cyclodextrin–albumin nanopreparations was calculated using Waterman chromatography paper (1 cm×10 cm) as a stationary phase carrier. A measurement of 3 µL of the nanopreparation was taken and added to 500 µL of serum or saline, and 3 µL of each mixture was taken and added dropwise 2 cm from the lower edge of the chromatography paper, left to dry and then unfolded with a system of ethanol/water/ammonia (2:5:1) as the unfolding agent. After sufficient unfolding (about 40 min), the chromatography paper was removed to dry, cut into a 1 cm wide strip and placed at the bottom of the test tube. The radioactivity was then measured with a radioactivity meter. 3.4 Stability test of nanoformulations. The radiochemical purity of the 131I–cyclodextrin–albumin nanopreparation in serum and saline was measured at 1, 6, 12, 24, 48, 72 and 96 hours, and the change rule of radiochemical purity with time was used to evaluate the radiochemical stability of the nanopreparation. 4. Preparation of SLC5A5 knockout B-CPAP cells The NIS-encoding gene, solute carrier family 5 member 5 (SLC5A5), is located on human chromosome 19 (19p13.11) and has an open reading frame of 1929 nucleotides with a coding region consisting of 14 introns and 15 exons capable of transcribing 3.9 kb of messenger RNA [ 13 ] . The SLC5A5 gene SLC5A5 gene encodes a highly specific and efficient 80–90 kDa transmembrane glycoprotein (i.e., NIS) that mediates the active transport of iodine from the blood to thyroid follicular cells. This translocation occurs through a sodium gradient generated by the Na+/K+-ATPase pump, where two sodium ions are exchanged for one iodine ion, a critical first step in thyroid hormone biosynthesis [ 14 ] . When SLC5A5 gene expression is decreased or NIS protein localization is abnormal, iodine uptake by thyroid cells is impaired, and radioactive iodine is unable to enter DTC cells, leading to the failure of 131 I treatment, which is the main mechanism of RAIR-DTC [ 4 – 5 ] . In this study, the third-generation gene editing technology CRISPR/Cas9 was utilized to knock down the SLC5A5 gene, which regulates the expression of NIS proteins, to construct a simulated RAIR-DTC cell model. 5. Preparation of fluorescein isothiocyanate–131I–cyclodextrin–albumin nanoformulations A measurement of 4 mL of the 131I–cyclodextrin–albumin nanopreparation was taken and added to a beaker, then 1 mL of aqueous solution containing 50 mg/L fluorescein isothiocyanate (FITC) was added, and the above mixture was stirred well for 2 hours with a magnetic stirrer at 25°C at 1500 rpm. The above mixture was added to a centrifuge tube, placed into a high-speed freezing centrifuge and centrifuged three times at a centrifugal radius of 20 cm and a speed of 5000 rpm/min for 1 hour each time until the solvent was colorless. After centrifugation, the FITC–131I–cyclodextrin–albumin nanopreparation solution was transferred into a centrifuge tube wrapped with aluminum foil and stored at 4°C. 6. Cell culture and cell uptake experiments 6.1. Cell culture. Mixed clonal cells and Nthy-ori3-1 human thyroid normal cells were cultured with RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin–streptomycin in a culture incubator at a temperature of 37 ℃, a humidity of 70%-80% and a CO2 concentration of 5%. 6.2 Cell uptake experiments. Mixed clonal cells and Nthy-ori3-1 cells at 1×105/mL were inoculated in coverslip-lined Petri dishes and cultured for 24 h. A total of 2 mL of the free FITC solution, FITC–131I–cyclodextrin–albumin nanopreparation and FITC–131I–cyclodextrin–albumin nanopreparation + albumin, each with an FITC concentration of 50 mg/L, were taken and added to the mixed clonal cells and incubated in the incubator for 4 h. A total of 2 mL of the FITC–131I–cyclodextrin–albumin nanopreparation with an FITC concentration of 50 mg/L was taken and added to the Nthy-ori3-1 cells, which were incubated for 4 h in the incubator. Finally, the cells were washed three times with PBS to remove the non-uptake material, and 1 mL of 4% formaldehyde solution was added. The cells were then fixed at room temperature for 20 min, washed three times with PBS and dehydrated with anhydrous ethanol, and the coverslips were removed and dried. The dried coverslips were observed with an inverted fluorescence microscope. The FITC–131I–cyclodextrin–albumin nanopreparation and FITC–131I–cyclodextrin–albumin nanopreparation + albumin solution were added to the mixed clonal cells, the FITC–131I–cyclodextrin–albumin nanopreparation was added to the Nthy-ori3-1 cells and their radioactivity was measured immediately by using an actinometer and again after the fixation and washing. A meter was used to measure their radioactivity separately and calculate the uptake rate. The formula for calculating the uptake rate is as follows: Uptake rate = radioactivity of the cells after fixation and washing/initial radioactivity of the cells after addition of the sample × 100%. 7. Statistical analyses All the experiments were repeated three times and statistically analyzed using SPSS 23.0 software, and data are expressed as mean ± standard deviation. The comparison of means between the two groups was performed using the paired t-test, and P < 0.05 was considered statistically significant. Results 1. 131I–cyclodextrin–albumin nanopreparation characterization results 1.1. Dynamic light scattering results. The hydrodynamic size, polydispersity index (PDI) and zeta potential values of the synthesized nanoformulations were detected using dynamic light scattering and the zeta potential meter. The average hydrodynamic diameter, PDI and zeta potential of the 131I–cyclodextrin–albumin nanopreparation were measured to be 132.80±1.60 nm, 0.21±0.02 and -29.20±0.30 mV, respectively, as shown in Fig. 1, which shows that there is only a single peak in the distribution of the nanopreparation, with a relatively centralized distribution of particle size. 1.2 Transmission electron microscopy results. The morphology and distribution characterization of the prepared nanopreparations was examined via transmission electron microscopy. As shown in Fig.2a, the 131I–cyclodextrin–albumin nanoparticles were circular or elliptical in shape, with no obvious adhesion phenomenon, relatively uniform distribution and particle sizes ranging from about 35 to 55 nm. 1.3. Encapsulation rate and radiochemical purity. The encapsulation rate of 131I–cyclodextrin–albumin nanoformulation was calculated as 68.70±2.79% according to the encapsulation rate formula, and the radiochemical purity of 131I–cyclodextrin–albumin nanoformulation was calculated as 91.70±1.67% according to the radiochemical purity formula using paper chromatography. 1.4. Stability of the nanoformulation. The 131I–cyclodextrin–albumin nanopreparation was added to serum or saline and left at 25°C. The radiochemical purity of the solution was measured with paper chromatography at 1, 6, 12, 24, 48, 72 and 96 h. The results are shown in Fig. 2b. The radiochemical purity of the 131I–cyclodextrin–albumin nanopreparation declined slower in serum, and it was still more than 90% at 48 h. The radiochemical purity of the nanopreparation declined faster in saline and had dropped below 85% at 48 hours, indicating that the 131I–cyclodextrin–albumin nanopreparation was more stable in serum than in saline (t=119.51, p<0.0001). 2. Results of SLC5A5 knockout in B-CPAP cells 2.1 Genotypic identification results of knockout cell lines. The DNA of wild-type cells and mixed clone cells (i.e., knockout cells) was extracted separately, and PCR amplification was performed. The PCR-amplified products were electrophoresed and sent to Hunan Starfish Biotechnology Co., Ltd., for sequencing. The results of PCR electrophoresis are shown in Fig. 3. The bands of wild-type cells at locus 1 are about 1055bp, those at locus 2 are about 873bp and those at locus 3 are undetectable. Those of the mixed clones are about 1055bp at locus 1, 873bp at locus 2 and 873bp at locus 3. This is because the wild-type cells are not cleaved at locus 1 or 2 and can be detected as intact, but the target gene is not knocked out, so the KO bands (i.e., knockout bands) cannot be amplified. The hybrid clones have various cell types (wild type, heterozygous and heterozygous) and are able to be detected at loci 1 and 2 and amplify the KO bands (i.e., knockout bands). The sequencing results are shown in Fig. 4. The comparison and analysis of the DNA sequencing results of the hybrid clonal cells with those of the wild-type cells using chromas 2.6.5 software revealed that the knockout peaks appeared in the hybrid clonal cells, while no knockout peaks appeared in the wild-type cells. It was also shown that the hybrid clonal cells had 340bp, 1055bp and 1340bp deletions of type 3 in the SLC5A5 gene, which, in combination with the electrophoresis results, indicates that the SLC5A5 gene fragment was successfully knocked down in B-CPAP cells. 3. Results of cellular uptake experiments As shown in Figure 5, the hybrid clonal cells and Nthy-ori3-1 cells grew well, after which the cellular uptake behavior was observed via inverted fluorescence microscopy. As shown by the cellular uptake graph in Figure 6, very little free FITC was observed after incubation of the hybrid clonal cells with free FITC for 4 h, while a large amount of the nanopreparation was observed after incubation of the hybrid clonal cells with the FITC–131I–cyclodextrin–albumin nanopreparation for 4 h. The uptake rate was calculated using the above formula to be approximately 80.60 ± 1.81%; however, after the addition of the albumin solution, a significant decrease in the amount of nanopreparation entering the cells was observed, with an uptake rate of approximately 43.70±1.75%, suggesting a competitive inhibitory effect of albumin (43.70% vs. 80.60%, t=241.57, p<0.0001). A small amount of nanopreparation was observed after incubation of Nthy-ori3-1 cells for 4 hr with the FITC–131I–cyclodextrin–albumin nanopreparation, with an uptake rate of approximately 20.50±2.17%, which was significantly lower than that of the mixed clonal cells (20.50% vs. 80.60%, t=288.71, p<0.0001). Discussion The prognosis of RAIR-DTC patients is extremely poor, with a 10-year survival rate of about 10% [ 15 ] . Currently, the therapeutic means for RAIR-DTC, mainly including systemic therapy and local therapy, have poor therapeutic efficacy. Local therapy includes surgical resection of metastatic foci, radioactive particle implantation and external radiotherapy; however, most patients with RAIR-DTC have multiple foci, and the efficacy of local therapy is limited. Systemic therapy includes targeted drug therapy and re-differentiation therapy. Most patients experience different degrees of adverse events after using targeted drugs [ 16 ] , such as those for hypertension, proteinuria and hand–foot syndrome, which are often resistant to targeted drug therapy, and drug resistance occurs with long-term use. Currently, data from several clinical trials of RAIR-DTC redifferentiation therapy show poor results. In recent years, targeted radionuclide therapy (TRT) has provided a novel treatment for patients with a variety of intermediate and advanced tumors. TRT targets molecules with high tumor-specific expression, and through the binding of ligands with specific, high-affinity binding, targeted transport radionuclides are aggregated at the tumor site and irradiated internally. However, a shortcoming of TRT is that a synthetic mixture of nuclides labeled on specific ligands, which has only a short plasma half-life, can only reach the tumor extracellular matrix. The nuclides also cannot participate in off-target metabolic processes. These factors have limited the application of targeted nuclide probe therapy to some extent. Therefore, radioactive iodine remains the most efficacious therapeutic strategy for DTC patients, and finding replacement NIS channels to deliver radioactive iodine into RAIR-DTC is a key point to address in RAIR-DTC therapy. With the continuous development of nanotechnology, nanoformulations have received extensive attention from the medical community [ 5 – 6 ] , and so far, researchers have developed various types of nanocarriers, including polymer nanoparticles, nanoprotein materials, metal-based nanoparticles and liposomes. Among them, human serum albumin has the advantages of good biodegradability, biocompatibility, low immunogenicity and high stability and is widely used in drug delivery systems [ 12 ] . Studies have shown that the binding of albumin to its receptor can activate the caveolin-1 (Cav-1)-mediated cytosolic pathway [ 17 ] , which results in the formation of caveolae in the cell membrane, which transport albumin and other plasma components into the cytoplasm. In addition to SPARC, receptors involved in albumin transport and distribution in the human body include Gp60, Gp18, Gp30 and FcRn [17–19 ] . Albumin receptors are highly expressed mainly in tumor cells [ 17 , 20 – 21 ] with natural active targeting and to a lesser extent in the normal liver, intestine and kidney. Therefore, we envisioned the utilization the biological behavior of albumin to transport radioactive iodine into the cell and solve the fatal pain point of RAIR-DTC's inability to uptake iodine through the NIS channel. Albumin has multiple sites for ligand binding, including covalent or non-covalent binding for drug loading [ 22 ] , but iodine does not bind directly to albumin to form nanoformulations. Therefore, the preparation of iodine-loaded albumin nanoformulations requires the addition of other carriers for loading 131I. Cyclodextrins are cyclic oligosaccharides consisting of multiple glucose units, which have the advantages of good biodegradability, biocompatibility and low immunogenicity [ 23 ] . β-Cyclodextrins have a lipophilic central vesicle lumen and hydrophilic outer surface, where the drug can enter and form a water-soluble inclusion complex, and β-Cyclodextrins can be used to form nanoformulations for drug loading with HSA as well as binding to iodine [ 24 ] . Through the above research ideas, we successfully prepared 131I–cyclodextrin–albumin nanopreparations, and the PDI of 131I–cyclodextrin–albumin nanopreparations was 0.21 ± 0.02, which, combined with the distribution curves, indicated that the distribution of the nanopreparations was relatively concentrated. The transmission electron microscopy results showed that the 131I–cyclodextrin–albumin nanoparticles were round or elliptical in shape, with no obvious adhesion phenomenon, and were more uniformly distributed, with a particle size of about 35–55 nm, which was smaller than the particle size measured with DLS (132.80 ± 1.60 nm). This was due to the dehydrated morphology of the nanopreparations shown in the transmission electron microscopy images, while DLS was used to detect the mean hydrodynamic particle size of the nanopreparations. The encapsulation rate of the 131I–cyclodextrin–albumin nanopreparation was 68.70 ± 2.79%, which may be related to the decay of 131I during the preparation process, but more importantly, the experimental conditions need to be further mapped to shorten the preparation time. The radiochemical purity of the 131I–cyclodextrin–albumin nanopreparation was 91.70 ± 1.67% as measured via paper chromatography. Radiochemical stability experiments verified that the 131I–cyclodextrin–albumin nanopreparation was more stable in serum than in saline (t = 119.51, p 90% in serum for 48 hours. In general, the stability of radiopharmaceuticals in saline is greater than the serum stability, but the opposite is true in the results of the present study, probably due to the fact that 131I has a certain protective effect within the dual carriers of albumin and cyclodextrins, which is mainly manifested by the biological properties of albumin in vivo. The cellular uptake assay showed that the mixed clonal cells were able to take up the 131I–cyclodextrin–albumin nanopreparation with an uptake rate of about 80.60 ± 1.81%, and after the addition of albumin solution, the nanopreparation entering the cells was significantly reduced, with an uptake rate of about 43.70 ± 1.75%. This indicated that there was a competitive inhibitory effect of albumin (43.70%VS80.60%, t = 241.57 and p < 0.0001). The uptake rate of Nthy-ori3-1 cells was about 20.50 ± 2.17%, which was significantly lower than that of the mixed clones (20.50% vs. 80.60%, t = 288.71, p < 0.0001). Albumin nanopreparations may utilize an enhanced permeability and retention effect (EPR) to stay in the interstitium of RAIR-DTC tumor cells [ 25 – 26 ] . In addition to this, it has been suggested that secreted protein that is acidic and rich in cysteine (SPARC) has a nanodrug-accumulating effect. Albumin nanodrugs can accumulate more drugs when combined with SPARC, which is highly expressed in tumor cells, and tend to achieve better efficacy in such tumors, including thyroid cancer cells [ 27 – 28 ] . Albumin nanoformulations that reach the tumor cell mesenchyme bind to receptors (SPARC or Gp60) that activate the Cav-1 pathway, allowing albumin-loaded drugs to accumulate within the tumor cell cytoplasm [ 17 , 27 – 28 ] . Therefore, it is tentatively suggested according to the uptake experiments that albumin nanopreparations can use this pathway to deliver radioactive iodine into the RAIR-DTC cytoplasm and kill tumor cells using rays released by the continuous decay of iodine. There are still some shortcomings of this study. First, the 131I–cyclodextrin–albumin nanopreparation was prepared for a long time, and the 131I decayed, which may have affected the experimental results to a certain extent, but we tried to use the same batch of the nanopreparation in each experiment to minimize the error. Second, the knockdown of SLC5A5 in B-CPAP cells was only verified at the gene level, the gene fragment was not verified at the protein level and no pure B-CPAP cells were screened for SLC5A5 knockdown. Furthermore, only the human thyroid gland was tested for the knockdown of the gene fragment, and no pure cells were screened. Only the B-CPAP cell line of human papillary thyroid carcinoma was investigated, neglecting other commonly used cell lines of human papillary thyroid carcinoma or human follicular thyroid carcinoma; these shortcomings will be further improved in the subsequent experiments. The aim of this study was to investigate the feasibility of albumin nanoformulations for the treatment of RAIR-DTC. Conclusion In this study, 131 I-cyclodextrin-albumin nanocomplex was successfully prepared. This novel nuclide complex can be absorbed by RAIR-DTC cells with high absorption rate. In conclusion, we have innovatively constructed a NTRT platform, and proposed a new strategy for clinical diagnosis and treatment of iodine-resistant thyroid cancer. Declarations Acknowledgements Not applicable. Author contributions ZZ, PW, FS and WP contributed to the theory behind experiments, contributed to sample preparation and data collection, and carried out the experiments. ZZ and PW drafted the manuscript together. HZ, JP and WT contributed to the data collection and analysis. FS has contributed to the conception of the study and the revision of the article. all the authors critically revised the paper and approved the submitted version of the manuscript. Funding This research was supported by the General Program of Natural Science Foundation of Hunan Province (2024JJ5241). Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethical approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References SUNG H, FERLAY J, SIEGEL R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J/OL]. CA: A Cancer Journal for Clinicians, 2021, 71(3): 209-249. DOI:10.3322/caac.21660. 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Metal-based nanopar- ticle in cancer treatment: lessons learned and challenges[J/OL]. Frontiers in Bioengineering and Biotechnology, 2024, 12: 1436297. DOI:10.3389/fbioe.2024.1436297. JIROFTI N, SARHADDI F, POORSARGOL M, et al. Lignin and Pluronic-based nanomicelles as promising amiodarone nanocarriers: Synthesis, physical characterization, biological, and in silico evaluations[J/OL]. Industrial Crops and Products, 2024, 216: 118703. DOI:10.1016/j.indcrop.2024.118703. SAFARI F, JALALIAN Y, ABDOUSS H, et al. Harnessing Nanotechnology for Idarubicin Delivery in Cancer Therapy: Current Approaches and Future Perspectives[J/OL]. BioNanoScience, 2024[2024-09-18]. https://link.springer.com/10.1007/s12668-024-01376-2. DOI:10.1007/s12668-024-01376-2. Spada A, Emami J, Tuszynski JA, et al. The Uniqueness of Albumin as a Carrier in Nanodrug Delivery. Mol Pharm. 2021;18(5):1862-1894. Ravera S, Reyna-Neyra A, Ferrandino G, et al. The Sodium/Iodide Symporter (NIS): Molecular Physiology and Preclinical and Clinical Applications. Annu Rev Physiol. 2017;79:261-289. Ravera S, Nicola JP, Salazar-De Simone G, et al. Structural insights into the mechanism of the sodium/iodide symporter. Nature. 2022;612(7941):795-801. Silaghi H, Lozovanu V, Georgescu CE, et al. State of the Art in the Current Management and Future Directions of Targeted Therapy for Differentiated Thyroid Cancer. Int J Mol Sci. 2022;23(7):3470. Published 2022 Mar 23. Schlumberger M, Tahara M, Wirth LJ, et al. Lenvatinib versus placebo in radioiodine-refractory thyroid cancer. N Engl J Med . 2015;372(7):621-630. Kumari N, Mathe VL, Dongre PM. Albumin nanoparticles conjugates binding with glycan - A strategic approach for targeted drug delivery. Int J Biol Macromol . 2019;126:74-90. Jordan SC, Ammerman N, Vo A. Implications of Fc Neonatal Receptor (FcRn) Manipulations for Transplant Immunotherapeutics. Transplantation . 2020;104(1):17-23. Sleep D. Albumin and its application in drug delivery. Expert Opin Drug Deliv . 2015;12(5):793-812. Ishima Y, Maruyama T, Otagiri M, Chuang VTG, et al. The New Delivery Strategy of Albumin Carrier Utilizing the Interaction with Albumin Receptors. Chem Pharm Bull (Tokyo) . 2022;70(5):330-333. Jiang S, Sun HF, Li S, Zhang N, et al. SPARC: a potential target for functional nanomaterials and drugs. Front Mol Biosci . 2023;10:1235428. Published 2023 Jul 28. Pilati D, Howard KA. Albumin-based drug designs for pharmacokinetic modulation. Expert Opin Drug Metab Toxicol . 2020;16(9):783-795. Tannous M, Caldera F, Hoti G, et al. Drug-Encapsulated Cyclodextrin Nanosponges. Methods Mol Biol . 2021;2207:247-283. Dattilo S, Spitaleri F, Aleo D, et al. Solid-State Preparation and Characterization of2-Hydroxypropylcyclodextrins-IodineComplexesasStable Iodophors. Biomolecules . 2023;13(3):474. Published 2023 Mar 3 Shinde VR, Revi N, Murugappan S, et al. Enhanced permeability and retention effect: A key facilitator for solid tumor targeting by nanoparticles. Photodiagnosis Photodyn Ther . 2022; 9:102915. Shi Y, van der Meel R, Chen X, et al. The EPR effect and beyond: Strategies to improve tumor targeting and cancer nanomedicine treatment efficacy. Theranostics . 2020; 0(17):7921-7924. Published 2020 Jun 25. Liang M, Jia J, Chen L, et al. LncRNA MCM3AP-AS1 promotes proliferation and invasion through regulating miR-211-5p/SPARC axis in papillary thyroid cancer. Endocrine . 2019;65(2):318-326. doi:10.1007/s12020-019-01939-4 Yu XZ, Guo ZY, Di Y, et al. The relationship between SPARC expression in primary tumor and metastatic lymph node of resected pancreatic cancer patients and patients' survival. Hepatobiliary Pancreat Dis Int . 2017;16(1):104-109. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7371870","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":500611364,"identity":"199611bb-894a-443d-8abd-1c1df2aebb53","order_by":0,"name":"Zhenyu Zou","email":"","orcid":"","institution":"Affiliated Cancer Hospital of School of Medicine Central South University Xiangya: Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Zou","suffix":""},{"id":500611365,"identity":"b1c132ed-23ac-468f-8523-2426c038ae4a","order_by":1,"name":"Peng Wen","email":"","orcid":"","institution":"chang de shi di yi ren min yi yuan: The First People's Hospital of Changde City","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Wen","suffix":""},{"id":500611366,"identity":"6af6ca48-0e26-45db-bb75-6b7edc5c8a2d","order_by":2,"name":"Wenjie Pan","email":"","orcid":"","institution":"Affiliated Cancer Hospital of School of Medicine Central South University Xiangya: Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjie","middleName":"","lastName":"Pan","suffix":""},{"id":500611367,"identity":"e7c7dc96-3ad4-4246-9afa-d273db542457","order_by":3,"name":"Haiqing Zhu","email":"","orcid":"","institution":"Affiliated Cancer Hospital of School of Medicine Central South University Xiangya: Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haiqing","middleName":"","lastName":"Zhu","suffix":""},{"id":500611368,"identity":"22b4a575-a96a-449a-9956-cff714efd9aa","order_by":4,"name":"Jing Peng","email":"","orcid":"","institution":"Affiliated Cancer Hospital of School of Medicine Central South University Xiangya: Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Peng","suffix":""},{"id":500611369,"identity":"39593a13-56ff-4e74-8977-70dad1e00c19","order_by":5,"name":"Wei Tan","email":"","orcid":"","institution":"Affiliated Cancer Hospital of School of Medicine Central South University Xiangya: Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Tan","suffix":""},{"id":500611370,"identity":"a5ceab8f-c778-41dd-aab0-4976881cb781","order_by":6,"name":"Feng Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACCcaGD0BKzoAULY0zgJQxKVoYGEFaEjcQrUN+dnNjw8cdtenbpZsffmCouGfXwH72AF4tBncONjbOPHM8d+ecY8YSDGeKkxt48hLwa5FIbH/M23Ysd8ONHKC/2hKSGSR48PtLfkZiY/PftmPpBjdymH8QpYXhBlALY1tNAlALG8gWO4JaDIBaGnvbDhhuuJFmZpFwJiGBjSeHkMPSHzb8bKuTN7iR/PjGh4oEe372M0TF0WEIlQCMoDZi1ANBHZxlT6SOUTAKRsEoGEEAAJGqSZ9QEK8sAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0000-0529-0420","institution":"Affiliated Cancer Hospital of School of Medicine Central South University Xiangya: Hunan Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Feng","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-08-14 08:55:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7371870/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7371870/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89971483,"identity":"cb0698e0-8ff5-4b0c-9d75-04e9b91f19cc","added_by":"auto","created_at":"2025-08-27 05:37:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29244,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution curve of nanopreparations measured using DLS.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/fa6ff20637b55dc74fc4ee18.png"},{"id":89971486,"identity":"9b78cc6e-a0a4-428c-bf80-334b797f2327","added_by":"auto","created_at":"2025-08-27 05:37:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69279,"visible":true,"origin":"","legend":"\u003cp\u003ea) Transmission electron microscopy image of nanopreparations. b) Radiochemical stability of nanopreparations.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/f6902de51cdf9156349a73f7.png"},{"id":89972553,"identity":"c0c03b08-18e9-4eee-ae30-f95ab092fc94","added_by":"auto","created_at":"2025-08-27 05:45:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171176,"visible":true,"origin":"","legend":"\u003cp\u003ePCR electrophoresis results (MIX: mixed clones, M: marker, WT: wild type).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/4a31a92154d098ef465dbdf9.png"},{"id":89971490,"identity":"e57808ae-a88e-41a9-bc25-5872b7e7af5d","added_by":"auto","created_at":"2025-08-27 05:37:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":312096,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing results of the SLC5A5 gene (the blue area is the position of gRNA-A2, the reverse sequencing result).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/2e0fcda9fbdc924273725c02.png"},{"id":89971492,"identity":"b806146e-1530-436b-982f-4d6577059fcb","added_by":"auto","created_at":"2025-08-27 05:37:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":171563,"visible":true,"origin":"","legend":"\u003cp\u003eNormal adherent growing cells (a: mixed clonal cells - ×100, b: Nthy-ori3-1 cells - ×100).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/c49427f0b5babdfe741e1653.png"},{"id":89974352,"identity":"3aa66d19-20b1-4134-ace6-517d46e7cc3a","added_by":"auto","created_at":"2025-08-27 05:53:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57009,"visible":true,"origin":"","legend":"\u003cp\u003eCellular uptake diagram (a) free FITC mixed clonal cells, (b) FITC–131I–cyclodextrin–albumin nanopreparation, (c) FITC–131I–cyclodextrin–albumin nanopreparation + albumin solution and (d) FITC–131I–cyclodextrin–albumin nanopreparation of Nthy-ori3-1 cells.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/03b74d000e36a10f03f8ea33.png"},{"id":90028835,"identity":"23788bcd-d796-4797-9af5-2844b711ccdd","added_by":"auto","created_at":"2025-08-27 14:39:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1582972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7371870/v1/e1fd0f16-b2b1-450b-ad0b-08d2b7e7f6a7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePreparation of \u003csup\u003e131\u003c/sup\u003eI-loaded albumin nanopreparations and uptake in radioiodine-refractory thyroid cancer cells\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eThyroid cancer is one of the most rapidly increasing tumor types \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, with the seventh highest incidence rate among our residents and the fourth highest among the female population, and it is expected to be the second most common cancer in women by 2030. Its most common form is differentiated thyroid cancer (DTC), which accounts for about 90% of all thyroid cancers, with a low degree of tumor malignancy and a good prognosis \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, some patients still develop metastasis or the loss of ability to concentrate iodine and eventually develop radioactive-iodine-refractory differentiated thyroid cancer (RAIR-DTC) \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. RAIR-DTC is currently a difficult clinical issue and pain point, and its definition is still disputed. According to the 2015 American Thyroid Association Guidelines for the Diagnosis and Management of Thyroid Nodules and Differentiated Thyroid Cancer, it is characterized by four main conditions \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e: a) Distant metastases do not take up iodine after initial 131I treatment. b) Distant metastases gradually lose iodine uptake after repeated treatments. c) Distant metastases show only partial iodine uptake after 131I treatment. d) Iodine uptake takes place in distant metastases after 131I treatment, but disease progression occurs within a certain period of time. Currently, most experts believe that the loss of iodine uptake in DTC is mainly related to the expression of Na/I symporter (NIS) \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. With the continuous development of nanotechnology, nanoformulations have received extensive attention from the medical community \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e],\u003c/sup\u003e and so far, researchers have developed various types of nanocarriers \u003csup\u003e[\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, including polymer nanoparticles, nanoprotein materials, metal-based nanoparticles and liposomes. Among them, human serum albumin has the advantages of good biodegradability, biocompatibility, low immunogenicity and high stability and is widely used in drug delivery systems \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the present study, we knocked out the NIS protein in DTC via the gene knockdown method, constructed a simulated RAIR-DTC model and innovatively combined radionuclides with a nanodelivery system to develop \u003csup\u003e131\u003c/sup\u003eI\u0026ndash;cyclodextrin\u0026ndash;albumin nanoparticles. The nanodelivery system incorporates radionuclides and a nanodelivery system that was used to develop a \u003csup\u003e131\u003c/sup\u003eI\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation, which was designed to solve the problem of RAIR-DTC iodine pump failure and the inability to continue to receive \u003csup\u003e131\u003c/sup\u003eI treatment as well as provide new ideas for the treatment of radioiodine-refractory thyroid cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eHuman serum albumin was supplied by Shanghai Yuanye Biotechnology Co., Ltd; Na 131I was supplied by Changsha Atomic High-Tech Pharmaceutical Co., Ltd; iodine and 2-hydroxypropyl-β-cyclodextrin were purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd; fluorescein isothiocyanate was purchased from Hunan HuibaiShi Bio-technology Co., Ltd; anhydrous ethanol was purchased from Hunan Huihong Reagent Co., Ltd; RPMI1640 was purchased from GE Co.; and fetal bovine serum was purchased from Zhejiang Tianhang Bio-technology Co., Ltd. The analytical-grade human papillary thyroid cancer cell line B-CPAP was purchased from Saibaikang (Shanghai) Biotechnology Co. and was used without further purification. A syringe pump was provided by Fresenius Kabi Jianyuan (Changsha) Medical Technology Co., Ltd, and a 0.45um microporous filter membrane was provided by Merck Millipore. The following instruments were also used in this study: a Tecnai G2 F20 S-TWIN transmission electron microscope purchased from FEI, a Carl Zeiss Axio Vert.A1 inverted fluorescence microscope purchased from ZEISS, a PL403 precision electronic balance provided by Mettler Toledo, an Eppendorf 5415 high-speed freezing centrifuge purchased from Eppendorf, a KQ-400/100DB ultrasonic cleaner purchased from Kunshan Ultrasonic Instrumentation Co., a PL403 precision electronic balance supplied by Mettler Toledo and an RM-905 radioactivity meter purchased from Shanghai HeYi Instrumentation Co., Ltd. An MPK5000 Neon\u0026trade; Transfection Instrument was purchased from Shanghai Hosheng Biochemical Technology Co., Ltd; a pure water system was purchased from ALGA; a PCR instrument and CO2 incubator were purchased from Thermo Fisher Scientific; a DNA electrophoresis instrument was supplied by Beijing Liuyi Biotechnology Co., Ltd.; a biosafety cabinet was purchased from Haier Company; and \u0026minus;\u0026thinsp;80℃, -20℃ and 4℃ refrigerators were purchased from Haier Company.\u003c/p\u003e\n\u003ch3\u003e2. Preparation of 131I–cyclodextrin–albumin nanoformulations\u003c/h3\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Preparation of \u003csup\u003e131\u003c/sup\u003eI\u0026ndash;cyclodextrin inclusion complex.\u003c/h2\u003e\u003cp\u003eA total of 0.6000g of 2-hydroxypropyl-β-cyclodextrin was precisely weighed and placed in a small beaker, and 5mL of distilled water was added to ensure complete dissolution. A precise measurement of 0.0500g of I2 was placed in another small beaker and mixed with 1ml of Na131I solution (about 46MBq), then 4mL of distilled water was added and stirred with a magnetic stirrer until dissolution. The mixture was then reacted to produce Na131I3 solution, which was then poured into the beaker of the above 2-hydroxypropyl-β-cyclodextrin solution before stirring with a magnetic stirrer for 4 hours at 0 ℃ until the 131I3 was fully contained. The resulting 131I\u0026ndash;cyclodextrin inclusion solution (hereinafter referred to as the \"inclusion\") was sealed in an EP tube and stored at 4 ℃.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation.\u003c/h2\u003e\u003cp\u003eA total of 0.0500 g of human blood albumin (HSA) was precisely weighed and placed in a small beaker containing 10 mL of distilled water. Then, 200 \u0026micro;L of inclusion solution was taken and added to the above solution. The above mixture was sufficiently mixed with a magnetic stirrer at 25\u0026deg;C at 1500 rpm. At the same time, 10 mL of anhydrous ethanol was extracted with a syringe mounted on a syringe pump and dropped into the above mixture at a flow rate of 2 mL/min until the solution became turbid, at which point the injection was stopped. To the above solution, 70 \u0026micro;L of 7% glutaraldehyde solution was added. The beaker was sealed with plastic wrap, and stirring was continued for 3 hours for chemical cross-linking and curing. Finally, the cured 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was dialyzed with distilled water, which was poured into an ordinary-type dialysis bag (molecular retention of 8000\u0026ndash;14000 Da), clamped and dialyzed for 24 hours, and the distilled water was replaced every 6\u0026ndash;8 hours to obtain the nanopreparation free of organic solvents and cross-linking agents. The obtained nanopreparation was packed in a centrifuge tube and stored at 4℃.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e3. Characterization of 131I–cyclodextrin–albumin nanoformulations\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Dynamic light scattering.\u003c/h2\u003e\u003cp\u003eA measurement of 2mL of the above nanopreparation was taken, subjected to ultrasonic dispersion for 5 minutes and filtered using a 0.45\u0026micro;m microporous filter membrane to produce a sample. A dynamic light scattering (DLS) particle size analyzer and zeta potential meter were then used to determine the size, potential and polydispersity index of the nanopreparation samples, and the measurements were repeated 3 times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Transmission electron microscopy.\u003c/h2\u003e\u003cp\u003eA total of 1mL of the above nanopreparation was taken with a pipette gun, added dropwise onto a copper mesh and left to stand for 3 minutes to ensure that the nanoparticles were deposited on the mesh. Then, the water was absorbed on the surface with a filter paper and air-dried naturally at room temperature. Next, the morphology of the nanoparticles was observed with a transmission electron microscope, and the particle size distribution of the particles was measured after taking pictures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Detection and calculation of encapsulation rate and radiochemical purity.\u003c/h2\u003e\u003cp\u003eThe radioactivity and radiochemical purity of the nanopreparations were measured with a radioactivity meter and Waterman chromatography paper, respectively, and the encapsulation rate and radiochemical purity were calculated. The calculation formula is as follows:\u003c/p\u003e\u003cp\u003eEncapsulation rate\u0026thinsp;=\u0026thinsp;activity of 131I loaded in the nanopreparation/activity of total 131I input x 100 percent\u003c/p\u003e\u003cp\u003eRadiochemical purity\u0026thinsp;=\u0026thinsp;131I activity loaded in the nanopreparation/total activity of the nanopreparation x 100%.\u003c/p\u003e\u003cp\u003eThe radiochemical purity of 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparations was calculated using Waterman chromatography paper (1 cm\u0026times;10 cm) as a stationary phase carrier. A measurement of 3 \u0026micro;L of the nanopreparation was taken and added to 500 \u0026micro;L of serum or saline, and 3 \u0026micro;L of each mixture was taken and added dropwise 2 cm from the lower edge of the chromatography paper, left to dry and then unfolded with a system of ethanol/water/ammonia (2:5:1) as the unfolding agent. After sufficient unfolding (about 40 min), the chromatography paper was removed to dry, cut into a 1 cm wide strip and placed at the bottom of the test tube. The radioactivity was then measured with a radioactivity meter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Stability test of nanoformulations.\u003c/h2\u003e\u003cp\u003eThe radiochemical purity of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation in serum and saline was measured at 1, 6, 12, 24, 48, 72 and 96 hours, and the change rule of radiochemical purity with time was used to evaluate the radiochemical stability of the nanopreparation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e4. Preparation of SLC5A5 knockout B-CPAP cells\u003c/h3\u003e\n\u003cp\u003eThe NIS-encoding gene, solute carrier family 5 member 5 (SLC5A5), is located on human chromosome 19 (19p13.11) and has an open reading frame of 1929 nucleotides with a coding region consisting of 14 introns and 15 exons capable of transcribing 3.9 kb of messenger RNA \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The SLC5A5 gene SLC5A5 gene encodes a highly specific and efficient 80\u0026ndash;90 kDa transmembrane glycoprotein (i.e., NIS) that mediates the active transport of iodine from the blood to thyroid follicular cells. This translocation occurs through a sodium gradient generated by the Na+/K+-ATPase pump, where two sodium ions are exchanged for one iodine ion, a critical first step in thyroid hormone biosynthesis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. When SLC5A5 gene expression is decreased or NIS protein localization is abnormal, iodine uptake by thyroid cells is impaired, and radioactive iodine is unable to enter DTC cells, leading to the failure of \u003csup\u003e131\u003c/sup\u003eI treatment, which is the main mechanism of RAIR-DTC \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In this study, the third-generation gene editing technology CRISPR/Cas9 was utilized to knock down the SLC5A5 gene, which regulates the expression of NIS proteins, to construct a simulated RAIR-DTC cell model.\u003c/p\u003e\n\u003ch3\u003e5. Preparation of fluorescein isothiocyanate–131I–cyclodextrin–albumin nanoformulations\u003c/h3\u003e\n\u003cp\u003eA measurement of 4 mL of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was taken and added to a beaker, then 1 mL of aqueous solution containing 50 mg/L fluorescein isothiocyanate (FITC) was added, and the above mixture was stirred well for 2 hours with a magnetic stirrer at 25\u0026deg;C at 1500 rpm. The above mixture was added to a centrifuge tube, placed into a high-speed freezing centrifuge and centrifuged three times at a centrifugal radius of 20 cm and a speed of 5000 rpm/min for 1 hour each time until the solvent was colorless. After centrifugation, the FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation solution was transferred into a centrifuge tube wrapped with aluminum foil and stored at 4\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003e6. Cell culture and cell uptake experiments\u003c/h3\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e6.1. Cell culture.\u003c/h2\u003e\u003cp\u003eMixed clonal cells and Nthy-ori3-1 human thyroid normal cells were cultured with RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin\u0026ndash;streptomycin in a culture incubator at a temperature of 37 ℃, a humidity of 70%-80% and a CO2 concentration of 5%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e6.2 Cell uptake experiments.\u003c/h2\u003e\u003cp\u003eMixed clonal cells and Nthy-ori3-1 cells at 1\u0026times;105/mL were inoculated in coverslip-lined Petri dishes and cultured for 24 h. A total of 2 mL of the free FITC solution, FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation and FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation\u0026thinsp;+\u0026thinsp;albumin, each with an FITC concentration of 50 mg/L, were taken and added to the mixed clonal cells and incubated in the incubator for 4 h. A total of 2 mL of the FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation with an FITC concentration of 50 mg/L was taken and added to the Nthy-ori3-1 cells, which were incubated for 4 h in the incubator. Finally, the cells were washed three times with PBS to remove the non-uptake material, and 1 mL of 4% formaldehyde solution was added. The cells were then fixed at room temperature for 20 min, washed three times with PBS and dehydrated with anhydrous ethanol, and the coverslips were removed and dried. The dried coverslips were observed with an inverted fluorescence microscope. The FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation and FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation\u0026thinsp;+\u0026thinsp;albumin solution were added to the mixed clonal cells, the FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was added to the Nthy-ori3-1 cells and their radioactivity was measured immediately by using an actinometer and again after the fixation and washing. A meter was used to measure their radioactivity separately and calculate the uptake rate. The formula for calculating the uptake rate is as follows:\u003c/p\u003e\u003cp\u003eUptake rate\u0026thinsp;=\u0026thinsp;radioactivity of the cells after fixation and washing/initial radioactivity of the cells after addition of the sample \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e7. Statistical analyses\u003c/h3\u003e\n\u003cp\u003eAll the experiments were repeated three times and statistically analyzed using SPSS 23.0 software, and data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The comparison of means between the two groups was performed using the paired t-test, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation characterization results\u003c/p\u003e\n\u003cp\u003e1.1. Dynamic light scattering results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe hydrodynamic size, polydispersity index (PDI) and zeta potential values of the synthesized nanoformulations were detected using dynamic light scattering and the zeta potential meter. The average hydrodynamic diameter, PDI and zeta potential of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation were measured to be 132.80\u0026plusmn;1.60 nm, 0.21\u0026plusmn;0.02 and -29.20\u0026plusmn;0.30 mV, respectively, as shown in Fig. 1, which shows that there is only a single peak in the distribution of the nanopreparation, with a relatively centralized distribution of particle size.\u003c/p\u003e\n\u003cp\u003e1.2 Transmission electron microscopy results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe morphology and distribution characterization of the prepared nanopreparations was examined via transmission electron microscopy. As shown in Fig.2a, the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanoparticles were circular or elliptical in shape, with no obvious adhesion phenomenon, relatively uniform distribution and particle sizes ranging from about 35 to 55 nm.\u003c/p\u003e\n\u003cp\u003e1.3. Encapsulation rate and radiochemical purity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe encapsulation rate of 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanoformulation was calculated as 68.70\u0026plusmn;2.79% according to the encapsulation rate formula, and the radiochemical purity of 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanoformulation was calculated as 91.70\u0026plusmn;1.67% according to the radiochemical purity formula using paper chromatography.\u003c/p\u003e\n\u003cp\u003e1.4. Stability of the nanoformulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was added to serum or saline and left at 25\u0026deg;C. The radiochemical purity of the solution was measured with paper chromatography at 1, 6, 12, 24, 48, 72 and 96 h. The results are shown in Fig. 2b. The radiochemical purity of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation declined slower in serum, and it was still more than 90% at 48 h. The radiochemical purity of the nanopreparation declined faster in saline and had dropped below 85% at 48 hours, indicating that the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was more stable in serum than in saline (t=119.51, p\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003e2. Results of SLC5A5 knockout in B-CPAP cells\u003c/p\u003e\n\u003cp\u003e2.1 Genotypic identification results of knockout cell lines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe DNA of wild-type cells and mixed clone cells (i.e., knockout cells) was extracted separately, and PCR amplification was performed. The PCR-amplified products were electrophoresed and sent to Hunan Starfish Biotechnology Co., Ltd., for sequencing. The results of PCR electrophoresis are shown in Fig. 3. The bands of wild-type cells at locus 1 are about 1055bp, those at locus 2 are about 873bp and those at locus 3 are undetectable. Those of the mixed clones are about 1055bp at locus 1, 873bp at locus 2 and 873bp at locus 3. This is because the wild-type cells are not cleaved at locus 1 or 2 and can be detected as intact, but the target gene is not knocked out, so the KO bands (i.e., knockout bands) cannot be amplified. The hybrid clones have various cell types (wild type, heterozygous and heterozygous) and are able to be detected at loci 1 and 2 and amplify the KO bands (i.e., knockout bands). The sequencing results are shown in Fig. 4. The comparison and analysis of the DNA sequencing results of the hybrid clonal cells with those of the wild-type cells using chromas 2.6.5 software revealed that the knockout peaks appeared in the hybrid clonal cells, while no knockout peaks appeared in the wild-type cells. It was also shown that the hybrid clonal cells had 340bp, 1055bp and 1340bp deletions of type 3 in the SLC5A5 gene, which, in combination with the electrophoresis results, indicates that the SLC5A5 gene fragment was successfully knocked down in B-CPAP cells.\u003c/p\u003e\n\u003cp\u003e3. Results of cellular uptake experiments\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 5, the hybrid clonal cells and Nthy-ori3-1 cells grew well, after which the cellular uptake behavior was observed via inverted fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003eAs shown by the cellular uptake graph in Figure 6, very little free FITC was observed after incubation of the hybrid clonal cells with free FITC for 4 h, while a large amount of the nanopreparation was observed after incubation of the hybrid clonal cells with the FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation for 4 h. The uptake rate was calculated using the above formula to be approximately 80.60 \u0026plusmn; 1.81%; however, after the addition of the albumin solution, a significant decrease in the amount of nanopreparation entering the cells was observed, with an uptake rate of approximately 43.70\u0026plusmn;1.75%, suggesting a competitive inhibitory effect of albumin (43.70% vs. 80.60%, t=241.57, p\u0026lt;0.0001). A small amount of nanopreparation was observed after incubation of Nthy-ori3-1 cells for 4 hr with the FITC\u0026ndash;131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation, with an uptake rate of approximately 20.50\u0026plusmn;2.17%, which was significantly lower than that of the mixed clonal cells (20.50% vs. 80.60%, t=288.71, p\u0026lt;0.0001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe prognosis of RAIR-DTC patients is extremely poor, with a 10-year survival rate of about 10% \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Currently, the therapeutic means for RAIR-DTC, mainly including systemic therapy and local therapy, have poor therapeutic efficacy. Local therapy includes surgical resection of metastatic foci, radioactive particle implantation and external radiotherapy; however, most patients with RAIR-DTC have multiple foci, and the efficacy of local therapy is limited. Systemic therapy includes targeted drug therapy and re-differentiation therapy. Most patients experience different degrees of adverse events after using targeted drugs \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, such as those for hypertension, proteinuria and hand\u0026ndash;foot syndrome, which are often resistant to targeted drug therapy, and drug resistance occurs with long-term use. Currently, data from several clinical trials of RAIR-DTC redifferentiation therapy show poor results. In recent years, targeted radionuclide therapy (TRT) has provided a novel treatment for patients with a variety of intermediate and advanced tumors. TRT targets molecules with high tumor-specific expression, and through the binding of ligands with specific, high-affinity binding, targeted transport radionuclides are aggregated at the tumor site and irradiated internally. However, a shortcoming of TRT is that a synthetic mixture of nuclides labeled on specific ligands, which has only a short plasma half-life, can only reach the tumor extracellular matrix. The nuclides also cannot participate in off-target metabolic processes. These factors have limited the application of targeted nuclide probe therapy to some extent. Therefore, radioactive iodine remains the most efficacious therapeutic strategy for DTC patients, and finding replacement NIS channels to deliver radioactive iodine into RAIR-DTC is a key point to address in RAIR-DTC therapy.\u003c/p\u003e\u003cp\u003eWith the continuous development of nanotechnology, nanoformulations have received extensive attention from the medical community \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, and so far, researchers have developed various types of nanocarriers, including polymer nanoparticles, nanoprotein materials, metal-based nanoparticles and liposomes. Among them, human serum albumin has the advantages of good biodegradability, biocompatibility, low immunogenicity and high stability and is widely used in drug delivery systems \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that the binding of albumin to its receptor can activate the caveolin-1 (Cav-1)-mediated cytosolic pathway \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, which results in the formation of caveolae in the cell membrane, which transport albumin and other plasma components into the cytoplasm. In addition to SPARC, receptors involved in albumin transport and distribution in the human body include Gp60, Gp18, Gp30 and FcRn \u003csup\u003e[17\u0026ndash;19 ]\u003c/sup\u003e. Albumin receptors are highly expressed mainly in tumor cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e with natural active targeting and to a lesser extent in the normal liver, intestine and kidney. Therefore, we envisioned the utilization the biological behavior of albumin to transport radioactive iodine into the cell and solve the fatal pain point of RAIR-DTC's inability to uptake iodine through the NIS channel. Albumin has multiple sites for ligand binding, including covalent or non-covalent binding for drug loading \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, but iodine does not bind directly to albumin to form nanoformulations. Therefore, the preparation of iodine-loaded albumin nanoformulations requires the addition of other carriers for loading 131I. Cyclodextrins are cyclic oligosaccharides consisting of multiple glucose units, which have the advantages of good biodegradability, biocompatibility and low immunogenicity \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. β-Cyclodextrins have a lipophilic central vesicle lumen and hydrophilic outer surface, where the drug can enter and form a water-soluble inclusion complex, and β-Cyclodextrins can be used to form nanoformulations for drug loading with HSA as well as binding to iodine\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThrough the above research ideas, we successfully prepared 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparations, and the PDI of 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparations was 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, which, combined with the distribution curves, indicated that the distribution of the nanopreparations was relatively concentrated. The transmission electron microscopy results showed that the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanoparticles were round or elliptical in shape, with no obvious adhesion phenomenon, and were more uniformly distributed, with a particle size of about 35\u0026ndash;55 nm, which was smaller than the particle size measured with DLS (132.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60 nm). This was due to the dehydrated morphology of the nanopreparations shown in the transmission electron microscopy images, while DLS was used to detect the mean hydrodynamic particle size of the nanopreparations. The encapsulation rate of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was 68.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79%, which may be related to the decay of 131I during the preparation process, but more importantly, the experimental conditions need to be further mapped to shorten the preparation time. The radiochemical purity of the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was 91.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67% as measured via paper chromatography. Radiochemical stability experiments verified that the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was more stable in serum than in saline (t\u0026thinsp;=\u0026thinsp;119.51, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the radiochemical purity of the preparation was still\u0026thinsp;\u0026gt;\u0026thinsp;90% in serum for 48 hours. In general, the stability of radiopharmaceuticals in saline is greater than the serum stability, but the opposite is true in the results of the present study, probably due to the fact that 131I has a certain protective effect within the dual carriers of albumin and cyclodextrins, which is mainly manifested by the biological properties of albumin in vivo.\u003c/p\u003e\u003cp\u003eThe cellular uptake assay showed that the mixed clonal cells were able to take up the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation with an uptake rate of about 80.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81%, and after the addition of albumin solution, the nanopreparation entering the cells was significantly reduced, with an uptake rate of about 43.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75%. This indicated that there was a competitive inhibitory effect of albumin (43.70%VS80.60%, t\u0026thinsp;=\u0026thinsp;241.57 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The uptake rate of Nthy-ori3-1 cells was about 20.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17%, which was significantly lower than that of the mixed clones (20.50% vs. 80.60%, t\u0026thinsp;=\u0026thinsp;288.71, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Albumin nanopreparations may utilize an enhanced permeability and retention effect (EPR) to stay in the interstitium of RAIR-DTC tumor cells \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In addition to this, it has been suggested that secreted protein that is acidic and rich in cysteine (SPARC) has a nanodrug-accumulating effect. Albumin nanodrugs can accumulate more drugs when combined with SPARC, which is highly expressed in tumor cells, and tend to achieve better efficacy in such tumors, including thyroid cancer cells\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Albumin nanoformulations that reach the tumor cell mesenchyme bind to receptors (SPARC or Gp60) that activate the Cav-1 pathway, allowing albumin-loaded drugs to accumulate within the tumor cell cytoplasm \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is tentatively suggested according to the uptake experiments that albumin nanopreparations can use this pathway to deliver radioactive iodine into the RAIR-DTC cytoplasm and kill tumor cells using rays released by the continuous decay of iodine.\u003c/p\u003e\u003cp\u003eThere are still some shortcomings of this study. First, the 131I\u0026ndash;cyclodextrin\u0026ndash;albumin nanopreparation was prepared for a long time, and the 131I decayed, which may have affected the experimental results to a certain extent, but we tried to use the same batch of the nanopreparation in each experiment to minimize the error. Second, the knockdown of SLC5A5 in B-CPAP cells was only verified at the gene level, the gene fragment was not verified at the protein level and no pure B-CPAP cells were screened for SLC5A5 knockdown. Furthermore, only the human thyroid gland was tested for the knockdown of the gene fragment, and no pure cells were screened. Only the B-CPAP cell line of human papillary thyroid carcinoma was investigated, neglecting other commonly used cell lines of human papillary thyroid carcinoma or human follicular thyroid carcinoma; these shortcomings will be further improved in the subsequent experiments. The aim of this study was to investigate the feasibility of albumin nanoformulations for the treatment of RAIR-DTC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, \u003csup\u003e131\u003c/sup\u003eI-cyclodextrin-albumin nanocomplex was successfully prepared. This novel nuclide complex can be absorbed by RAIR-DTC cells with high absorption rate. In conclusion, we have innovatively constructed a NTRT platform, and proposed a new strategy for clinical diagnosis and treatment of iodine-resistant thyroid cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZZ, PW, FS and WP contributed to the theory behind experiments, contributed to sample preparation and data collection, and carried out the experiments. ZZ and PW drafted the manuscript together. HZ, JP and WT contributed to the data collection and analysis. FS has contributed to the conception of the study and the revision of the article. all the authors critically revised the paper and approved the submitted version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the General Program of Natural Science Foundation of Hunan Province (2024JJ5241).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSUNG H, FERLAY J, SIEGEL R L, et al. 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The relationship between SPARC expression in primary tumor and metastatic lymph node of resected pancreatic cancer patients and patients\u0026apos; survival. \u003cem\u003eHepatobiliary Pancreat Dis Int\u003c/em\u003e. 2017;16(1):104-109.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Radioiodine-refractory thyroid cancer, NIS, Radionuclide therapy,131I","lastPublishedDoi":"10.21203/rs.3.rs-7371870/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7371870/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eRadioiodine-refractory thyroid cancer is currently a difficult clinical issue and pain point. This may be related to a combination of factors such as cytokines, signaling pathways and the cell microenvironment causing damage to the cell's iodine uptake channels, mainly manifested as the decreased expression of Na/I symporter（NIS） and the loss of the best treatment strategy. This study innovatively combined targeted radionuclide therapy (TRT) with nano delivery system to build a nano targeted radionuclide therapy (NTRT) platform to solve the problem of NIS failure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe nanopreparations were characterized according to their particle size, potential, morphology, encapsulation rate, radiochemical purity and stability, and the uptake of \u003csup\u003e131\u003c/sup\u003eI-cyclodextrin-albumin nanoparticles by SLC5A5 knockout B-CPAP cells was analyzed. the average hydrodynamic diameter, PDI and zeta potential of the prepared \u003csup\u003e131\u003c/sup\u003eI-cyclodextrin-albumin nanoparticles were 132.80±1.60nm, 0.21±0.02 and -29.20±0.30mV. The results of transmission electron microscopy showed that the \u003csup\u003e131\u003c/sup\u003eI-cyclodextrin-albumin nanoparticles were round or elliptical, without an obvious adhesion phenomenon. The distribution was relatively uniform, and the particle size was about 35-55nm. The encapsulation rate of the nanopreparation was 68.70±2.79%, and the radiochemical purity was 91.70±1.67%. SLC5A5 knockout B-CPAP cells can ingest \u003csup\u003e131\u003c/sup\u003eI-cyclodextrin-albumin nanopreparations, and the uptake rate is about 80.60±1.81%. It is concluded that we successfully prepared a \u003csup\u003e131\u003c/sup\u003eI-cyclodextrin-albumin nanoparticle preparation that can be taken up by RAIR-DTC cells with a high uptake rate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWe have successfully developed a preliminary NTRT platform, which will be a promising alternative therapy for RAIR-DTC patients who have lost radioiodine therapy due to NIS failure.\u003c/p\u003e","manuscriptTitle":"Preparation of 131I-loaded albumin nanopreparations and uptake in radioiodine-refractory thyroid cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:37:43","doi":"10.21203/rs.3.rs-7371870/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3b1f57df-5c79-4241-9a2c-2d31b3869b25","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-27T14:31:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 05:37:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7371870","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7371870","identity":"rs-7371870","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}