NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer

doi:10.21203/rs.3.rs-3194973/v1

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NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer

https://doi.org/10.21203/rs.3.rs-3194973/v1

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Adoptive cellular immunotherapy is critical for future clinical applications as a promising and alternative cancer therapy platform. Natural killer (NK) cells have attracted attention as an important type of innate immune regulatory cell that can rapidly kill multiple adjacent cancer cells. However, these cells are significantly less effective in treating solid tumors than in treating hematological tumors. Herein, we report the synthesis of a Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobe labeled with NK-92 cells that can be used for adoptive cellular immunotherapy, photodynamic therapy and dual-modality imaging-based in vivo fate tracking. The labeled NK-92 cells specifically target the tumor cells, which increases the amount of cancer cell apoptosis in vitro. Furthermore, the in vivo results indicated that the labeled NK-92 cells can be used for tumor magnetic resonance imaging and fluorescence imaging, adoptive cellular immunotherapy, and photodynamic therapy after tail vein injection. These data show that the developed multifunctional nanostructure is a promising platform for efficient innate immunotherapy, photodynamic treatment and the noninvasive therapeutic evaluation of breast cancer.

multimodal nanomaterials

NK-92 cells

magnetic resonance imaging

adoptive cellular immunotherapy

photodynamic therapy

In women, breast cancer is the most common type of cancer and the second leading cause of cancer-related death¹. Conventional treatment for breast cancer includes surgical resection, radiotherapy, chemotherapy and hormone therapy. Patients with aggressive forms of this disease have significantly shortened disease-free survival and overall survival (OS) and increased resistance to radiotherapy and chemotherapy^2,3. Currently, adoptive cellular immunotherapy has developed into a clinically validated therapy for many diseases, especially cancer⁴. Immune cell-mediated cancer therapy has been actively investigated and exhibits promising antitumor efficacy in both hematological cancers and solid tumors^5,6, five chimeric antigen receptor (CAR) T cells have been approved by the U.S. Food and Drug Administration (FDA)⁷. Compared with T cells, Natural kill (NK) cells have distinctive mechanisms to target and eliminate cancer cells, including the capacity to destroy tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC)⁹, available cell line for adoptive transfer in humans to decrease therapeutic cost¹⁰. However, the treatment effects of these cells for solid tumors are significantly worse than those on hematological tumors, which may be due to certain limitations, such as inadequate targeting and an insufficient local concentration of NK cells in the tumor area.

In addition, photodynamic therapy (PDT) has been recently adopted as a treatment strategy, which can be achieved by applying photosensitizers and visible light in combination with oxygen to produce reactive oxygen species (ROS) to destroy tumor cells¹¹. Furthermore, PDT can synergistically boost the efficiency of immunotherapy^12,13. Chlorin e6 (Ce6) is a second-generation photosensitizer that can be used as a second-generation photosensitizer against various cancers¹⁴. It also has the abilities to accumulate more effectively in tumors than in normal cells, absorb light more strongly at longer wavelengths (660 nm) for faster clearance from organisms, and the capability to be used as an ideal fluorescent label for optical imaging. Therefore, combining PDT with immunotherapy has the potential to be applied in cancer treatment.

Herein, Fe₃O₄ NPs were coated with a layer of polyethylene glycol (PEG) to improve biocompatibility¹⁵, and the surface of these PEGylated Fe₃O₄ NPs (Fe₃O₄-PEG-COOH) were further decorated with bevacizumab (Avastin®), anti-CD56 antibody and Ce6 to form a nanocomposite (Fe₃O₄-PEG-CD56/Avastin@Ce6). Bevacizumab is a clinic available monoclonal immunoglobulin G antibody targeting VEGF¹⁶. CD56 is abundantly expressed on NK cells^17,18. Therefore, the yielded Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs were synthesized to label NK-92 cells for targeted delivery to breast cancer cells. NK-92 cells labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6 were used for in vitro and in vivo breast cancer adoptive cellular immunotherapy and PDT as well as MR/optical dual molecular imaging(Fig. 1).

Materials

FeSO₄·7H₂O and ferric citrate were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Amine-PEG_2k-carboxyl was purchased from Shanghai Yare Biotechnology Co., Ltd. (Shanghai, China). Hoechst 33342 was purchased from Sigma-Aldrich Chemical Co., Ltd. (St Louis, MO, USA). N-Hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and Ce6 were obtained from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Cell culture medium, horse serum, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco (USA). Recombinant IL-2 was from PeproTech (USA). Bevacizumab (Avastin®) was obtained from Roche (Genentech, South San Francisco, CA). The anti-CD56 antibody was obtained from BD (USA). The CCK-8 assay materials were purchased from NCM Biotech Co., Ltd. (Suzhou, China).

Synthesis of targeted NPs: Fe₃O₄ NPs were produced according to our previous study¹⁹. After that, EDC and NHS were separately mixed with sodium borate buffer, pH = 9 (SBB 9) (c = 3.125 mg/mL), followed by the addition of 0.1 mL of Fe₃O₄-COOH NPs. Then, 10 mg of PEG was dissolved in 100 µL of deionized water and mixed well with 0.3 mL of the SBB 9/Fe₃O₄-COOH NPs mixture, which was maintained at room temperature for 12 h. After the reaction was finished, the product solution was purified with an ultrafiltration device (Millipore, Mw = 100 kDa) for 10 min at 2000 r.p.m., and the product (Fe₃O₄-PEG-COOH) was collected. To activate the carboxyl group on the Fe₃O₄-PEG NPs, EDC and NHS (200 µL, 6.25 mg/mL) were added for 20 min of incubation. Then, 50 µL of CD56 and 50 µL of Avastin were added to the activated Fe₃O₄-PEG NPs for 12 h of reaction. Next, the product solution was purified with an ultrafiltration device (Millipore, Mw = 100 kDa). Then, 500 µL of a 5 mg/mL solution of Ce6 dissolved in 0.01 M NaHCO₃ (pH 8.3) was mixed into the aforesaid concentrated solution, and this mixture was left at 37°C for 24 h on a shaking bed. Finally, the product was collected and dialyzed against water in a dialysis bag (MWCO: 3500) for three days, changing the water every four hours. The samples were kept at 4°C for further use.

Characterization techniques

The morphology and size of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes were examined by TEM (JEM-2100 electron microscope, JEOL, Japan). One hundred NPs were randomly selected and analyzed by ImageJ software. TGA of the Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs were acquired by a Thermo gravimetric analyzer (Pyris 1 TGA, Perkin Elmer, America) under a N₂ atmosphere at a heating rate of 20°C/min. The UV-visible absorption spectrum was acquired with a Varian Cary 50 spectrophotometer. FTIR spectroscopy was performed with a PerkinElmer GX spectrophotometer. The T2 relaxation times of increasing concentrations of the nanoprobe were measured with a 3.0 T MRI system.

Cell culture: 4T1 cells and MCF-7 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO₂. The base medium for the NK-92 cell line was α-MEM without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate. The following components were added to the base medium to make complete growth medium: 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid and 100–200 U/mL recombinant IL-2; additionally, the final concentrations of horse serum (HS) and FBS were each adjusted to 12.5%. Cells were cultured in complete growth medium at 37°C with 5% CO₂.

Cytotoxicity evaluation: A CCK-8 cell viability assay was used to determine whether the NPs cause adverse effects on the biological characteristics of NK-92 cells,. Briefly, NK-92 cells were cultured overnight in 96-well plates. Then, 100 µL of culture medium containing different concentrations of the nanoprobes was added for incubation for an additional 12 h, 24 h or 48 h. Next, 20 µL of CCK-8 reagent were added. After incubation for 3 h, the optical density (OD) values were obtained at 450 nm using a microplate reader. Cell viability was determined according to the following formula: cell viability (%) = (A_{experimental group} − A_blank)/(A_{control group}− A_blank) × 100%. The values from triplicate wells were measured for each group. FCM was performed to evaluate cell apoptosis and the cell cycle after incubation with different concentrations of Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs. For cell apoptosis analysis, labeled NK-92 cells were washed 3 times with binding buffer. 5 µL of FITC-annexin V and 100 µL of binding buffer were added to the cells. After 15 min of incubation in the dark, 5 µL of propidium iodide (PI) solution and 400 µL of binding buffer were added to each tube. Finally, the fluorescence signal was detected by FCM. For cell cycle analysis, we fixed labeled NK-92 cells in 75% ethanol for 24 h, stained the cells with PI and RNase (BD) and then washed them with PBS. The cell cycle was analyzed by measuring the DNA content by FCM.

Cell labeling and NP localization

NK-92 cells were incubated with Fe₃O₄-PEG-CD56/Avastin@Ce6 (10 µg/mL) at 37°C for 24 h in complete medium. Labeled NK cells were purified by density-gradient centrifugation, and the unincorporated NPs were removed from the solution. The sample was washed with PBS and fixed with electron microscope fixative. To determine whether the NPs were internalized or mainly adsorbed on the cell surface, NP localization was observed by TEM. FCM was used to examine cellular uptake efficiency. NK-92 cells were seeded in 24-well cell culture plates at a density of 2 × 10⁵ cells per well and then cultured in fresh complete medium containing Fe₃O₄-PEG-CD56/Avastin@Ce6 or Fe₃O₄-PEG-Avastin@Ce6 at concentrations of 0, 5, 10, 15 and 20 µg/mL. After 4 h of incubation, the cells were washed with PBS 3 times and resuspended in 500 µL of PBS for FCM analysis on a Becton Dickinson FACScan analyzer. The FL3 fluorescence of 10,000 cells was measured, and the mean fluorescence of the gated viable cells was quantified. Confocal microscopy was also used to analyze the specific uptake of NPs by NK-92 cells. Cell suspensions were placed on microscope slides and the solution was evaporated. Samples were fixed with 4% paraformaldehyde and stained with Hoechst 33342 followed by confocal microscopy observation. Emission was detected separately through either 420–460 nm or 650–700 nm barrier filters.

MR and optical imaging of NK-92 cells in vitro: NK-92 cells were cultured in fresh complete medium containing Fe₃O₄-PEG-CD56/Avastin@Ce6 or Fe₃O₄-PEG-Avastin@Ce6 at concentrations of 0, 10 and 20 µg/mL. After 4 h of incubation, the cells were washed with PBS and then resuspended in 500 µL of 1% agarose. MRI was performed using a 3.0 T MRI system with a wrist coil. T2WI were acquired using the following scanning parameters: repetition time (TR) = 2500 ms, echo time (TE) = 60 ms, field of view (FOV) = 130 mm×104 mm, slice thickness = 3 mm, and slice gap = 0.1 mm. The T2 signal intensities were measured within the region of interest (ROI). Optical images were performed using a Caliper Life Sciences imaging system. (excitation: 630 nm, emission: 700 nm)

Apoptosis assay

Labeled NK-92 cell toxicity was measured by CCK-8 assay in 4T1 and MCF-7 target cells. 4T1 cells were seeded into 6-well plates at 5 × 10⁵ cells per well in DMEM containing 10% FBS and incubated for 24 h (37°C, 5% CO₂). Then, NK-92 cells labeled with Fe₃O₄-PEG-CD56@Ce6 or NK-92 cells labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6 were coincubated with the target cells for 24 h at 37°C. The cells were collected in Eppendorf tubes, and after washing twice with PBS, the cells were stained with an Annexin V-FITC Apoptosis Detection Kit. The staining procedure was conducted at room temperature in the dark. The stained cells were analyzed by FCM.

Western blot: 4T1 and MCF-7 cells in 6-well plates at a density of 5 × 10⁴ cells per well were treated as indicated. Proteins were isolated from the cells by utilizing RIPA buffer with the help of a Protease/Phosphatase Inhibitor Cocktail. A modified BCA Protein Assay Kit was used to assess protein purity. The protein samples were then subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA). The primary antibodies listed below were utilized to probe the specific target proteins: anti-Bax, anti-Bcl-2, anti-cleaved caspase-3, anti-cleaved PARP and anti-GAPDH, all of which were purchased from Cell Signaling Technology. After incubation with the secondary antibodies (CST), the target bands were developed with an EasyBlot ECL kit.

Establishment of the mouse breast cancer model

Animal experiments and animal care were conducted according to protocols approved by the institutional committee. Female 5-week-old BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China) and maintained in a specific pathogen-free (SPF) environment. The tumor-bearing mouse model was established by subcutaneously injecting 100 µL of a 4T1 cell suspension containing 1 × 10⁶ cells into each mouse in the right mammary fat pad.

In vivo MR and optical dual-modal imaging: MR and optical dual-modal imaging and biodistribution studies were performed when the tumor diameters were 5–10 mm. NK-92 cells labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6 were injected into tumor-bearing mice via the tail vein. In vivo MRI was carried out using a 3.0 T MR system with a high-resolution animal coil before injection and 2 h, 4 h, 6 h, 12 h and 24 h after intravenous injection. The T2WI used the following scanning parameters: TR = 2000 ms, TE = 97 ms, flip angle = 90°, FOV = 60 mm×80 mm, slice thickness = 2 mm, and slice gap = 0 mm. Optical images were obtained using a Caliper Life Sciences imaging system before injection and 6 h, 12 h and 24 h after intravenous injection (excitation: 630 nm, emission: 700 nm).

In vivo antitumor effects

The tumor-bearing mice were randomly divided into four groups and treated with PBS, NK-92 cells (2 × 10⁷ cells/0.1 mL of PBS), NK + NPs or NK + NPs + L. The NK + NPs + L group was irradiated with a laser for 15 min at 6 h, 12 h and 24 h after injection. The tumor volumes and mouse body weights were recorded daily. The longest (a) and shortest (b) diameters of the tumors were measured with a digital Vernier caliper to determine the volume using the formula (a × b²)/2. After 15 days, all animals were sacrificed and the main organs were excised for H&E staining.

Statistical analysis

All quantitative data are expressed as the mean ± standard deviation (SD). SPSS 25.0 statistical software (SPSS Institute, Cary, North Carolina, USA) was used for statistical analyses. Data among multiple groups were compared using one-way ANOVA followed by the least significant difference (LSD) test. P values < 0.05 were considered statistically significant.

3.1 Synthesis and characterization of Fe₃O₄-PEG-CD56/Avastin@Ce6

Transmission electron microscopy (TEM) and histogram images showed the size distribution of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes (Fig. 2A and B). The shape of the formed Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes was similar to a sphere. The average particle size of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes was 6.37 nm. After two weeks, no obvious changes in the morphology or diameter of the NPs were observed, indicating that the nanomaterial was stable in structure (Fig. S1). Various characterizations were undertaken to confirm the successful synthesis of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes. UV-vis absorption spectroscopy of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes showed the distinct characteristic absorption peak of Ce6 at 405 nm, and those from PEG, the anti-CD56 antibody and Avastin between 200 and 300 nm (Fig. 2C). As shown by Fourier transform infrared (FTIR) spectroscopy (Fig. 2D), the peaks from the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes (curve d in Fig. 2D) at 1105 nm and 2963 nm originated from the bending vibrations of the C-O-C bond and C-H bond of PEG. Moreover, the strong absorption peaks at 1601 nm and 1712 nm corresponded to the carboxyl stretching vibration of the amide group (-CO) and the bending vibration of the N-H bond, indicating successful conjugation of PEG, the anti-CD56 antibody and Avastin. We also acquired T2-weighted MR images(T2WI) of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes at different Fe concentrations to evaluate the ability of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes to act as contrast agents. As shown in Fig. 2E, the signal intensity from the T2WI gradually decreased with increasing Fe concentration. After generating a plot with the Fe concentration as the abscissa and the T2 relaxation time as the ordinate, a linear relationship was found (Fig. 2F). The thermal stability and decomposition behavior of Fe₃O₄ were evaluated by thermogravimetric analysis (TGA) under a nitrogen atmosphere, as shown in Fig. S2. The weight loss from 100 to 400°C corresponded to the removal of unstable oxygen-containing groups from the organic species. Further weight loss from 450 to 750°C was related to the removal of the relatively stable oxygen-containing groups. When the temperature was above 750°C, the oxygen-containing groups on the surface of Fe₃O₄ were completely removed. The total weight loss of 11.8% revealed that the surface of the synthesized Fe₃O₄ NPs contains a large number of carboxyl groups.

(A) TEM image of the synthesized Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs. (B) Size distribution of the Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs determined from the TEM images. (C) UV-visible absorption spectrum. (D) FTIR spectrum. a. Fe₃O₄ NPs, b. Fe3O4-PEG, c. Fe₃O₄-PEG-CD56/Avastin, and d. Fe₃O₄-PEG-CD56/Avastin@Ce6. (E) T2-weighted MR images of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes in aqueous solutions at various Fe concentrations. (F) T2 relaxation rates of the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes.

3.2 Cell viability assay

To investigate the effects of the Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs on the physiological characteristics of NK-92 cells, cell apoptosis and cell cycle analyses were performed. The cell apoptosis rate was measured by flow cytometry (FCM). Figure 3A shows that the NK-92 cells in different treatment groups exhibited the same number of early and late apoptotic cells. These findings suggest that even high concentrations of Ce6 (20 µg/mL) do not induce NK-92 cell apoptosis. As shown in Fig. 3B, the cell cycle did not significantly change at different Ce6 concentrations. Then, Cell Counting Kit-8 (CCK-8) assays were also performed to investigate the effects of the nanoprobes on NK-92 cell activity. Fig. S3 shows that cell viability gradually decreased with increasing Ce6 concentration. However, more than 60% of the cells survived at the highest vector concentration after 48 h. Taken together, these results suggested that these NPs possessed good biocompatibility within this concentration range and could be safely used for further study.

3.3 Cellular uptake assay

TEM was used to investigate the internalization of the Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs into NK-92 cells (Fig. 4A). The TEM images of the NK-92 cells revealed that the NPs appeared with high electron densities inside the cell cytoplasm. The cellular uptake of the nanoprobes with and without the anti-CD56 antibody was measured in vitro by FCM (Fig. 4B, C and D). As the Ce6 concentration increased, the average value of the fluorescence intensity increased. In addition, we found that the median fluorescence intensity value of the cells incubated with Fe₃O₄-PEG-CD56/Avastin@Ce6 was significantly higher than that of the cells treated with Fe₃O₄-PEG-Avastin@Ce6 at the same Ce6 concentration. This result indicated that the anti-CD56 antibody increased the uptake of these NPs, which will help with cancer therapy effectiveness. These results confirmed nanoprobe internalization by NK-92 cells.

3.4 MR and optical imaging in vitro in NK-92 cells

To determine the ability of Fe₃O₄-PEG-CD56/Avastin@Ce6 to target NK-92 cells, we measured the T2 signal intensity in the NK-92 cells after incubation with NPs at different concentrations of Ce6 (Fig. 5A). The results showed that the T2 signal intensity decreased significantly as the Fe₃O₄ concentration in the NPs increased. The MRI signal intensity of the NK-92 cells labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6 decreased more significantly than that of the cells labeled with Fe₃O₄-PEG-Avastin@Ce6 at the same Ce6 concentration (10 µg/mL) (Fig. 5B). Furthermore, optical imaging was performed. As shown in Fig. 5C, NK-92 cells incubated with the Fe₃O₄-PEG-CD56/Avastin@Ce6 nanoprobes exhibited stronger fluorescence intensity than cells treated with Fe₃O₄-PEG-Avastin@Ce6, and the fluorescence signal in the cytoplasm was also significantly enhanced with increasing NP concentration.

3.5 In vitro therapeutic efficacy

The targeting effects of NK-92 cells labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6 (NK + NPs) was demonstrated by FCM using MCF-7 and 4T1 breast cancer cells. Figure 6A and Fig. S4 indicate that the NK + NPs were the most cytotoxic to the 4T1 and MCF-7 cells compared to the other groups. The apoptotic activity of the NK + NPs (32.0%) was higher than that of NK-92 cells alone (19.3%) or NK-92 cells loaded with Fe₃O₄-PEG-CD56@Ce6 (NK + nNPs; 26.1%). These results indicate that NK + NPs could guide localization into tumor cells.

Furthermore, we investigated the effects of NK + NPs on the expression of apoptotic markers in breast cancer cells. Western blotting was performed to identify the expression of Bax, Bcl-2, cleaved caspase-3 and cleaved PARP. In the NK + NP group, Bcl-2 was downregulated and Bax, cleaved caspase-3 and cleaved PARP were upregulated (Fig. 6B). Collectively, these results indicated that NK + NPs more successfully induced tumor cell apoptosis and Avastin improved the effectiveness of cancer therapy.

3.6 In vivo MR and optical dual-modal imaging

To investigate the tumor-specific targeting properties of the NK + NPs, optical imaging was applied to monitor the intrinsic near infrared (NIR) fluorescence signal of Ce6 released by the nanoprobes. When the tumor nodules of nude mice reached a size of 60–80 mm³, NK + NPs were intravenously injected for the real-time detection of drug distribution in vivo. Figure 7A shows the changes in the fluorescence signals in the nude mice before injection and 6 h, 12 h and 24 h later. The fluorescence signal at the tumor site gradually increased with time, which implies that the NK + NPs can target the tumors. To further investigate whether NK + NPs can be used as an effective imaging agent, MRI was carried out before and 2 h, 4 h, 6 h, 12 h and 24 h after intravenous injection (Fig. 7B). The MR signal in the tumor mass was found to gradually decrease over time. This result indicates that the NK + NPs localized in the tumor, which is consistent with the fluorescence imaging results. These results indicate that the NPs are biocompatible, promote internalization and target tumors, thus contributing to efficient innate immune cell therapy and PDT.

3.7 In vivo cancer therapy

Therapeutic efficacy was evaluated via 4T1 tumor xenografts in BALB/c nude mice. The experimental mice were divided into four groups: PBS, NK-92 cells, NK + NPs and NK + NPs + L. The changes in tumor volume were monitored over 15 days (Fig. 8A and B). Over the 15 days, all treated groups showed tumor growth inhibition compared to the PBS group, suggesting that NK-92 therapy majorly contributed to tumor suppression. Tumor growth in the NK + NP group showed a more efficient lag than that found in the NK-92 group. This finding indicates that the application of Fe₃O₄-PEG-CD56/Avastin@Ce6 may enhance the therapeutic effects by improving the local number of NK-92 cells in the tumor region. Notably, tumor growth in the NK + NPs + L group was significantly inhibited compared with the other three groups, illustrating the effectiveness of immunotherapy combined with PDT. These results demonstrate that the magnetic targeting method effectively enhances tumor treatment efficacy and provides safer NK-92 cell delivery.

To confirm and investigate the potential toxicity of NK + NPs in mice, the changes in the weights of the tumor-bearing mice were monitored as an indicator of poisonous side effects. As shown in Fig. 8C, no noticeable changes in mouse weight were observed in any of the four groups. On day 15, the major organs and tissues (heart, liver, spleen, lungs, and kidneys) were collected from the mice in the four groups and sliced, stained with hematoxylin and eosin (H&E) according to the standard protocol, and then observed by microscopy. Notably, no pathological abnormalities or other conditions were observed after histological examination of the major organ tissues from the mice in our experiments (Fig. 8D). Thus, no noteworthy signs of toxic side effects from the NK + NPs + L were observed in vivo, as further uncovered by histological examination after PDT treatment.

In summary, Fe₃O₄-PEG-CD56/Avastin@Ce6 NPs were synthesized and shown to be taken up by NK-92 cells. Within a certain concentration range, the physiological properties of the NK-92 cells remained unaffected by the NPs, and their ability to kill the target cells was significantly enhanced when the NK-92 cells were labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6. 4T1 xenograft tumors in mice were detected by MRI and optical imaging after a single intravenous administration of the nanoprobes. In addition, the antitumor therapeutic efficiency of NK-92 cells labeled with Fe₃O₄-PEG-CD56/Avastin@Ce6 was significantly enhanced by PDT under laser irradiation. These findings strongly suggest that the designed multifunctional Fe₃O₄-PEG-CD56/Avastin@Ce6 nanocomposites can be potential nanoprobes for targeting innate immune cells, administering PDT and MR/optical dual-mode imaging. This study also showed that it is possible to observe and assess the curative effects of immunotherapy through imaging technologies in patients with breast cancer without the need for invasive, damaging or time-consuming methods to optimize and individualize breast cancer therapy for future clinical applications.

Acknowledgements

This research is financially supported by the National Natural Science Foundation of China (KAL 12090024, 81972872, 11826020 and GG 81671737), the Shanghai Pujiang Project (KAL 2014PJD028, GG18PJD020), the Shanghai Jiaotong University Cross-cooperation Project (KAL YG2015MS31, YG2019QNB31), the Shanghai Science and Technology Commission Project (KAL 17441900700), and the Shanghai Shenkang Project (KAL 6CR3091B).

Conflict of Interest: The authors declare no conflicts of interest.

Availability of data and materials: All relevant data are within the manuscript and supplement files.

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NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer

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Abstract

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1. Introduction

2. Materials and methods

3. Results and Discussion

3.1 Synthesis and characterization of Fe₃O₄-PEG-CD56/Avastin@Ce6

3.2 Cell viability assay

3.3 Cellular uptake assay

3.4 MR and optical imaging in vitro in NK-92 cells

3.5 In vitro therapeutic efficacy

3.6 In vivo MR and optical dual-modal imaging

3.7 In vivo cancer therapy

4. Conclusions

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Supplementary Files

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Version 1

NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer

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Abstract

Figures

1. Introduction

2. Materials and methods

3. Results and Discussion

3.1 Synthesis and characterization of Fe3O4-PEG-CD56/Avastin@Ce6

3.2 Cell viability assay

3.3 Cellular uptake assay

3.4 MR and optical imaging in vitro in NK-92 cells

3.5 In vitro therapeutic efficacy

3.6 In vivo MR and optical dual-modal imaging

3.7 In vivo cancer therapy

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

3.1 Synthesis and characterization of Fe₃O₄-PEG-CD56/Avastin@Ce6