Injectable Biocompatible Nanocomposites of Prussian Blue Nanoparticles and Bacterial Cellulose as a Safe and Effective Photothermal Cancer Therapy

doi:10.21203/rs.3.rs-3012905/v1

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Research Article

Injectable Biocompatible Nanocomposites of Prussian Blue Nanoparticles and Bacterial Cellulose as a Safe and Effective Photothermal Cancer Therapy

https://doi.org/10.21203/rs.3.rs-3012905/v1

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published 05 Oct, 2023

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Photothermal therapy (PTT) is a novel cancer treatment using a photoabsorber to cause hyperthermia to kill tumors by laser irradiation. Prussian blue nanoparticles (PB NPs) are considered as next-generation photothermal agents due to the facile synthesis and excellent absorption of near-infrared light. Although PB NPs demonstrate remarkable PTT capabilities, their clinical application is limited due to their systemic toxicity. Bacterial cellulose (BC) has been applied to various bio-applications based on its unique properties and biocompatibility. Herein, we design composites with PB NPs and BC as an injectable, highly biocompatible PTT agent (IBC-PB composites). Injectable bacterial cellulose (IBC) is produced through the trituration of BC, with PB NPs synthesized on the IBC surface to prepare IBC-PB composites. IBC-PB composites show in vitro and in vivo photothermal therapeutic effects similar to those of PB NPs but with significantly greater biocompatibility. Specifically, in vitro therapeutic index of IBC-PB composites is 26.5-fold higher than that of PB NPs. Furthermore, unlike PB NPs, IBC-PB composites exhibit no overt toxicity in mice as assessed by blood biochemical analysis and histological images. Hence, it is worth pursuing further research and development of IBC-PB composites as they hold promise as safe and efficacious PTT agents for clinical application.

prussian blue nanoparticles

bacterial celluloses

nanocomposites

photothermal therapy

improved biocompatibility

Photothermal therapy (PTT) is an emerging cancer treatment strategy that has been extensively researched in recent years. ^1–4 This method involves the use of near-infrared (NIR) laser to irradiate photothermal agents accumulated in cancer lesions. These agents then convert the absorbed light into heat, causing a rise in local tissue temperature and thereby causing apoptosis or necrosis of tumor cells. ^5–8 Numerous materials, including carbon-based materials ^{9, 10}, gold ^11–13, silver ^14–16, iron oxide ^{17, 18}, and germanium nanocrystals ^{19, 20}, are currently being developed as potential agents for PTT. Also, a recent phase 1 clinical trial in patients with prostate cancer demonstrated the feasibility of PTT using a gold nanoshell, with successful tumor ablation and no serious complications observed in 94% (15/16) of patients. ²¹ This study demonstrates the promise of PTT agents for clinical applications, but the high cost of gold nanoparticles is considered as a potential limitation hindering large-scale clinical trials. ²²

Prussian blue (PB) is an ancient low-cost dye that can be easily prepared. ²³ Due to its strong optical absorbance in the NIR region and good photostability, Prussian blue nanoparticles (PB NPs) have been developed as potential photothermal agents for cancer. ^24–26 PB NPs have been widely utilized in the biomedical field to enable theranostics and drug delivery owing to its facile synthesis, controllable size and shape, easy surface modification for multifunctionality, and cost-effectiveness. ^27–30 In practice, the US FDA approved PB NPs as a clinical medicine for oral administration to treat patients with internal contamination of thallium (Tl⁺) or radiocesium (Cs⁺) poisoning in 2003. ³⁰ However, PB NPs have disadvantages as well, such as the accumulation in normal organs when they are systemically administered. ³¹ In particular, Chen et al. reported that intravenously injected PB NPs in mice caused acute liver injury, an outcome attributed to the substantial accumulation of the nanoparticles in Kupffer cells, the resident macrophages in the liver. ³²

Bacterial cellulose (BC) has many favorable properties as a biomaterial, such as biocompatibility, flexibility, and porosity. Based on these properties, BC is used in various biomedical applications, such as diagnostic sensors ^{33, 34}, tissue engineering ³⁵, drug delivery systems ^36–38, wound dressing ^39–42, and artificial skin ⁴³. Nata de coco, a type of BC invented in 1949, is made by fermenting coconut water through Komagataeibacter xylinus. ⁴⁴ Bacterial cellulose can be used by itself, but it is also used to make composites with various additives such as biopolymers ^{45, 46}, quantum dots ^{47, 48}, nanoparticles ^{39, 40, 49, 50} and nano carbons. ^{51, 52} Nanocellulose reportedly has an excellent stabilizing effect for metal nanoparticles, which can promote the nucleation of nanoparticles as well as prevent their agglomeration. ⁵³

Herein, we successfully manufactured and synthesized a composite of injectable bacterial cellulose with PB NPs that is biocompatible, locally injectable, and is amenable with repeated PTT sessions. The injectable BC composite with PB NPs (IBC-PB composites) went through a simple reduction reaction. The PB NPs were directly grown onto BC fragmented on the nanoscale. We hypothesized that IBC-PB composites can exert a potent PTT effect while remaining less toxic compared to PB NPs due to the biocompatibility of BC and the enhanced stability realized here.

2.1 Injectable bacterial cellulose (IBC) purification and fragmentation from nata de coco

Impurities such as remaining bacteria and sugar of the nata de coco were ideally removed by flowing D.W. for a few days. For an accurate quantitative experiment, physical grinding and lyophilization were conducted. Schweitzer’s reagent was used to reduce the nanofiber structure of BC to obtain an injectable bacterial cellulose. After the pre-treatment, the injectable quantified bacterial cellulose was successfully obtained (Figure S1).

2.2 Synthesis of PB NPs and IBC-PB composites

For the PB and IBC-PB composites, they were simply synthesized by the reduction of K₃Fe(CN)₆ under an acidic condition at 80 ℃ with the addition of PVP or IBC. The synthesized IBC-PB composites were dispersed at 50 ml D.W. and kept in a refrigerator at 5 ℃ to prevent contamination (Fig. 1).

2.3 Characterizations of the PB NPs and IBC-PB composites

The morphology of IBC-PB composites were examined by SEM, and PB NPs with a cubic structure were found to be evenly distributed on the IBC sheet (Fig. 2a-d). The ratio of PB NPs per area and particle size increases according to the concentration of potassium ferricyanide (0.25, 0.5, 0.75, and 1 mmol respectively). The PB NP size of the IBC-PB composites were 150 ~ 275 nm, and we synthesized 200 nm PB NPs as a control (Figure S2). Pristine PB NPs were utilized as a control in all further in vitro and in vivo studies.

TGA analysis was conducted under an air condition to measure the volume of PB nanoparticles (Fig. 2e). From the results of the TGA analysis of BC, only 0.8% of the current weight of cellulose remained after calcination at a temperature that exceeded 600 ℃, implying that all cellulose was removed at this high temperature. According to this result, the remaining important material of IBC-PB composites after TGA was confirmed to be iron oxide, which was produced by the calcination of PB, with the level proportional to the amount of potassium ferricyanide from 0 mmol to 1 mmol.

The XRD patterns of PB and IBC-PB composites are matched with that of a face-centered cubic lattice and are in good agreement with the standard data of PB (ICDD PDF2 01-073-0687). The peaks at 2-theta values of 17.4, 24.7, 35.1, 39,4, 43.5, 50.8, 54.1 and 57.3° can be assigned correspondingly to the (200), (220), (400), (420), (422), (440), (600) and (620) planes of the PB (Fig. 2f).

SEM, XRD, TGA data showed that Prussian blue nanoparticles were synthesized proportionally to the amount of the initial PB precursor and were uniformly distributed on the BC sheet.

2.4 Photothermal effect of the IBC-PB composites

The absorbance spectra of the IBC-PB composites according to a UV-visible spectrometer showed absorption bands at 600 ~ 900 nm with a high peak value at 800 ~ 900 nm. Due to its strong NIR absorption, IBC-PB composites had noticeable photothermal effects. The absorbance intensity increased in a linearly proportional manner relative to the initial concentration of potassium ferricyanide from 0.25 to 0.75 mmol. The absorbance of the IBC-PB composites (1 mmol) was mostly in accord with IBC-PB composites (0.75 mmol), allowing us to confirm that saturation accrued at 0.75mmol (Fig. 3a).

During the photothermal test, the temperature of the IBC-PB composites in each case was increased by irradiation using an 808nm-laser with at an intensity level of 1 W cm^− 2 by 20.5 ℃ in five minutes (Fig. 3b). The photothermal effect of IBC-PB composites increased with the amount of PB from 0.25 to 0.75 mmol, with the results for IBC-PB composites (1 mmol) in good agreement with those of IBC-PB composites (0.75 mmol). This is consistent with the UV-vis spectra data, where the photothermal effect was saturated at 0.75 mmol of K₄[Fe(CN)₆. Therefore, IBC-PB composites (0.75 mmol) was selected as the leading group for all further in vitro and in vivo studies.

2.5 Repetitive photothermal ability of IBC-PB composites

In the repetition demonstration, the laser was irradiated five times for five minutes and the temperature rise was maintained by 25 ℃. As a result, the photothermal effect of IBC-PB composites was maintained after multiple laser irradiation trials (Fig. 3c). After five cycles, the UV-vis spectra confirmed that the absorbance at NIR was maintained (Fig. 3d).

2.6. In vitro photothermal therapeutic effect in the 4T1 breast cancer cell line

The in vitro PTT effects of the BC, PB NPs and IBC-PB composites were evaluated using an 808-nm NIR laser (Fig. 4a). BC had no overt cytotoxicity with or without laser irradiation (Fig. 4b). The unirradiated IBC-PB composites showed no cytotoxicity at all treated concentrations. The laser-irradiated IBC-PB composites showed a dramatic photothermal therapeutic effect with more than 82% cell killing ability from a PB concentration of 20 µg 200 µL^− 1(Fig. 4d). Live 4T1 cells were scarcely observed after in vitro PTT at PB concentrations above 20 µg 200 µL^− 1 in the IBC-PB composites. However, the cells were intact in the IBC-PB composites without PTT (Fig. 4e). On the other hand, despite the fact that the PB NPs displayed a high PTT effect of more than 80% cell death when irradiated by an 808-nm laser at a concentration of 20 µg 200 µL^− 1, the unirradiated PB NPs also exhibited increased cytotoxicity as their concentration was increased. This suggests that cell death was caused not only by the PTT effect but also by the inherent cytotoxicity of the PB NPs (Fig. 4c).

2.7. In vivo retention at the tumor region of PB NPs and IBC-PB composites

We also conducted an in vivo PTT experiment with 4T1 tumor-bearing mice. We injected PB NPs and IBC-PB composites peritumorally as earlier studies reported that doing so resulted in better positioning and an improved PTT effect compared to an intratumoral injection. [30, 56] We compared the retention profiles of the PB NPs and the IBC-PB composites after a peritumoral injection around the subcutaneously located 4T1 tumor tissue region with 4T1 tumor-bearing mice (n = 3) for seven days (Figure S3). The peritumoral injections of the PB NPs and IBC-PB composites were clearly visible around the tumor region immediately after the injection (day 0). However, the PB NPs rapidly disappeared and were difficult to detect 24 hours post-injection due to systemic absorption and were not visible in the resected tumor images seven days after the injection. In contrast, the IBC-PB composites remained at the site of the injection up to seven days post-injection, as evidenced by images taken of resected tumors. It should be noted that the IBC-PB composites were not fully absorbed by the body, suggesting that the IBC-PB composites could be repeatedly targeted by an 808-nm laser for complete tumor removal via repeatable PTT and that they can also be considered more biocompatible than PB NPs.

2.8. In vivo photothermal imaging and therapy in 4T1 cancer-bearing mice

For the in vivo photothermal imaging and therapy, NS, PB NPs, and IBC-PB composites were peritumorally injected into 4T1 tumor bearing mice. After the injection in each group, an 808-nm NIR laser was irradiated under the condition of 2 W cm^− 2 for five minutes only once. The three different injected materials were separated into two groups, referred to the laser irradiated and unirradiated groups (NS, PB NPs, IBC-PB composites, NS + Laser, PB NPs + Laser, and IBC-PB composites + Laser). In vivo photothermal imaging was conducted and the temperature distribution at the tumor sites was observed (Fig. 6a). The temperature at the NS-injected tumor area increased by only 6.2°C from 32.7°C to 38.9°C after 808-nm laser irradiation. In contrast, the temperature in the tumors injected with PB NPs and IBC-PB composites increased dramatically, reaching 47.7°C (ΔT: 16.2°C) and 48.1°C (ΔT: 20.4°C), respectively. The IBC-PB composites showed a slightly larger temperature change of 4.2°C compared to that of the PB NPs (Fig. 6b,c). This demonstrates that the IBC-PB composites has a potent photothermal effect in vivo.

The tumor tissue growth in the unirradiated groups and the NS + laser group increased proportionally with time. In contrast, the PB + Laser and IBC-PB composites + Laser groups showed significantly smaller tumor sizes compared to the control groups (NS, PB NPs, and IBC-PB composites) (P < 0.05 for all comparisons at day 18) (Fig. 6d). The suppressed tumor growth was evaluated by examining the tumor volume up to 18 days (Fig. 6e). The IBC-PB composites showed a 71.5% tumor growth suppression rate, demonstrating greater tumor-growth inhibition ability compared to the control groups, particularly the PB NPs (68.5% tumor-growth suppression effect). The body weight up to 18 days after treatment was not significantly reduced in all groups (Fig. 6f).

2.9. In vitro cytotoxicity test in the RAW 264.7 macrophage cell line

Given the limitations in the toxicity of PB NPs, we undertook a further evaluation of the toxicity profiles of both the PB NPs and the IBC-PB composites. To do this, we conducted an MTT assay using RAW 264.7 macrophage cells (Fig. 7a). The PB NPs demonstrated an increasing toxic effect as the PB concentration was increased. The PB NPs started to show cell viability of less than 72% at a PB concentration of 50 µg mL^− 1 and showed cell viability of 19% at a concentration of 2500 µg mL^− 1. Meanwhile, the IBC-PB composites showed no noticeable cytotoxic effect up to 2500 µg mL^− 1, and the viability of IBC-PB composites-treated cells was 4.4-fold higher than that of PB NPs-treated cells at that concentration. It was clear that IBC-PB composites were far less toxic than PB NPs in RAW 264.7 macrophage cells. We also measured the in vitro therapeutic window (Fig. 7b) based on the cell viability presented in Fig. 4c and d and Fig. 7a. We defined the in vitro therapeutic window as a concentration range from the concentration at which 50% of cancer cells survived with laser irradiation (therapeutic dose) to the concentration at which 80% of macrophage cells survived without laser irradiation (non-toxic dose) (Figure S4). It should be noted that IBC-PB composites showed 84% cell viability even at the highest dose (2500 µg mL^− 1); accordingly, 2500 µg mL^− 1 was considered as a non-toxic dose of IBC-PB composites. We found that the in vitro therapeutic window for PB-NPs ranges from 76.7 ug ml^− 1 to 97.7 ug ml^− 1, while for IBC-PB composites, it ranges from 72.7 ug ml^− 1 to 2500 ug ml^− 1. Also, we defined the therapeutic index (TI) as the ratio between the therapeutic dose and non-toxic dose. The TIs for the PB-NPs and IBC-PB composites were 1.3 and 34.4, respectively.

2.10. In vivo toxicity test

The biocompatibilities of the IBC-PB composites and PB NPs were assessed through a biochemical analysis 24 hours after the subcutaneous injection in the right thighs and a blood draw of normal Balb/c-nude mice (n = 3) (Fig. 7c). The NS group served as a control group. The biocompatibility in each group was evaluated according to BUN, Cr, ALT, and AST, which are representative indicators of the kidney and liver function, respectively. In the BUN and Cr indices, the PB NPs and IBC-PB composites groups showed little difference numerically when compared with the NS group. The IBC-PB composites showed no significant difference compared to the NS group in terms of ALT and AST. However, in the PB NPs group, ALT was 1.30-fold higher and AST was 1.61-fold higher compared to that of the IBC-PB composites. The H&E-stained tissues from major organs (heart, liver, spleen, and kidney) in each group were imaged to confirm systemic toxicity (Fig. 7d). There were no significant signs of damage in any of the major organs, including the livers of the NS and IBC-PB composites groups. Meanwhile, prominent hepatocytic vacuolation was observed in the liver image of the PB NPs group, indicating acute liver toxicity ^{55, 56}, unlike the liver tissue images of the other two groups. We speculate that for the IBC-PB composites, PB NPs were directly bound to BC, remaining at the injection site and not affecting the body, whereas the PB NPs were absorbed into the body 24 hours after the injection and affected the liver function, leading to acute liver damage.

Several studies have described the use of engineered PB NPs to achieve efficient PTT effects, theranostic properties, or improved biocompatibility. Wang et al. describes the development of a boracic-acid-modified graphite carbon nitride and PB nanohybrid for theranostic applications, offering targeted Raman recognition and synergistic photothermal/photodynamic therapy in the near-infrared region. ²⁷ A NaDyF4:50%Lu@PB nanocomposite was developed by Liu et al. as a biocompatible and effective PTT agent, which also possesses the ability to be used for magnetic resonance imaging. ²⁹ Jing et al. developed a multifunctional nanoplatform by modifying hollow PB NPs with hyaluronic acid grafting polyethylene glycol and loaded with 10-hydroxycamptothecin for tumor-specific thermochemotherapy. ⁵⁷ Chen et al. developed PEGylated PB NPs loaded with doxorubicin as a photothermal-chemo therapy for cancer. ⁵⁸ Due to variations in therapeutic techniques, such as differences in laser settings and the use of additional therapeutic agents, it is challenging to make direct comparisons of therapeutic effects across studies. However, we were able to evaluate and compare the safety profile of the nanocomposites here, as presented in Table 1. Of the PB-containing nanocomposites, our IBC-PB composites exhibited the most biocompatible profile.

Table 1

Viability of cells treated with various forms of PB NPs. PB@ Bg-C₃N₄: a boracic-acid-modified graphite carbon nitride and Prussian blue nanohybrid, HCPT@HPBNs@PAA/PAH/HA-g-PEG: 10-hydroxycamptothecin loaded hyaluronic acid grafting polyethylene glycol modified hollow Prussian blue nanoparticles, NaDyF₄:50%Lu@PB: Prussian blue-coated NaDyF₄ doped with Lu³⁺ ions nanocomposites, PEGylated PB NPs: polyethylene glycol-attached Prussian blue nanoparticles.
Particle name	Method	Cell viability at a certain concentration	Ref
PB@Bg-C₃N₄	In situ reduction	95.6% at 200 µg mL^− 1	27
NaDyF₄:50%Lu@PB	Solvothermal method	86% at 1000 µg mL^− 1	29
HCPT@HPBNs@PAA/PAH/HA-g-PEG	Self-etching reaction and PEGylation by ECD-NHS reaction	56% at 30 µg mL^− 1	57
PEGylated PB NPs	Precipitation, thin film hydration method	84.2% at 40 µg mL^− 1	58
IBC-PB composites	Trituration and self-etching reaction	83.59% at 2500 µg mL^− 1	Our study

Various routes have been investigated for the administration of therapeutic nanoparticles, including intravenous, intramuscular, subcutaneous, intralesional, and perilesional types ⁵⁹. Among these routes, the localized administration of nanoparticles through intramuscular, subcutaneous, intralesional, and perilesional routes has been deemed ineffective for treating cancer due to the disseminating nature of cancer. However, several studies of cancer therapies based on locally injectable PB NPs have been reported, showing synergistic therapeutic effects when combined with other therapy strategies such as chemotherapy, radiotherapy, and immunotherapy. A gellan-based nanocomposite (NC) hydrogel embedding combretastatin A4 (CA4), a tubulin polymerization inhibitor for tumor growth suppression in cancer chemotherapy, and PB NPs showed a strong synergistic therapeutic effect as a type of NIR-triggered PTT and disrupting tumor vascular due to the continuous release of the CA4 ⁶⁰. An injectable PB-NPs-encapsulated agarose hydrogel, termed a PB reservoir and release controller (PRC) nanosystem, functioned as an improved combination therapy with PTT and radiotherapy (RT) given its excellent photothermal characteristics upon 808-nm NIR laser irradiation and with the catalytic capabilities of a radiosensitizer by converting endogenous hydrogen peroxide into oxygen for reactive oxygen species production by X-ray mediated RT ⁶¹. A localized therapy using nanoparticles was also found to improve the efficacy of immunotherapy significantly, as the destruction of cancer cells in the nanoparticle-mediated therapy results in a release of cancer antigens, which in turn stimulates the adaptive immune system.⁶² Juliana et al. used photothermal immunotherapy to enhance an immunotherapeutic effect with anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4), well known as an immune checkpoint inhibitor (ICI), by means of PTT based on pH-dependent and intratumorally injectable PB NPs ⁶³. The studies discussed above suggest that IBC-PB composites could have broader applications, including disseminated cancer treatments, when used in combination with other cancer therapy systems, particularly immunotherapy.

We developed a highly biocompatible, repeatable PTT agent by directly growing PB NPs on BC with a simple thermal reduction process. The synthesized IBC-PB composites had a monodispersed PB-NP-decorated BC morphology, a strong hyperthermal effect, an excellent PTT effect, and good recyclability. The IBC-PB composites showed a PTT effect similar to that of PB NPs but with a considerably higher safety profile compared to PB NPs. Consequently, the IBC-PB composites developed here is a promising nanomaterial based on PB that can function as a highly biocompatible and repeatable PTT agent.

Materials

Bacterial cellulose (nata de coco) was obtained from Vietnam Coco Food Co., Ltd. (Tang Nhon Phu, Vietnam). Potassium ferricyanide (K₃Fe(CN)₆·3H₂O), thiazolyl blue tetrazolium bromide, and Roswell Park Memorial Institute (RPMI) 1640 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cu(OH)₂ was purchased from Alfa Aesar (Haverhill, MA, USA). The ammonia solution, sodium hydroxide (NaOH), hydrochloric acid (HCl), and diethyl ether used here were purchased from Samchun (Gangnam, Korea). Polyvinylpyrrolidone (PVP, K30) was purchased from Kanto Chemical Co. (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from GE Healthcare Life Sciences (Buckinghamshire, UK), and 4% paraformaldehyde (PFA) was obtained from Biosesang (Seongnam, Korea) to fix the major organs. Female Balb/c nude mice (6 ~ 8 weeks) were purchased from Orient Bio (Seongnam, Korea) for the in vivo PTT experiment. The BD vacutainer® SST™ system used in the study was purchased from BD Bioscience (New Jersey, USA).

Preparation of PB NPs

PB NPs were synthesized according to a method reported in the literature. ⁵⁴ PVP (3g) and K₃Fe(CN)₆ (132 mg) were dissolved in a HCl solution (0.01 M, 40 mL) under magnetic stirring. After 30 min of stirring, a clear solution was obtained, which was then placed in an oven at 80 ℃ for 24 h. The precipitates were collected by centrifugation and washed in distilled water, ethanol, and diethyl ether several times. After drying at 60 ℃ in an oven for 24 h, PB nanoparticle powder was obtained.

Synthesis of IBC

The impurities of nata de coco (1 kg) were removed by dialysis with deionized water (D.W.) for three days. After the washing process, the nata de coco was physically pulverized through a grinder and filtered through a sieve several times. Water was completely removed from the physically treated nata de coco through freeze-drying for more than four days. Schweitzer’s reagent was produced by dissolving copper hydroxide (100 mg) in an ammonia solution (5 ml) and stirring sufficiently. Lyophilized nata de coco (50 mg) was added to Schweitzer’s reagent (5 ml) and reacted for two hours at room temperature under vigorous stirring. Subsequently, 20 ml of a 10% HCl solution was added for neutralization and the mixture was stirred sufficiently until the color no longer changed. The neutralized bacterial cellulose was filtered by a glass filter and washed with D.W. several times until the pH reached approximately 7. After washing, the collected bacterial cellulose was dispersed in D.W. (20 ml) and stored in a refrigerator at 5 ℃.

Synthesis of IBC-PB composites

In 20 ml of the IBC solution, D.W. (70 ml), 1M HCl (10 ml) and K₃Fe(CN)₆·3H₂O (0.25 mmol, 0.5 mmol, 0.75 mmol, 1 mmol) were added, reaching a total solution amount of 100 ml. This was then reacted for 24 hours at 80 ℃. The IBC-PB composites were collected using a glass filter and washed with D.W. several times until the pH was approximately 7. After washing, the collected IBC-PB composites were dispersed in 50 ml D.W. and stored in a refrigerator at 5 ℃.

Characterization of IBC-PB composites

The produced composites were characterized using a field-emission scanning electron microscope (FE-SEM, Hitachi S-4800). The crystal structure of the composites was confirmed by a powder X-ray diffraction analysis using a Bruker D8 Advance device (Cu Ka1 radiation, 5°·min^− 1). A thermogravimetric analysis (TGA) was conducted using a TGA/DSC 1 analyzer (Mettler Toledo) with a heating rate of 5 ℃·min^− 1 in air. An inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis was conducted in 6000 K Ar plasma with a range of 167–782 nm (OPTIMA 8300, Perkin-Elmer, USA). The absorbance was measured by a microplate reader (SYNERGY H1, BioTek, Winooski, VT, USA). An 808-nm NIR laser (FC-W-808-10W, CNI, Changchun, China) was used for photothermal imaging and PTT studies. A thermal imaging camera (HT-18, HT Instruments, Faenza, Italy) was used for real-time hyperthermal imaging. A blood biochemistry analyzer (Hitachi 7020, Tokyo, Japan) was utilized for the blood biochemical analysis.

Photothermal performance test

The IBC-PB composites (0.2 mg 1.5 ml^− 1) dispersed in D.W. was added to a 4 ml vial, a stirring bar was added, and the surroundings were wrapped with Styrofoam. An 808-nm NIR laser (1 W cm^− 2) was irradiated five minutes onto each sample and the temperature was measured every 30 seconds. According to the experimental results, we collected the best sample, and it was used for a retention test. The retention test was conducted by repeating the five-minute laser irradiation, which was followed by cooling to the initial temperature five times.

The evaluation of in vitro cytotoxicity and photothermal therapeutic effect

4T1 breast cancer cell and RAW 264.7 macrophage cell lines were authenticated and obtained from the Korean Cell Line Bank, Korea). The cells were cultured, added to a 96-well microplate (10³ cells 200 µL^− 1 of RPMI 1640 or DMEM), and incubated at 37 ℃ with 5% CO₂ for 24 hours. After incubation, the cell media were discarded and the cells were washed with DPBS three times to remove remained media, followed by an exchange for new cell media in each well. The PB NPs and IBC-PB composites were incubated with RAW 264.7 cells with PB concentration gradients (0, 25, 50, 100, 250, 500, 1000, and 2500 µg mL^− 1). The BC and IBC-PB composites were added to the 4T1 cancer cells with the series of BC concentrations (0, 10, 50, 100, 200, and 500 µg mL^− 1). All nanomaterial treated cells were incubated overnight. The IBC-PB composite-treated 4T1 cells were irradiated with an 808-nm laser at 2 W cm^− 2 for five minutes and another 24 hours of incubation was conducted. Subsequently, 0.5 mg mL^− 1 of MTT solution was added to each well after incubation and a DPBS washing step for two hours for the MTT assay. After the incubation of the MTT solution, cell images were obtained by an optical microscope of the IBC-PB composite-treated groups before the DMSO solvent exchange step. Each well was measured at 540 nm by the microplate reader to acquire its absorbance. Cell viability was calculated at a ratio relative to untreated control samples and the data were evaluated by a t-test. In vitro cytotoxicity test was carried out in triplicate.

Preparation of the 4T1 tumor-bearing mouse model

The 4T1 cancer cells were cultured and collected with PBS (10⁵ cells 15 µL^− 1 PBS). The condensed 4T1 cells were injected subcutaneously into the right thigh of female Balb/c nude mouse. Further in vivo experiments with the mouse tumor model were performed when the tumor size reached approximately 50 to 100 mm³. All animal studies were approved by the Institutional Animal Care and Use Committee of Woojung Bio Inc.

In vivo photothermal imaging

Normal saline (NS), the PB NPs, and the IBC-PB composites were used for a peritumoral injection (p.i.) at the tumor region in the 4T1 tumor bearing mice. After each injection, an 808-nm laser was irradiated at 2 W cm^− 2 for five minutes, in vivo photothermal imaging was conducted, and photothermal images were obtained in each group during laser irradiation.

In vivo photothermal therapy study

First of all, the mice were randomized into three groups. The NS, PB NPs, and IBC-PB composites underwent a p.i. procedure around the tumor region in the 4T1 tumor bearing mice (n = 3). The 808-nm laser was irradiated under a condition identical to that used for the in vivo photothermal imaging step after the injection. The treated and untreated groups underwent a follow-up assessment for 18 days that involved measuring the tumor sizes and weights. The data were evaluated statistically by an ANOVA test.

The assessment for tumor retention of in vivo PB NPs and IBC-PB composites

The PB NPs and IBC-PB composites were inserted by peritumoral injection at the 4T1 tumor region. Photographic images were obtained at various time points post injection (0, 1, and 7 days). The tumor tissues were resectioned and internal photographic images were obtained.

Biocompatibility test of IBC-PB composite and PB NPs

The NS, PB NPs, and IBC-PB composites were subcutaneously injected into the right thigh region in each case. Blood samples with a volume of 500 µL were collected with a SST vacutainer in each group 24 hours after the injection. The obtained blood samples were centrifuged, and the supernatant was re-collected for each sample. The supernatant was analyzed for indices that represent liver and kidney functions (alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cr), and blood urea nitrogen (BUN)) by a blood biochemistry analyzer. After the blood draw, major organs in each group were collected, stored in a 4% PFA solution, stained with hematoxylin and eosin (H&E) staining, and paraffin-sectioned for tissue imaging.

Acknowledgements

This research was supported by the National Research Foundation of Korea (NRF) (NRF-2019M2D2A1A01058210, NRF-2020R1C1C1009000, NRF-2021M2E8A1039564, NRF-2022R1A6A1A03063039); by the Korea Evaluation Institute of Industrial Technology (KEIT) funded by the Korean government (MOTIE) (No. 20018522); and by the Korea Drug Development Fund funded by the Ministry of Science and ICT, Ministry of Trade, Industry and Energy and the Ministry of Health and Welfare (HN22C0632) of Korea. Schematic illustrations were created with BioRender.

Author contributions

Hwichan Hong, Wooseung Lee, and Chaedong Lee contributed to the study concept. Hwichan Hong prepared the most of nanoparticles, and Wooseung Lee performed the most of in vitro and in vivo experiments. Hwichan Hong, Wooseung Lee, and MinKyu Kim analyzed and curated the data from experiments, and wrote the manuscript. Also, MinKyu Kim edited and finalized the manuscript. Miyeon Jeon and Hoonsub Kim helped formal analysis for this study. Hyung-Jun Im and Yuanzhe Piao supervised and administrated this study.

Funding

National Research Foundation of Korea (NRF) (NRF-2019M2D2A1A01058210, NRF-2020R1C1C1009000, NRF-2021M2E8A1039564, NRF-2022R1A6A1A03063039); Korea Evaluation Institute of Industrial Technology (KEIT) funded by the Korean government (MOTIE) (No. 20018522); and by the Korea Drug Development Fund funded by the Ministry of Science and ICT, Ministry of Trade, Industry and Energy and the Ministry of Health and Welfare (HN22C0632) of Korea.

Availability of data and materials

All data during the current study are available by corresponding authors on reasonable request.

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee of Woojung Bio Inc.

Consent from publication

All authors has been informed about the publication of this manuscript.

Competing interests

Hyung-Jun Im is the co-founder and the Chief Scientific Officer of Portrai. The authors declare that they have no competing interests.

Author details

¹Department of Applied Bioengineering, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, 08826, Republic of Korea. ²Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, 08826, Republic of Korea. ³Cancer Research Institute, Seoul National University, 03080 Seoul, Republic of Korea. ⁴Advanced Institutes of Convergence Technology, Seoul National University, Suwon-si, Gyeonggi-do, Republic of Korea.

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No competing interests reported.

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published 05 Oct, 2023

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Editorial decision: Major revision
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You are reading this latest preprint version

Injectable Biocompatible Nanocomposites of Prussian Blue Nanoparticles and Bacterial Cellulose as a Safe and Effective Photothermal Cancer Therapy

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Journal Publication

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Abstract

Figures

1. Introduction

2. Results and discussion

2.1 Injectable bacterial cellulose (IBC) purification and fragmentation from nata de coco

2.2 Synthesis of PB NPs and IBC-PB composites

2.3 Characterizations of the PB NPs and IBC-PB composites

2.4 Photothermal effect of the IBC-PB composites

2.5 Repetitive photothermal ability of IBC-PB composites

2.6. In vitro photothermal therapeutic effect in the 4T1 breast cancer cell line

2.7. In vivo retention at the tumor region of PB NPs and IBC-PB composites

2.8. In vivo photothermal imaging and therapy in 4T1 cancer-bearing mice

2.9. In vitro cytotoxicity test in the RAW 264.7 macrophage cell line

2.10. In vivo toxicity test

3. Conclusion

4. Experimental Section

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1