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. 2022 Oct 14;5(12):1267–1278. doi: 10.1021/acsptsci.2c00159

NFL-TBS.40-63 Peptide Gold Complex Nanovector: A Novel Therapeutic Approach to Increase Anticancer Activity by Breakdown of Microtubules in Pancreatic Adenocarcinoma (PDAC)

Qiqian Liu †, Hui Liu ‡, Audrey Griveau §, Xiaowu Li ‡,*, Joel Eyer §, Celia Arib †,*, Jolanda Spadavecchia †,*
PMCID: PMC9745895  PMID: 36524008

Abstract

graphic file with name pt2c00159_0011.jpg

The role of the NFL-TBS.40-63 peptide is to destroy the microtubule network of target glioma cancer cells. Recently, we have conceived a gold-complex biotinylated NFL-TBS.40-63 (BIOT-NFL) to form a hybrid gold nanovector (BIOT-NFL-PEG-AuNPs). This methodology showed, for the first time, the ability of the BIOT-NFL-PEG-AuNPs to target the destruction of pancreatic cancer cells (PDAC) under experimental conditions, as well as detoxification and preclinical therapeutic efficacy regulated by the steric and chemical configuration of the peptide. For this aim, a mouse transplantation tumor model induced by MIA-PACA-2 cells was applied to estimate the therapeutic efficacy of BIOT-NFL-PEG-AuNPs as a nanoformulation. Our relevant results display that BIOT-NFL-PEG-AuNPs slowed the tumor growth and decreased the tumor index without effects on the body weight of mice with an excellent antiangiogenic effect, mediated by the ability of BIOT-NFL-PEG-AuNPs to alter the metabolic profiles of these MIA-PACA-2 cells. The cytokine levels were detected to evaluate the behavior of serum inflammatory factors and the power of BIOT-NFL-PEG-AuNPs to boost the immune system.

Keywords: BIOT-NFL peptide, hybrid gold nanoparticles, PDAC, immune system

Pancreatic ductal adenocarcinoma (PDAC) is characterized by several cancer signaling pathways that boost tumorigenesis.1−3 The particularly abundant desmoplastic (i.e., fibrous) stroma of PDAC has been recognized as a key player in encouraging the cell adhesion-mediated drug resistance (CAM-DR) of this disease.2 The fixation of pancreatic cancer cells to fibronectin, collagen types I and IV, and laminin was shown to reduce the cytotoxicity of anticancer drugs.4,5 The dense mass surrounding the tumor tissue results, in fact, in increased interstitial pressure, subsequent capillary compression, vessel collapse, and impaired blood perfusion, which ultimately hinders drug delivery at the targeted site. For this problem, targeted therapies offer the opportunity to tailor a strategy to the unique properties of a patient′s individual tumor.6 Selecting targets restricted to cancer or cancer-associated cells and not healthy cells, these therapies potentially minimize side effects associated with conventional chemotherapeutic strategies that employ cytotoxic agents.7,8

Despite the recent progress in cancer diagnosis and treatment, pancreatic cancer and its most prevalent form, the PDAC, constantly have a bleak diagnosis with a 5-year survival rate below 6%; great systemic drug resistance is a result of dense stroma that drugs cannot penetrate and complex cellular processes.9 Further powerful drugs and treatment systems targeted at PDAC are needed to refine its clinical achievement.10 Research to increase the survival rate has been carried out in recent years, in particular, by probing the prognostic markers and the original mechanisms of PDAC carcinogenesis.11 Recently, microtubules have played a key role in the overtures in pancreatic cancer pharmacology and targeted therapy due to their main function in mitotic cell division.12

Therefore microtubule inhibitors include most classes of anticancer drugs and have been entirely applied in the treatment of hematopoietic and solid tumors.12

In the last few years, Eyer and his team have found the sequences ≪tubulin binding sites (TBS)≫ capable of binding free tubulin. They have then obtained a peptide called NFL-TBS.40-63 (neurofilament low subunit-tubulin binding site 40-63, also called the NFL peptide), which interacts specifically in several glioblastoma cell lines reducing the glioblastoma viability.13

This NFL-TBS.40-63 peptide gives us an idea to overcome the stroma barrier of PDA and to interact with the microtubule of PDAC cells. Anyway, several studies prove that the stability of cell-penetrating peptides requires an evaluation.14 In this context, gold nanoparticles have been considered good candidates for peptide stability and remarkable therapeutic tools in biomedical applications.15−17 Recently, Spadavecchia et al. have realized a synthetic method of hybrid gold nanoparticles in which drugs and/or biomolecules are functionalized with a biopolymer (“Methodology IN”18−22) to protect the biological degradation decreasing the side effects of drugs. The major relevance of the NFL-TBS.40-63 peptide is to target cancer cells and their stem cells (e.g., glioblastoma), where it destroys their microtubule network.13 Overall, there is an urgent need to develop novel therapies, consider new combinations, and appropriately select those patients who may benefit. Recently, we showed that this peptide can target pancreatic cancer cells and pancreatic tumors arising in animal models, allowing the intracellular uptake of nanoparticles in pancreatic cancer cells (Figure 1A,B). This recently developed technology (P7391FR00-50481 LIV) has many advantages, including selective and targeted destruction of cancer cells (PDAC), hyperthermia, excellent biological and chemical stability under experimental conditions, and detoxification and preclinical therapeutic efficacy modulated by the steric and chemical configuration of the peptide.22

Figure 1.

Figure 1

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(A) Representation of BIOT-NFL-PEG-AuNPs internalization into MIA-PACA-2 cells. (B) Electron micrographs of MIA-PACA-2 cancer cells treated with BIOT-TAT-PEG-AuNPs;22 (c: 100 μM; t: 24 h); N: nucleus, V: vacuole. (C) Schematic mechanism of BIOT-NFL-PEG-AuNPs translocation on microtubules and destruction.

The present work proposes a starting study (PoC) to develop novel strategies based on peptide nanocarriers designed to specifically attack pancreatic cancer. The therapeutic effect in vivo of BIOT-NFL-PEG-AuNPs was evaluated after the administration of MIA-PACA-2 cell lines onto the mouse transplantation model. All organs and tumors were experimentally treated as previously described,23 and the cytokines were used to detect the serum inflammatory factors. These results show that BIOT-NFL-PEG-AuNPs significantly decreases tumor growth without consequences on the body weight of mice. Moreover, BIOT-NFL-PEG-AuNPs boosts the immune system and might influence the angiogenic process. We assume that this study is very important to develop an innovative peptide therapy to understand the mechanisms of adverse effects of other chemotherapeutic drugs.

Results and Discussion

Cytotoxicity

The cytotoxicity of BIOT-NFL-PEG-AuNPs was evaluated on MIA-PACA-2 (pancreatic cells) using MTT cytotoxic tests with increasing concentration of the BIOT-NFL peptide in nanoparticles from 1 nM to 1 μM (Figure S1 in the Supporting Information).

MIA-PACA-2 cells were exposed to BIOT-NFL-PEG-AuNPs dilutions, with the BIOT-NFL peptide and PEG-AuNPs (as controls) in DMEM+10% FBS. The percentage (%) of living MIA-PACA-2 cells was evaluated with UV–vis spectroscopy after 24 h exposure to BIOT-NFL-PEG-AuNPs, BIOT-NFL peptide, and PEG-AuNPs alone. In Figure S1, we observed that in the presence of PEG-AuNPs and the BIOT-NFL peptide, the cell viability does not change with different concentrations; it is more than 50%. Contrarily, in the presence of BIOT-NFL-PEG-AuNPs, the cell viability decreases with the increase of peptide concentration. This means that the therapeutic effect of the peptide is optimized with AuNPs, which shows the efficiency of our nanovector.

In Vivo Antitumor Efficacy

Microtubules are protein polymers of about 25 nm in diameter formed by the combination of α and β tubulin heterodimers.12,24 Many studies have demonstrated the roles of tubulin in PDAC and their wide expression in pancreatic cancer (PC) tissues.12,25 Compounds that inhibit microtubule polymerization and reduce microtubule polymer bulk are known as microtubule-destabilizing agents (MDAs).26,27 Among the MDA class, Eyer and co-workers have discovered a peptide located on the neurofilament light subunit (NFL-TBS.40-63), able to fix tubulin dimers on specific sites,13 inhibiting the proliferation of glioma cells by altering their microtubule network.

Recently, we conceived a nanoformulation composed of the biotinylated NFL-TBS.40-63 peptide (BIOT-NFL) complexed to gold nanoparticles (BIOT-NFL-PEG-AuNPs), and we have investigated their power of internalization on PDAC cells (P7391FR00-50481 LIV), including the cytotoxicity activity of cancer cells (PDAC)22 (Figure S1 in the Supporting Information). Based on these findings, we investigated, for the first time, the capacity of the BIOT-NFL-PEG-AuNPs to target mouse transplantation tumor model PDAC to understand the capacity of BIOT-NFL-PEG-AuNPs to destroy microtubules on PDAC tissues and thus improving their anticancer efficacity.

The therapeutic effect of BIOT-NFL-PEG-AuNPs compared to the BIOT-NFL peptide and PEG-AuNPs as controls on PDAC cancer was established using the mouse transplantation tumor model in which human MIA-PACA-2 cells were hypodermically injected to generate subcutaneous pancreatic cancer. As shown in Figure 2A, BIOT-NFL-PEG-AuNPs did not affect the body weight of pancreatic cancer in model mice; however, the mice in the 5-FU group (positive control drug) showed the phenomenon of weight loss after one week of administration. Compared with the model group on the 22nd, 25th, 28th, and 31st days, this is statistically significant (p < 0.05). Compared with the 5-FU group, the body weight of the mice in the BIOT-NFL-PEG-AuNPs group increased significantly on day 31 (p < 0.05). This result shows that the BIOT-NFL-PEG-AuNPs does not have toxic effects on mice compared with the conventional 5-FU treatment. Compared with the model group, the BIOT-NFL-PEG-AuNPs significantly reduced the tumor volume of the pancreatic cancer model mice (p < 0.05) (Figure 2B). On the 28th and 31st days, the 5-FU treatment significantly reduced the tumor size of pancreatic cancer mice as well (p < 0.05), but the therapeutic effect of the BIOT-NFL-PEG-AuNPs was better than the 5-FU treatment. Here, we should take into consideration that between the two groups, there is no statistical difference. The experimental results are shown in Figure 2C.

Figure 2.

Figure 2

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(A) Effect of gold nanoparticles on the body weight of pancreatic cancer in mice. Body weight is shown as mean ± SD. The data are analyzed by two-way ANOVA; the 5-FU group compared with the model group, #p < 0.05; the 5-FU group compared with the gold peptide nanoparticle group, *p < 0.05 and **p < 0.05. (B) Effect of BIOT-NFLPEG-AuNPs on the tumor volume of pancreatic cancer in mice. Tumor volume (mm3); (C) inhibition rate (%). Data are shown as mean ± SD and analyzed by two-way ANOVA. Compared with the model group, #p < 0.05, ##p < 0.01, and ###p < 0.001.

Compared with the PEG-AuNPs, BIOT-NFL peptide, and BIOT-NFL-PEG-AuNPs groups, the inhibition rate of BIOT-NFL-PEG-AuNPs was significantly increased (p < 0.05), and the BIOT-NFL-PEG-AuNP inhibition rate is much better than that of the 5-FU treatment. At the end of the experiments, the tumor-bearing mice (Figure S2A in the Supporting Information) and the tumor tissues were dissected (Figure S2B in the Supporting Information) and photographed; the tumor index of each group was counted. The experimental results are shown in Figure S2C in the Supporting Information. Compared with the model group, BIOT-NFL-PEG-AuNPs reduced the tumor index of pancreatic cancer mice, and this effect was better than that of 5-FU. The presence of gold nanoparticles confers better stability to the peptide,1528 and exerts a better effect on microtubules to stoically clutter and destroy them (Figure 1C).

BIOT-NFL-Gold Complex as an Angiogenic Modulator: Hypothesis

A great effect for angiogenesis in the prediction of cancer has been accomplished previously for several types of cancer.29,30 Besides peculiar biomarkers, CD31 and vascular endothelial growth factor (VEGF) are extensively studied.31−33 CD31 is really expressed on the surface of endothelial cells to detect vessel density in cancerous tissues. PDAC displays a rich vascular endothelial cell defined by high expression of CD31 with upregulated immune response-related pathways.34,35

Previous studies have proved the ability of novel small molecules, i.e., peptides, capable of linking VEGF, closing its interaction with receptors with consequent inhibition of angiogenesis and tumorigenesis.36 The antiangiogenic role of AuNPs has been discussed in several studies.37 AuNPs decreased vascular density, as proved by staining tumor tissues with the CD31 vessel marker.37 Several studies have shown that AuNPs could increase angiogenesis in animal models.37 Additionally, AuNPs improved the expression of CD31 endothelial markers and enhanced angiogenesis in an orthotopic implantation model of pancreatic cancer.38 In our study, we carried out immunohistochemical CD31 staining in the presence of the BIOT-NFL peptide before (Figure 3 panels K–O) and after conjugation onto gold nanoparticles (Figure 3 panels P–T) to understand their role in the process of angiogenesis.

Figure 3.

Figure 3

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Immunohistochemical CD31 staining. The effect of BIOT-NFL-PEG-AuNPs (P–T) on tumor angiogenesis in mice with pancreatic cancer, compared to BIOT-NFL (K–O), PEG-AuNPs (F–J), model (A–E), and 5-FU (U–Y) as controls.

After three weeks of treatment, HES staining put in evidence the presence of many blood vessels in the core of control tumors, while only a poor big vessel was checked at the periphery of tumors of the treated mice (Figure 3 panels A–E; panels U–Y).

After injection of BIOT-NFL-PEG-AuNPs (Figure 3 panels P–T), we found that it is a complete disappearance of brown-yellow particles and a consequent inhibition of the formation of tumor blood vessels in pancreatic cancer mice. These findings established the reduction in vessel density, which is likely associated with the diminution of proliferation and improvement of apoptosis of endothelial cells. As shown in Figure 3, the inhibition effect of angiogenesis is better than that in the 5-FU group (Figure 3 panel U–Y). We believe that after injection in the tumor tissue, BIOT-NFL-PEG-AuNPs released BIOT-NFL as a gold complex, interacting with receptors and inhibiting angiogenesis and tumorigenesis (Scheme 1). This very encouraging result should be fixed on the task of CD31 to promote vascular stability and immune responses.

Scheme 1. Picture of Hypothetic Angiogenic Mechanism of BIOT-NFL-PEG-AuNPs on Cancer Cells.

Scheme 1

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ROS Production

The NFL-TBS.40-63 peptide derives from the NFL subunits, which serve as a reservoir of unpolymerized free tubulin to regulate microtubule (MT) dynamics.39 However, the NFL subunit is also capable of interacting with molecules responsible for mitochondrial dynamics. Additionally, tubulin has been shown to regulate mitochondrial metabolism by modulating the membrane potential of mitochondria was.40,41 Previously, the effect of the peptide on the biology and dynamics of glioblastoma cell mitochondria was established, confirming the ability of the peptide to alter the biogenesis and function of the mitochondria of human glioblastoma cells through a direct reduction in mitochondrial respiration.13 Other studies demonstrated that NFL can interact directly with mitochondria, such as vimentin, to regulate mitochondrial motility.42 This interaction takes place directly via a site located on the N-terminal domain of vimentin. At the same time, it was established that the NFL-TBS.40-63 peptide also reduces mitochondrial motility, interacting directly or indirectly with the mitochondria to modulate their dynamics. On the basis of previous studies, we have demonstrated the exceptional activity of the NFL-TBS.40-63 peptide in biotinylated form complexed to gold nanoparticles (BIOT-NFL-PEG-AuNPs) toward PDAC. The idea and consequent choice of PDAC are due to the presence of the dense desmoplastic stroma that is responsible for the poor internalization of therapeutics like BBB in GBM. Our studies have shown that ROS can oxidatively damage tumor cells and promote tumor cell apoptosis. The experimental results are shown in Figure 4 panel A. By comparison with the model group, both BIOT-NFL-PEG-AuNPs and 5-FU significantly increase the ROS content in the tumor cells of pancreatic cancer in mice (p<0.01) (Figure 4 panel B).

Figure 4.

Figure 4

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(A) Statistical and (B) schematic behavior of BIOT-NFL-PEG-AuNPs on the ROS content of the MIA-PACA-2 cancer cell after i.v. onto mice. Data are shown as mean ± SD and analyzed by one-way ANOVA; *p < 0.05, **p < 0.01, and ***p < 0.001.

Peripheral Blood: Reaction to Disease

Hematology screening is an important analysis of both clinical medicine and biomedical research.43 It was estimated that the peculiar WBC count in mice is 2000–10,000/μL.43

The blood cell analysis is an important quality-control check to understand and elucidate severe leukopenia associated with responses to stress or excitement, infectious diseases, and acute inflammation.43

Compared with the model group, BIOT-NFL-PEG-AuNPs can significantly increase the number of WBC, NE, and LY (p < 0.01) and significantly reduce the content of MCV and MCH (p < 0.01), while 5-FU decreases the number of WBC, NE, and LY and significantly increases the number of PLT (p < 0.001); the PLT parameter plays a key role in the state of the immune system.44 Platelet activation in mice can be spontaneous and strain-dependent.45,46

The main function of platelets is primary hemostasis, and platelet production can increase due to inflammatory diseases, myeloproliferative diseases, neoplasia, and iron deficiency.44 Conversely, platelet production can decrease due to myeloproliferative diseases, neoplasia, and erythropoietin administration; compared with the 5-FU group, the BIOT-NFL-PEG-AuNPs group showed significantly increased number of WBC, NE, and LY (p < 0.01). The experimental results are shown in Figure S3 and Table S1 in the Supporting Information.

Footprint of BIOT-NFL-PEG-AuNPs on Cytokine Expression

The antitumor activity is influenced by the good functioning of the immune system as a defense.47 Immunotherapy represents an antitumor immune reaction through the high production of cytokines, chemokines, and several immune cells into the tumor microenvironment (TME). Some anticancer therapeutic approaches boost the immune cells to promote the damage of cancer cells and the immunologic memory that is essential to prevent the reappearance of primary tumors and/or metastases.48 Cytokines, including IL-6, IFN-γ, and TNF-α, play a great role in the modulation of antitumor immune responses and have been extensively studied preclinically in vitro and in vivo and among patients in the clinic for immunotherapy of cancer diseases.49 TNF-α has great antitumor activity in vitro and in vivo through apoptotic cell death and tumor necrosis.50 IL-6 is a cytokine implicated in the regulation of several processes and immune activation.51 Although we did not measure the serum concentrations of IL-18, this study found that IL-18 has anticancer activity in preclinical models and increases the serum concentrations of IFN-γ.52 In particular, cytokines, including IL-6, IFN-γ, and TNF-α, play a great role in the decrease of tumor growth regulating immune cell proliferation, differentiation, and preclinical tests in vitro and in vivo. The cytokines IL-6 improve the proliferation of responsive T cells. TNF-α has been established to play a key role in the antitumor activity to induce apoptotic cell death and tumor necrosis.47,53,54 In addition, several recent studies have also shown clear evidence that IFN-γ promoted specific immune responses through immunological processes on the growth of tumor cells.55 Many studies have indicated that GM-CSF has inhibitory effects on tumor progression. GM-CSF as an adjuvant has been shown to promote antitumor immunity in mice and humans.56 Our results found that BIOT-NFL-PEG-AuNPs had significantly increased levels of serum IL-6, IFN-γ, and TNF-α. Compared with the model group, 5-FU basically did not affect the change of levels of serum IL-6, IFN-γ, and TNF-α. Several conventional chemotherapeutics employ their cytotoxic mechanism of action by interposing with many proteins that influence the DNA synthesis and replication with consequent genotoxic stress and cell death.

GM-CSF is also a cytokine and hematopoietic growth factor that favors the differentiation from human and mouse hematopoietic progenitors in vitro.57−59 Several human pancreatic cancer cells show a mutation in the KRAS gene.60,61 Previous studies have demonstrated that in mice, mutations in KRAS upregulated the production of GM-CSF, with immunosuppressive cells into the tumor.62 By repressing the production of GM-CSF, the researchers demonstrated a reduction in the growth of cells containing mutated KRAS, which was mediated by cytotoxic T cells.62

In our in vivo preliminary study (POC), after injection of BIOT-NFL-PEG-AuNPs into the PDAC tumor-bearing mice, the levels of serum IL-6, IFN-γ, and TNF-α (p < 0.05) were strongly improved. This result, opposite to the 5-FU effect, proved that BIOT-NFL-PEG-AuNPs plays an antitumor role by stimulating the secretion of cytokines in the PDAC tumor-bearing mice (Model: C57BL/6j). Among these results and contrary to the 5-FU effect, BIOT-NFL-PEG-AuNPs decreases the level of GMF-CSF (granulocyte-macrophage colony-stimulating factor), a white blood growth factor (Figure 5). Although the remaining factors, including GM-CSF, had no significant difference among the groups, 5-FU increases the GM-CSF content compared to the BIOT-NFL-PEG-AuNPs group. The study found that the amounts of secreted GM-CSF in HFCL/EG cells exposed to 5-FU improved as compared to the non-5-FU group. Fluorouracil-inducible GM-CSF gene therapy was regulated by an Egr-1 promoter in the tumor-bearing mice.63 Therefore, we still think that these deviations do not affect the conclusions. The effect of immunotherapy is better than that of 5-FU. On the basis of these results, we also assume that BIOT-NFL-PEG-AuNPs could help design valid immunotherapeutic strategies against pancreatic cancer.

Figure 5.

Figure 5

Figure 5

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Effect of BIOT-NFL-PEG-AuNPs on the content of inflammatory factors in pancreatic cancer mice. Data are shown as mean ± SD and analyzed by one-way ANOVA; *p < 0.05 and **p < 0.01.

Biodistribution

It was established that ICP-MS was applied to check the distribution of gold nanoparticles in the organs after administration.64 The experimental results are shown in Figure 6. The content of BIOT-NFL-PEG-AuNPs in the heart, lung, kidney, tumor, and brain tissues was significantly increased (p < 0.001). In the liver, the content of BIOT-NFL-PEG-AuNPs was significantly lower than PEG-AuNPs (p < 0.0001); we also observed an increase in the BIOT-NFL-PEG-AuNPs (p < 0.05) in the spleen tissue.

Figure 6.

Figure 6

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Biodistribution of the gold nanoparticle content in the organs of pancreatic cancer mice. Data are shown as mean ± SD and analyzed by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

It is known that after intravenous administration, the amount of 30-99% of nanoparticles accumulates in the liver of the body.64 The pegylation strategy of nanoparticles65,66 is also applied to reduce their absorption in the organs, and they are confined with an important amount in the liver and spleen after intravenous injection. In the present study, we confirmed once again the increased accumulation of PEG-AuNPs (control) in the liver and spleen (Figure 6). Exceptionally and contrary to previous works in the literature, after bioconjugation of the peptide (BIOT-NFL), our nanoparticle system (BIOT-NFL-PEG-AuNPs) is almost absent in the liver and more accumulated in the brain, kidneys, and lungs, with a high accumulation in the pancreatic tumor model confirming its excellent therapeutic efficacy. The presence of BIOT-NFL-PEG-AuNPs in the lungs and kidneys confirms the lack of translocation into the circulatory system and secondary organs.67 These results suggest that BIOT-NFL-PEG-AuNPs has low liver cytotoxicity while exhibiting improved lung bioavailability to control excessive inflammation. Furthermore, the rapid excretion of nanoparticles via the kidney could reduce their risk of long-term toxicity. This indicates a reduction in the risk of long-term toxicity.

The high accumulation of BIOT-NFL-PEG-AuNPs in the brain was also agreeably observed.

We can assume an excellent dual behavior to overtake a stroma barrier on the tumor model of pancreatic cancer and BBB of brain cancer (i.e., GBM) (Scheme 2). The positive surface charge of BIOT-NFL-PEG-AuNPs (P7391FR00-50481 LIV) also influences its quick clearance via the kidneys, as discussed previously.68

Scheme 2. BIOT-NFL-PEG-AuNPs’ Behavior to Overtake a Stroma Barrier on the Tumor Model of Pancreatic Cancer (PDAC) and BBB of Brain Cancer (i.e., GBM).

Scheme 2

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We also assume that BIOT-NFL-PEG-AuNPs can influence multiple factors of pathogenesis simultaneously to control the acute inflammatory responses in the lung, including inhibition of GM-CSF and reduction of neutrophil infiltration. We can conclude that our system (BIOT-NFL-PEG-AuNPs) has a good biodistribution and metabolic profile to control acute inflammation.

Experimental Section

Materials and Methods

Tetrachloroauric acid trihydrate (HAuCl4* 3H2O; 99%), sodium borohydride (NaBH4; 98%), dicarboxylic poly(ethylene glycol) (PEG)-600 (PEG), sodium chloride NaCl (0.9; 99.5%), phosphate-buffered saline (PBS) solution, EDTA, 5 fluorouracil (5-FU), isoflurane, and paraformaldehyde were purchased from Sigma-Aldrich at maximum purity grade. All solvents were used without any further purification. Experiments were carried out at room temperature if not specified otherwise.

BIOT-NFL peptide was purchased by the Polypeptide Group (Strasbourg, France).

Synthesis of BIOT-NFL-PEG-AuNPs

Biotinylated NFL-TBS.40-63 (BIOT-NFL) peptide nanoparticles were synthesized from a chemical protocol described recently22 (P7391FR00-50481 LIV).

Synthesis of Polymeric Nanoparticles (PEG-AuNPs)

Polymeric gold nanoparticles (PEG-AuNPs), as a control, were produced as described previously.66

Cell Lines

Cell lines obtained from American Tissue Culture Collection (ATCC) were used in this study. MIA-PACA-2 cells, which are a human pancreatic cancer cell line, were cultured in DMEM (Dulbecco′s modified Eagle′s medium; Gibco, Biosciences Ltd., Ireland) supplemented with 10% FBS (fetal bovine serum; Sigma-Aldrich), 1% antibiotics (penicillin at 50 IU/mL and streptomycin at 50 μg/mL), and l-glutamine (2 mmol/L).

Stability

The stability of the BIOT-NFL peptide conjugated onto gold nanoparticles was realized by UV–vis spectroscopy and then checked during 12 months as described previously.22

Measurements of Cellular Mitochondrial Activity

To investigate the effects of gold nanoparticles complexed or not with the BIOT-NFL-PEG-AuNPs peptide on cell viability, a (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay (Abcam, Paris, France) measuring the mitochondrial activity of cells was performed. Briefly, cells were seeded in 96-well plates with 4000 cells per well and incubated for 24 h at 37 °C and 5% CO2. The samples in different treatments were exposed to increasing concentrations of gold nanoparticles complexed or not with the BIOT-NFL peptide (between 50 and 1000 μmol/L) for 24 h at 37 °C and 5% CO2 (each sample was duplicated 6 times (n = 6)). At the end of the treatment, 10 μL of MTT reagent (5mg/mL in water) was added to each well for 3 h, and resulting formazan crystals were dissolved with DMSO. The absorbance at 570 nm was measured using a SpectraMax M2 spectrophotometer (Molecular Devices, San Jose, California).

Spectroscopic Characterization

All nanoparticles were characterized as described previously.66

UV–Vis Measurements

Absorption spectra were recorded using a double-beam Varian Cary 500 UV–vis spectrophotometer (Agilent, France). Absorption spectra of the AuNPs were recorded in water at a concentration of 10–4 M in the 200–900 nm spectral range.

Transmission Electron Microscopy (TEM)

BIOT-NFL-PEG-AuNPs was observed by transmission electron microscopy (TEM) at the Service Commun d′Imageries et Analyses Microscopiques (SCIAM; University of Angers, France) as described previously.22

Experimental in vivo test (see the paragraph in the Supporting Information section).

Conclusions

This paper shows a novel nanomedicine and its potential development in the field of PDAC therapy. The advanced antitumor efficacy of BIOT-NFL-PEG-AuNPs shows not only the reduction of tumor growth but also the stimulation of the immune system. Our work provides the preliminary way for the development of a theragnostic platform to realize the detection of cancer biomarkers and the simultaneous therapeutic effect through the enhancement of the immune system and the suppression of the angiogenic phenomenon.

Acknowledgments

This work has been partly performed on the CNanoMat platform of the University Paris 13 and supported by Shenzhen International Cooperative Research Project [grant nos. GJHZ20200731095210030], Guangdong Basic and Applied Basic Research Foundation [grant nos. 2021A1515012161], Guangdong Province Regional Joint Fund-Key Projects [no. 2020B1515120096], and Sanming Project of Medicine in Shenzhen [no. SZSM202003009]

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00159.

  • Experimental description, instrument set up, and main reagents used during the in vivo test; cytotoxicity MTT tests with PDAC cell lines (MIA-PACA-2) (Figure S1); data and picture concerning the tumor weight (Figure S2); experimental data about the effect of BIOT-NFL-PEG-AuNPs on blood cells in mice with pancreatic cancer (Figure S3); and hematology screening and table of blood cell values after BIOT-NFL-PEG-AuNPs administration (Table S1) (PDF)

The authors declare no competing financial interest.

A personal conflict was inadvertently published on October 14, 2022, but this has been corrected and removed as of November 18, 2022.

Supplementary Material

pt2c00159_si_001.pdf (817.6KB, pdf)

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