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Fluorescent bovine serum albumin-silver nanoclusters loaded with paclitaxel can traverse the blood-brain barrier to inhibit the migration of glioma

Liu, Yua; Liu, Yub; Zhang, Yinic; Xie, Weichangc; Yang, Yalanc; Hu, Kaigengc; Xin, Hongboc,∗; Hu, Pingc,∗; Ai, Fanronga,∗

Author Information
doi: 10.1097/JBR.0000000000000082

Abstract

Introduction

Glioma is a very high-risk tumor that occurs in the brain, associated with a very high metastasis rate and high mortality. At present, few specific anti-tumor drugs exist to treat this tumor.[1,2] Glioma treatment can be difficult due to the presence of the blood-brain barrier (BBB).[3] The BBB is able to prevent most classical chemotherapy drugs from accessing the brain; therefore, a drug carrier system that is able to traverse the blood-brain barrier is essential for the effective delivery of drugs to the brain. Several systems have been developed for this purpose, with excellent results.[4–8] However, many of these systems were complicated to generate, the efficiency of delivery beyond the BBB was not very high, and the materials used in these systems without therapy effect on glioma.[9] Few studies have examined the effects of carrier materials on tumor cells. The materials in the drug-delivery system comprise a large proportion of the whole treatment that is delivered.[10] Optimally, the developed material system would be able to inhibit the growth and migration of tumor cells, have no toxic effects against ordinary cells, and be quickly metabolized and removed from the body after treatment.[11] The development of such a delivery system would improve tumor treatment.[12–14]

Noble metal nanoclusters (NCs) represent a new type of nanomaterial, consisting of a few to dozens of atoms. In particular, silver nanoclusters (AgNCs) have attracted attention due to their ultra-small size, excellent biocompatibility, and high fluorescence properties and can be used as promising biosensing probes.[15–17] AgNCs have demonstrated great potential advantages for use in biological imaging and the evaluation of catalytic activity. However, few reports have examined the application of AgNCs to nano-drug carrier systems.[18]

Silver clusters are easy to prepare and, because of their ultra-small size, can easily enter the brain.[19,20] Therefore, we used AgNCs as the core of the drug-loading system, with bovine serum albumin (BSA) as the system medium. We then loaded BSA-AgNCs with paclitaxel (PTX), and the BSA-AgNC-PTX nanoparticle (NP) drug-delivery system was developed for the subsequent treatment of cerebral glioma.

Materials and methods

Materials

The aqueous solution was prepared from ultrapure water, silver nitrate (AgNO3), sodium borohydride (NaBH4), sodium hydroxide (NaOH), bovine serum albumin (BSA), paclitaxel (PTX), methanol (CH3OH), and phosphate-buffered saline (PBS), which were purchased from Macklin (Shanghai, China). Harmine was purchased from Aladdin (Shanghai, China). Dulbecco's modified Eagle medium (DMEM) was purchased from HyClone (Beijing, China). A calcein-acetoxymethyl ester (calcein-AM)/propidium iodide (PI) dual staining kit and a cell counting kit 8 (CCK-8) were purchased from Bestbio Biotech Co., Ltd. (Shanghai, China). All reagents were analytically pure and were used without further purification.

Characterization

A transmission electron microscope (TEM; JEM-2100, 200 kV, Japan) was used to characterize the morphologies and structures of BSA-AgNCs and BSA-AgNC-PTX NPs. A fluorescence spectrophotometer (Techcomp/3077–00, China) was used to detect fluorescence intensity. The hydrodynamic sizes and zeta potentials of BSA-AgNCs and BSA-AgNC-PTX NPs were measured using a particle size and potential tester (Zetasizer Nano ZS-90 instrument, Malvern, UK) at 25°C. An X-ray photoelectron spectrometer (XPS; ESCALAB250Xi, USA) was used to analyze the properties of BSA-AgNCs, and an ultraviolet-visible spectrophotometer (Shimadzu UV-2600, Japan) was used to detect the absorption spectrum. The in vivo fluorescence images of live animals were obtained using a live animal imager (IVIS Lumina II, PerkinElmer, USA). All experiments were performed at least three times.

Synthesis of BSA-AgNCs

At room temperature, 200 μL 10 mM silver nitrate solution was added to 0.1 g (1.5 μM) BSA (66.4kD) powder in 10 mL distilled water and was stirred for 30 minutes. The pH was adjusted to approximately 12 through the addition of 0.1 mL NaOH (1 M), followed by stirring for 15 minutes. Then, 90 μL 112 mM NaBH4 solution was added, dropwise, until the solution changed from colorless to reddish-brown, indicating the formation of various amounts of clusters. The solution continued to be stirred for 2 hours. The samples were purified 3–5 times using an ultrafiltration tube (3 kD). Then, the BSA-AgNCs were collected and diluted to a final volume of 4 mL with distilled water.

Preparation of BSA-AgNC-PTX NPs and BSA-PTX

A stock solution was prepared by dissolving 2 mg PTX (853.9 kD) in 100 mg methanol to obtain a concentration of 20 mg/mL (23.4 mM). A stock solution of BSA was prepared by dissolving 0.1 g (1.5 μM) BSA in 4 mL (0.375 mM) distilled water. A 40 μL volume of PTX, which was pre-dissolved in methanol (20 mg/mL; 23.4 mM), was added to both 2.5 mL BSA-AgNCs, in aqueous solution and 2.5 mL BSA solution. After stirring overnight, BSA-AgNC-PTX NPs and BSA-PTX samples were obtained after dialysis against PBS (molecular weight cut off of 14 kD) to remove any unbound molecules and organic solvents.

Encapsulation efficiency of PTX

BSA-AgNC-PTX NP solution was prepared, with the mole ration of BSA to PTX were 1:1, 1:2, 1:4, 1:8, respectively. The BSA-AgNC-PTX NP solution was centrifuged, and the lower layer (free PTX) was removed and dissolved in a mixed solution (acetonitrile:water = 60:40). For the mixed solution, high-performance liquid chromatography was used to determine PTX solubility. The UV detection wavelength was 227 nm. The concentration of the dissociated drug was calculated and used in the following equation to calculate the Encapsulation Efficiency:Encapsulationefficiency(%)=(W0W1)/W0×100

where W0 represents the total amount of the drug, and W1 represents the amount of the dissociated drug.

In vitro BBB model

We established an in vitro BBB model using a Transwell cell-culture system. First, mouse-derived brain microvascular endothelial cells (2 × 105 cells/well) were inoculated on Transwell porous biofilms pre-coated with gelatin (2%), and fetal bovine serum (FBS) medium (10%) was added at the same time. Then, the integrity of the system was assessed using a Millicell-ERS Voltmeter (Millipore) to determine the integrity of the system. When the transepithelial electrical resistance (TEER) was measured at greater than 300 Ω cm2, we believed that the BBB model was successfully established for use in subsequent research. Then, we added various experimental materials to the upper chamber. After 6 hours, the supernatant in the upper chamber and the filtrate in the lower chamber were sampled and placed in separate Eppendorf (EP) tubes. The absorbance values of the AgNCs in both the supernatant and filtrate for each experimental group were measured using an ultraviolet spectrophotometer. Finally, we determined the total intracellular contents by adding the concentration of AgNCs in the lower chamber to that in the upper chamber.

Construction of U251-GFP-Luc lentiviral cell line

U251 cells were inoculated into a 60-mm culture dish, cultured until they reached 80% to 90% confluence, and infected with the lentiviral vector LV-GFP-Puro-LUC, which encoded a green fluorescent protein (GFP) and luciferase (LUC, Hanbio, Shanghai) fusion protein. After 4 hours of infection at 37°C, 4 mL DMEM (HyClone, Beijing, China) medium was added, which was replaced with fresh DMEM medium after 24 hours. After an additional 48 hours, fresh complete DMEM medium containing 2 μg/mL puromycin was used to select successfully transformed colonies. DMEM medium was replaced every 2 days until the selection was complete. The expression of GFP fluorescence was detected under an inverted fluorescence microscope (IX83, Olympus, Japan) to evaluate the lentiviral infection efficiency. The selected cells that stably expressed GFP-Luc were referred to as U251-GFP-Luc cells.

Glioma treatment

Six-week-old BALA/c male nude mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). All surgical procedures were approved by the Animal Ethics Committee of Nanchang University (SYXK2019-0003) on December 29, 2019. A stereotactic implant device (RWD Life Science, Shenzhen, China) was used to inject U251-GFP-Luc cells into the striatum of each mouse. The mice were anesthetized with sodium pentobarbital and placed in a stereotactic frame for the entire operation, and a heating pad was used to maintain their body temperature. A burr hole was drilled in the skull, 1.0 mm from the front of the skull, and 2.0 mm from the prefrontal region of the brain, and then 1 × 106 cells (10 μL) were injected stereotactically to a depth of 2.5 mm. After fully recovering from anesthesia, the mice were returned to their cages. One week after the injection, the experimental group was intraperitoneally injected daily with BSA-AgNC-PTX NPs (2 mg/kg, n = 6). Simultaneously, a second group was injected with sterile saline daily to serve as a blank control group (n = 6). Finally, Hamine was administered daily (2 mg/kg, n = 6) to the drug control group. All treatments were injected directly through the tail vein, and each treatment cycle was maintained for 4 weeks. Changes in each mouse were observed and recorded weekly, including the body weights and survival rates in each group of mice. After 4 weeks, brain tumors were evaluated using an IVIS Lumina II bioluminescence imaging system (PerkinElmer, USA), and the treatment effects were compared according to the real-time fluorescence values.

Cell culture

All cell lines, including a hippocampal neuronal cell line (HT-22), normal human fibroblasts (NHF), and brain microvascular endothelial cells (BMECs), were purchased from the Cell Culture Center of the Chinese Academy of Sciences (CBTCCCAS, Shanghai, China), and were passaged and frozen in accordance with relevant regulations. The cells were cultured in DMEM medium supplemented with 4.5 g/L glucose, 10% fetal bovine serum (FBS), and 1% glutamine. The cells were cultured at 37°C in a humidified incubator containing 5% CO2. The LUC-GFP lentiviral transfection system (Hanbio, Shanghai, China) was used for cell transfections.

Cytotoxicity assessment

The cytotoxicity of the experimental materials was analyzed using a cell counting kit 8 (CCK-8). HT-22 cells, NHFs, and BMECs were seeded into 96-well plates overnight, at a density of 1 × 104 cells per well. Then, the cells were treated with BSA-AgNCs at concentrations ranging from 0 to 200 μg/mL, followed by the addition of 10 μL CCK-8 to each well, incubated continuously for 2 hours. An enzyme-linked immunosorbent assay plate reader (Synergy 2, Bio-TEK, Vermont, USA) was used to measure the optical density (OD) at a reference wavelength of 450 nm.

Cell scratch assay

U251 cells were evenly plated in a six-well plate. When the cells reached 90% to 95% confluency, a sterilized ruler was used to draw a straight line through the middle of each well, using a 200-μL pipette tip. The marked wells were washed with PBS 2 to 3 times to remove dissociated floating cells, followed by the addition of 2% serum medium containing the drug. White light photos were obtained for each scratched well under an optical microscope (Olympus CX41, Tokyo, Japan) at 0, 12, and 24 hours, and the pictures were stored digitally. ImageJ software (NIH, Bethesda, MD, USA) was used to measure the widths of the scratches in each image.

Quantitative real-time PCR

Total cellular RNA from U251 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed to obtain cDNA, according to the manufacturer's instructions from Invitrogen (12594025, SuperScript IV One-Step RT-PCR System, Carlsbad, CA, USA). The PCR primers used for the analysis were as follows: matrix metalloproteinase (MMP)-2: 5’-CCG TCG CCC ATC ATC AAG TTC-3’ (sense) and 5’-GCA GCC ATA GAA GGT GTT CAG G-3’ (anti-sense), which amplified a 90-bp fragment; MMP-9: 5’-TGG TCC TGG TGC TCC TGG TG-3’ (sense) and 5’-GCT GCC TGT CGG TGA GAT TGG-3’ (anti-sense), which amplified a 111-bp fragment; vascular endothelial growth factor (VEGF): 5’-CTG GGC TGT TCT CGC TTC G-3’ (sense) and 5’-CTC TCC TCT TCC TTC TCT TCT TCC-3’ (anti-sense), which amplified a 140-bp fragment; MMP-7: 5’-GAGTGAGCTACAGTGGGAACA-3’ (sense) and 5’-CTA TGA CGC GGG AGT TTA ACA T-3’ (anti-sense), which amplified a 158-bp fragment; transforming growth factor (TGF)-β: 5’-GTA GCT CTG ATG AGT GCA ATG AC’ (sense) and 5’-CAG ATA TGG CAA CTC CCA GTG-3’ (anti-sense), which amplified a 132-bp fragment; metastasis associated (MTA)-1: 5’-TGT ACC GCT ATG GTT ACA CTC G-3’ (sense) and 5’-GGC AGG GAC AGT TGC TTC T-3’ (anti-sense), which amplified a 97-bp fragment; NM23: 5’-AAG GAG ATC GGC TTG TGG TTT-3’(sense) and 5’CTG AGC ACA GCT CGT GTA ATC-3’ (anti-sense), which amplified a 60-bp fragment; glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5’-ACA ACT TTG GTA TCG TGG AAG G-3’ (sense), 5’-GCC ATC ACG CCA CAG TTT C-3’ (anti-sense), which amplified a 101-bp fragment. The 20 μL PCR system contained 2 μL cDNA, 10 μL SYBR Premix Ex Taq II, 0.4 μL ROX Reference Dye II, 0.8 μL 10 μM sense primer, 0.8 μL 10 μM anti-sense primer, and 6 μL ddH2O. The amplification protocol was 95°C for 30 seconds, 40 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 34 seconds, elongation at 95°C for 15 seconds, and extension at 60°C for 1 minute. At least three independent experiments were conducted, and the samples were assessed in triplicate for each experiment.

Blood biochemical analysis

After 4 weeks of injection, we fixed the mice in a supine position on a mouse board. Scissors were used to cut off the precordial hair, and the skin was disinfected with alcohol. The heartbeat was located using by placing an index finger in the third to fourth intercostal space on the left side of the mouse. A syringe with a 4-gauge needle was used to puncture the strongest part of the heartbeat to obtain blood. The blood sample was then centrifuged at a low speed (1000 ×g for 5 minutes), and the supernatant was removed and stored at −80°C. All blood samples were sent to Wuhan Seville Technology Co., Ltd. to perform liver and kidney function tests [alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine].

HE staining

After sampling blood from the mouse heart, we sacrificed all mice using a high concentration of carbon dioxide and removed the mouse brain, heart, liver, spleen, lung, and kidney. Tissues were fixed in formaldehyde and then dehydrated using a gradient of low to high concentrations of alcohol. Specimens were embedded in wax blocks, and the wax blocks were cut into thin slices using a microtome (approximately 5 mm thick). Slices were deparaffinized twice with xylene (10 minutes each), attached to glass slides, and rinsed with ultrapure water three times for 5 minutes each. The slides were stained in hematoxylin and eosin (HE) stain for 8 minutes, differentiated with 5% acetic acid for 1inute, rinsed in running water, and dehydrated using an alcohol gradient. The slides were dried under a hood, covered in neutral resin, coverslipped, and analyzed under an optical microscope.

Statistical analysis

Graph Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) and Origin 9 (OriginLab Corporation, Northampton, MA, USA) software were used to perform statistical analyses. The data were expressed as the mean ± standard deviation (SD). Comparisons between groups were analyzed using one-way analysis of variance (ANOVA) and Student's t-test. P < 0.05 was considered significant.

Results

Preparation and characterization of BSA-AgNCs and BSA-AgNC-PTX NPs

In this study, due to the presence of 17 disulfide bonds and 1 free cysteine, BSA was chosen as the template for the synthesis of AgNCs. Interestingly, BSA has a strong affinity for inorganic metal salts, which makes it suitable for use as a stabilizer during the preparation of AgNCs. Figure 1A and B show the excitation and emission spectra of BSA-AgNCs, respectively. The excitation spectrum, which was monitored at 605 nm, is composed of broad excitation bands, ranging from 400 to 500 nm. Upon excitation at 400 to 450 nm, the recorded emission spectrum showed the strongest red emissions at 605 nm. An ultraviolet-visible spectrum analysis was also performed, and the spectrum revealed a clear absorption peak at 406 nm, which was the absorption of AgNCs (Additional Fig. 1, http://links.lww.com/JR9/A24). TEM analysis was used to characterize the morphologies and size distributions of the obtained BSA-AgNCs. As shown in Figure 1C, the average size of the obtained BSA-AgNCs was approximately 4 nm, and they presented a nearly spherical shape. Energy dispersive spectroscopy (EDS) analysis detected the presence of four basic elements (C, O, N, and Ag) in BSA-AgNCs (Fig. 1D). Dynamic light scattering (DLS) was also performed, and the results showed that the size of the nanoclusters was primarily distributed between 1 nm and 3 nm, which was consistent with the TEM results (Fig. 1E). XPS analysis was further performed to examine the valence states of the Ag atoms in AgNCs (Additional Fig. 2, http://links.lww.com/JR9/A24). Two peaks were clearly observed, at 368.06 eV and 374.1 eV, which corresponded to Ag3d5/2 and Ag3d3/2, respectively. Next, to prepare the organic-inorganic nanomedicine composite material, freshly prepared PTX was added to a dilute aqueous dispersion of synthesized BSA-AgNCs, under constant stirring conditions at room temperature, and then aged for 2 hours. The details of the synthesis have been described in Materials and Methods. TEM examination showed that the synthesized, spherical BSA-AgNC-PTX NPs had a particle size of 102.6 ± 30.5 nm (Fig. 1F), which represented the most thermodynamically stable structure and shape for the given reaction conditions. However, the DLS-based hydrodynamic size measurement of the BSA-AgNC-PTX NPs revealed a size of 177.2 ± 18.3 nm, which may be due to the protein in the liquid medium and the hydrated layer (Fig. 1H). In addition, to clarify the mechanism of BSA-AgNC-PTX NP formation, DLS-based zeta potential measurement was performed. The zeta potential data showed that the potential of the synthesized BSA-AgNCs was −16.97 ± 1.62 mV. The potential of PTX was 5.72 ± 0.98 mV. After adding the positively charged PTX, the zeta potential of the final BSA-AgNC-PTX NP complex increased to −9.05 ± 0.68 mV, indicating the presence of electrostatic interactions between the two substances (Fig. 1G). However, H-bond interactions and π-π stacking interactions between PTX and the hydrophilic-hydrophobic sites in the protein may also be the basis of the interaction. In addition, high-performance liquid chromatography was performed to measure the drug loading rate of BSA-AgNCs at different BSA to PTX ratios. The results showed that when the ratio of BSA:PTX was 1:1, a maximal drug loading rate of 96 ± 3% was observed (Fig. 1I).

Figure 1
Figure 1:
Characterization of BSA-AgNCs and BSA-AgNC-PTX NPs. (A) The excitation spectrum of BSA-AgNCs with emission fixed at 605 nm. (B) The emission spectrum of BSA-AgNCs with excitation between 400 and 450 nm. The highest intensity was obtained at 450 nm. (C) TEM image of BSA-AgNCs. (D) The energy dispersive spectrum analysis of BSA-AgNCs. The inset graph shows the element mapping results for BSA-AgNCs. (E, H) DLS particle size analyses for AgNCs (E) and BSA-AgNC-PTX NPs (H) in aqueous solution. (F) TEM image of BSA-AgNC-PTX NPs. (G) Zeta potential analysis. (I) PTX drug-loading rates at different ratios of BSA to PTX (the inset shows the digital photo of BSA:PTX at different ratios). Error bars represent the standard deviation of three separate measurements. AgNCs = silver nanoclusters, BSA = bovine serum albumin, NPs = nanoparticles, PTX = paclitaxel.

Cytotoxicity of BSA-AgNCs

The in vitro cytocompatibility of BSA-AgNCs was evaluated using cell viability (Fig. 2A–C) and living/dead cell assays (Fig. 2D–F) after co-culturing BSA-AgNCs with BMEC, HT-22, and NHF cell lines for 1, 3, and 7 days. Compared with the control group for each cell line, no obvious influence on cell growth was observed when treated with BSA-AgNCs at various concentrations. Living/dead cell assays showed no significant differences in the numbers of live cells between BSA-AgNC-cultured and control groups (P > 0.05), indicating that BSA-AgNCs showed excellent biocompatibility.

Figure 2
Figure 2:
Cytotoxicity and dead/live cell staining following BSA-AgNC treatment. (A–C) Cytotoxicity of BSA-AgNCs in BMECs (A), HT-22 (B), and NHF cells (C) at 1, 3, and 7 days. (D–F) Dead/live cell staining after BSA-AgNCs were incubated with BMECs (D), HF-22 (E), and NHF cells (F) for 1 and 3 days and stained after 7 days. The living cells were green fluorescent, and the dead cells were red fluorescent. Error bars represent the standard deviation of three separate measurements. AgNCs = silver nanoclusters, BMECs = brain microvascular endothelial cells, BSA = bovine serum albumin, HT22 = hippocampal neuronal cell line, NHFs = normal human fibroblasts, OD = optical density.

BSA-AgNCs and BSA-AgNC-PTX NPs traverse the BBB with high efficiency in vitro

To evaluate the ability of the experimental materials to pass through the BBB, in vitro, a Transwell model was used to simulate the BBB structure. Gelatin and BMECs were placed on the bottom surface of the Transwell insert (Fig. 3A). Then, the in vitro BBB transmission efficiencies of BSA-AgNCs, BSA-AgNC-PTX NPs, BSA, and BSA-PTX were measured. The upper and lower chamber fluids for the various experimental groups were collected at 3, 6, and 24 hours, and the sample concentrations were determined using a micro-protein analysis tester to determine the intracellular contents (Fig. 3B and C). The BSA-AgNC and BSA-AgNC-PTX NP groups achieved very high transmission efficiencies after 3 hours, after which they maintained reverse concentration differences between the upper and lower chambers. Meanwhile, in the BSA and BSA-PTX groups, similar concentration differences were maintained between the upper and lower chambers at various time points. However, the BBB transmission efficiencies for the groups loaded with PTX were lower than those for groups without PTX. Particle size has been speculated to play an important role in whether a material successfully passes through the in vitro BBB model, and AgNCs appeared to play a vital role in increasing the penetration efficiency of the BBB.

Figure 3
Figure 3:
BSA-AgNC-PTX NPs successfully traverse the BBB in vitro. (A) An in vitro BBB model was established using mouse brain microvascular endothelial cells in a Transwell system. (B, C) Quantitative analysis of the effects of AgNCs and loaded paclitaxel drugs on the penetration of the BBB, in vitro. Penetration was assessed at 3, 6, and 24 hours. Error bars represent the standard deviation of three separate measurements. AgNCs = silver nanoclusters, BBB = blood-brain barrier, BMECs = brain microvascular endothelial cells, BSA = bovine serum albumin.

BSA-AgNC-PTX NPs traverse the BBB in vivo

The in vivo BBB permeability of BSA-AgNC-PTX NPs was verified using nude mice. The BSA-AgNC-PTX NPs were tail-vein-injected into the mice, and the distribution of fluorescent BSA-AgNC-PTX NPs was evaluated using an IVIS optical imaging system at 1 and 3 hours post-injection. In addition, quantitative data were obtained by performing region-of-interest analyses on the fluorescent images of the excised individual organs, such as the liver, kidney, and brain. AgNCs were abundantly enriched in the liver, which featured a higher concentration than those measured in the brain and kidney 1 hour post-injection. The accumulation of BSA-AgNC-PTX NPs in the brain could be clearly visualized using fluorescent images, as determined by the brightening of the brain observe 3 hours post-injection (Fig. 4A). To further determine whether AgNCs played a critical role in passing through the BBB and to determine in which brain regions AgNCs were distributed, BSA-PTX and BSA-AgNC-PTX NPs were separately injected into nude mice via the tail vein. After the mice were perfused, the brain tissues were extracted, sliced, and observed under a confocal microscope (Fig. 4B). Brain slice images revealed that the fluorescent BSA-AgNC-PTX NPs accumulated in the regions of the hippocampus and the medial prefrontal cortex (Fig. 4B).

Figure 4
Figure 4:
BSA-AgNC-PTX NPs traverse the blood-brain barrier in vivo and accumulate in the hippocampus and the medial prefrontal cortex. (A) AgNCs were injected into normal nude mice, and the bioluminescence imaging system was used at 1 and 3 hours after injection. The liver, kidney, and brain tissues of AgNC-injected nude mice were extracted at the corresponding time points for observation. (B) After perfusion, mice were injected with BSA-PTX and BSA-AgNC-PTX NPs, and the indicated brain regions were observed (circles). Original magnification, 40×. AgNCs = silver nanoclusters, BSA = bovine serum albumin, HIP = hippocampus, MPFC = medial prefrontal cortex, PTX = paclitaxel.

BSA-AgNCs inhibit the proliferation and migration of U251 tumor cells

To analyze the performance of BSA-AgNCs during the treatment of glioma cells, different concentrations of BSA-AgNCs and U251 brain tumor cells were co-cultured for 1, 3, and 7 days. The results showed that BSA-AgNCs were able to inhibit the proliferation of U251 tumor cells, and this inhibitory effect increased in a dose-dependent manner (Fig. 5A). To further explore the effects of BSA-AgNCs on brain tumor cells, BSA-AgNCs and U251 brain tumor cells were co-cultured for 24 hours, and then the cell scratch test was performed. The results showed that BSA-AgNC treatment inhibited the migration of U251 brain tumor cells (Fig. 5B). Moreover, a variety of genes related to tumor migration were selected for quantitative PCR experiments (Fig. 5C). The results showed that BSA-AgNCs inhibited the expression of multiple U251 tumor cell migration genes, which indicated that BSA-AgNCs could inhibit the migration of U251 tumor cells.

Figure 5
Figure 5:
Cell proliferation, cell scratch assay, and qPCR tests verified that BSA-AgNCs inhibited the proliferation and migration of U251 cells. (A) Cell proliferation of U251 cells co-cultured with BSA-AgNCs. (B) Cell scratch assay of U251 cells co-cultured with BSA-AgNCs. (C) Expression levels of various tumor metastasis suppressor genes by qPCR. Error bars represent the standard deviation of three separate measurements. MMP = matrix metalloproteinase, OD = optical density, TGF = transforming growth factor, VEGF = vascular endothelial growth factor.

Inhibitory effects of BSA-AgNC-PTX NPs on glioma metastasis

Using a small animal stereotaxic instrument, the constructed LUC-GFP-U251 cell lines were implanted into the brains of nude mice. After implantation for 1 week, BSA-AgNC-PTX NPs, Hamine (a type of small molecule that can directly permeate the BBB and inhibit tumor drugs), and saline were tail-vein-injected into nude mice. After 4 weeks, the tumors of each group were observed using the IVIS optical imaging system (Fig. 6A). Mice in the Hamine and control groups showed the systemic metastasis of glioma, whereas the tumors in the BSA-AgNC-PTX NP group showed no systemic metastasis. Furthermore, a lateral comparison of the three groups of nude mice was performed (Fig. 6B), and the quantitative data obtained from the region-of-interest analyses are shown in Figure 6C. The weight changes (Fig. 6D) and survival rates (Fig. 6E) of nude mice were measured throughout the experiment. BSA-AgNC-PTX NPs were not only effective for the treatment of cerebral glioma through the BBB but also demonstrated a significant inhibitory effect against glioma metastasis during treatment, resulting in a longer-term survival rate for BSA-AgNC-PTX NP-treated mice compared with those of the other two groups.

Figure 6
Figure 6:
Comparison of glioma metastasis between different groups after the injection of U251 cells and changes in the body weights and survival curves of mice. (A) Changes in the fluorescence intensity of glioma biotin in the BSA-AgNC-PTX NP, Harmine, and control groups. (B, C) One mouse was selected from each of the three groups for comparison and observations of the brain, neck, and back metastases (B) and quantitative analysis was performed (C). (D) Changes in the body weights of mice in the different treatment groups. (E) Comparison of the survival rates among mice in different groups. Error bars represent the standard deviation of three separate measurements. AgNCs = silver nanoclusters, BSA = bovine serum albumin, HM = Harmine, NP = nanoparticle, PTX = paclitaxel.

In vivo toxicity of BSA-AgNC-PTX NPs

Toxicity is a critical issue for the in vivo application of BSA-AgNC-PTX NPs. We assessed the acute toxicity of BSA-AgNC-PTX NPs by performing daily tail vein injections into nude mice for 4 weeks. Serum biochemistry assays were conducted to assess potential toxicity after the injection of BSA-AgNC-PTX NPs. Primary liver function markers, including ALT and AST, and kidney function markers, including BUN and serum creatinine, were measured (Fig. 7A) 7 days post-injection. No hepatic or kidney disorders were observed in the treatment group compared with the control group. In addition, nude mice were dissected to obtain the major organs (heart, liver, spleen, lung, kidney, and brain), which were sliced and stained using HE for histological analysis (Fig. 7B). The results further revealed a lack of noticeable tissue damage in all major organs.

Figure 7
Figure 7:
Biochemical analysis and HE staining showed that BSA-AgNC-PTX NPs have good biological safety profiles in vivo. (A) Blood samples from control and BSA-AgNC-PTX NPs mouse groups were obtained for biochemical analyses (ALT, AST, BUN, and creatinine). Error bars represent the standard deviation of three separate measurements. (B) The mice in the experimental group (BSA-AgNC-PTX NP group) and the control group were dissected, HE staining was performed, and images were obtained. Scale bars = 50 μm. AgNCs = silver nanoclusters, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BSA = bovine serum albumin, BUN = blood urea nitrogen, NPs = nanoparticles.

Discussion

Our experimental results showed that the BSA-AgNC drug-loading system had very high permeation efficiencies for both the in vitro BBB model and the nude mouse in vivo BBB model. Simultaneously, no obvious toxicity was observed for the BSA-AgNC drug-loading system when examined using the in vitro cell compatibility test or in vivo vital organ HE staining. The BSA-AgNC drug-loading system demonstrated the ability to inhibit the proliferation and metastasis of glioma cells.

AgNCs have been previously used as a drug-delivery system for some traditional small molecule drugs, such as PTX, which was able to traverse the BBB and was successfully used to treat cerebral gliomas.[21,22] The results of the present study showed that AgNCs were able to easily penetrate the BBB, both in vitro and in vivo, and accumulated in the cerebral cortex. This region is also a common site for cerebral gliomas. Subsequent animal experiments demonstrated that the AgNC system inhibited the growth of gliomas in the brain while also inhibiting glioma metastasis,[23–25] which is exciting because poor tumor prognosis requires both glioma destruction and the control of glioma metastasis. A drug delivery system with efficient BBB penetration and tumor metastasis inhibitory effects represent a significant development for the treatment of clinical gliomas.[26–28] AgNCs have a high penetration efficiency for BBB, therefore, AgNCs can also be used as carriers to combine with other small molecules to treat a series of brain diseases, including some neurodegeneratives diseases, such as whether it can be combined with furoxetine to treat depression. In addition, related literature reports that AgNCs, as a kind of ultra-small nanoparticles, also have excellent antibacterial properties and can be used as a safe and non-toxic antibacterial additive.

Although the current research results are encouraging, some limitations remain to be addressed. For example, the long-term biological safety of BSA-AgNC nano-drug carrier systems in vivo must still be assessed. Future research should focus on two aspects: building an intelligent drug delivery system for the early detection and treatment of glioma and improving the targeting performance of the system for application to other deep tumors.

Conclusion

The as-synthesized BSA-AgNC novel nano-drug carriers show a high penetration efficiency for BBB, which can be used as drug carriers for treatment of brain diseases, including glioma. Paclitaxel (PTX) was loaded into BSA-AgNCs through electrostatic and hydrophobic interactions to formulate spherical BSA-AgNC-PTX nanoparticles. Meanwhile, both in vitro and in vivo experiments revealed the as-synthesized BSA-AgNC-PTX NPs can efficiently pass through the BBB, showing significant inhibition of tumor growth and migration, and prolonging the survival of the mice. The BSA-AgNC nano-drug carrier system could be a promising candidate for effective brain tumor therapy.

Acknowledgments

None.

Author contributions

All authors contributed to the study design, data analysis and manuscript preparation, and performed the study, and approved the final version of the manuscript.

Financial support

This work was funded by the National Natural Science Foundation of China (No. 31960207 to FA); China Postdoctoral Science Foundation (No. 2017M610402 to FA); Postdoctoral Science Foundation of Jiangxi Province of China (No. 2017KY06 to FA); and Nanchang Municipal Key Laboratory of 3D Bioprinting Technology and Equipment (No. 2019NCZDSY001 to FA).

Institutional review board statement

All surgical procedures were approved by the Animal Ethics Committee of Nanchang University, China (SYXK2019-0003) on December 29, 2019.

Conflicts of interest

The authors declare no conflict of interest.

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Keywords:

blood-brain barrier; glioma; paclitaxel; renal clearable; silver nanoclusters

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