Bioinspired Lipoproteins of Furoxans–Gemcitabine Preferentially Targets Glioblastoma and Overcomes Radiotherapy Resistance

Abstract Radiotherapy (RT) resistance is an enormous challenge in glioblastoma multiforme (GBM) treatment, which is largely associated with DNA repair, poor distribution of reactive radicals in tumors, and limited delivery of radiosensitizers to the tumor sites. Inspired by the aberrant upregulation of RAD51 (a critical protein of DNA repair), scavenger receptor B type 1 (SR‐B1), and C‐C motif chemokine ligand 5 (CCL5) in GBM patients, a reduction‐sensitive nitric oxide (NO) donor conjugate of gemcitabine (RAD51 inhibitor) (NG) is synthesized as radio‐sensitizer and a CCL5 peptide‐modified bioinspired lipoprotein system of NG (C‐LNG) is rationally designed, aiming to preferentially target the tumor sites and overcome the RT resistance. C‐LNG can preferentially accumulate at the orthotopic GBM tumor sites with considerable intratumor permeation, responsively release the gemcitabine and NO, and then generate abundant peroxynitrite (ONOO−) upon X‐ray radiation, thereby producing a 99.64% inhibition of tumor growth and a 71.44% survival rate at 120 days in GL261‐induced orthotopic GBM tumor model. Therefore, the rationally designed bioinspired lipoprotein of NG provides an essential strategy to target GBM and overcome RT resistance.

Cell culture.The murine GL261 cells and the human U87 cells were provided by the Cell Bank of Shanghai, Chinese Academy of Sciences (CAS, Shanghai, China).The GL261 cells with S3 stable expression of luciferase were provided by iCell Bioscience Inc (Shanghai, China).Cells were cultured with high glucose Dulbecco's modified eagle medium (DMEM) (Hyclone) containing 10% FBS (Inner Mongolia Opcel) and 1% penicillin-streptomycin (Hyclone) in a humidified atmosphere that contained 5% CO2 at 37 °C.

Bioinformatic analysis of GBM patients.
The expression of RAD51, SR-B1, and CCL5 in GBM patients (n = 153) and healthy people (n = 1157) was assessed in TCGA and GTEx databases using visualization tools in Sangerbox 3.0 (http://sangerbox.com/home.html).The data were transformed as log2 (TPM + 1) and n is the number of biologically independent samples.The effect of RAD51 expression level on the survival benefits of GBM patients on the placebo arm of the AVAglio dataset was analyzed based on publicly available data (GSE84010) from GEO datasets.Furthermore, the effect of RAD51 expression level on the survival benefits of GBM patients with radiotherapy was analyzed based on publicly available data (GSE186057) from GEO datasets (https://www.ncbi.nlm.nih.gov/geo).Survival curves of GBM patients were generated by an R language package and presented with Kaplan-Meier plots.Immunofluorescence assays of tumor and peritumor tissues from GBM patients.The tumor and peritumor tissues from GBM patients were collected under routine surgical resections.To detect the expression of RAD51, sections were treated with Fixation/Permeabilization solution (BD Cytofix/Cytoperm), stained with primary antibodies of RAD51 (Abcam, ab133534, 1:200), and followed by Alexa Fluor® 647 antibody (Beyotime, A0468, 1:200).In contrast, the sections were counterstained with DAPI for visualization under a confocal laser scanning microscope (CLSM, LSM710.Carl Zeiss, Germany).The expression of indicators exhibited from the confocal image was further analyzed by Image J software.

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Preparation and characterization of C-LNG.C-LNG was fabricated with DPPC, DSPE-PEG-CCL5, NG, and ApoA1 peptide.In brief, DPPC, DSPE-PEG-CCL5, and NG (6: 3: 1, w/w) were accurately weighed, dissolved in methanol, evaporated to form a thin film in a round flask, dispersed with purified water, and sonicated with a probe for 1 min.Then, 1 mg of ApoA1 peptide was added to the mixed solution and performed three heat-cooling cycles between 50 °C and 4 °C to obtain C-LNG.In contrast, the counterpart formulation of LG (DPPC, DSPE-PEG, and 14 C-Gem, 6: 3: 1, w/w) and LNG (DPPC, DSPE-PEG, and NG, 6: 3: 1, w/w) were fabricated in the same preparation process.
The morphology of LG, LNG, and C-LNG was measured using transmission electron microscopy (TEM) (Tecnai G2 S-Twin, FEI) after staining with saturated uranyl acetate solution.The particle size distribution of LG, LNG, and C-LNG was measured using a dynamic light scattering (DLS) analysis on a Nano ZS 90 Zetasizer (Malvern, UK).The encapsulation efficiency (EE) of 14 C-Gem in LG, NG in LNG, and NG in C-LNG was respectively determined using high-performance liquid chromatography (HPLC) analysis (Agilent, 1290II-6460) under the following condition: mobile phase, acetonitrile-0.1% formic acid water (45/55, v/v); column, BEH-C18 1.7 μm (2.1×100mm); flow rate, 0.3 mL /min; detect wavelength: 269 nm.The unloaded drug was separated from the formulations using ultrafiltration tubes (30 KD, Vivaspin500, Sartorius) by centrifuging at 4 °C for 10 min.The drug amount was determined by the HPLC method to calculate the EE and drug loading capacity in the bioinspired lipoprotein system.To further evaluate their stability in the physiological fluids, these formulations were incubated in phosphate buffer solution (PBS) (pH 7.4) and PBS (pH 7.4) plus 10% FBS for 48 h, and measured by DLS (Malvern, UK).Moreover, C-LNG was incubated in the PBS (pH 7.4) and PBS (pH 7.4)+10% FBS at 37 °C for 48 h.At predetermined time points, the EE values of NG from C-LNG was monitored by the aforementioned HPLC method to evaluate their stability.

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To investigate the reduction-sensitive properties, these formulations were respectively incubated in PBS (pH 7.4) +10 mM GSH at 37 °C.The morphologies of LG, LNG, and C-LNG were detected by TEM, and the particle size distribution was measured by DLS analysis at 48 h of incubation.Moreover, the degraded products were analyzed by liquid chromatographyelectrospray ionization mass spectrometry (Agilent Technologies, Inc. EP-C18).To determine the time-dependent drug release profiles, C-LNG was incubated in PBS (pH 7.4) +10 mM GSH for 48 h at 37 °C.At predetermined time point, the NG amount remained in the C-LNG was quantified by the aforementioned HPLC method to calculate the percentage of drug release.To verify the NO release from these formulations, they were incubated in PBS (pH 7.4) + 10 mM GSH at 37 °C with a NO fluorescence probe of DAR-1 (Abcam, ab145388, 20 μM).At predetermined time intervals, samples were analyzed using a microplate reader (Ex560nm, Em595nm) (Bio-Tek Instrument Inc., USA).

NO production in GL261 cancer cells. The NO production in GL261 cells was examined by
CLSM and flow cytometry analysis (BD FACS Vantage SE, USA).In brief, GL261 cells (1×10 5 cells per well) were seeded into the confocal dish or 6-well plate and cultured overnight.
Cells were pretreated with a NO fluorescence probe of diaminofluorescein-FM diacetate (DAF-FM DA, Beyotime, 1:1000) for 1 h at 37 °C, then replaced with fresh culture media containing LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations for a further 24 h of incubation.Afterward, cells from each treatment were harvested for CLSM detections and flow cytometry examinations.
To detect their impact on cellular GSH in GL261 cells, cells were seeded into a 6-well plate (1×10 5 cells per well), cultured for 12 h, and incubated with LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations for 24 h.The intracellular level of GSH was quantified according to the protocol of a GSH assay kit (Beyotime, S0052).S7 ONOO -production and characterization of lipid peroxidation.Regarding the efficient NO generation from LNG and C-LNG groups in vitro, we further measured the production of ONOO -upon X-ray radiation.GL261 cells (1×10 5 cells per well) were seeded into the confocal dish and cultured overnight.Then, they were incubated with free Gem, LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations.After 4 h of incubation, cells from each treatment were exposed to X-ray radiation at 2 Gy (6 MV, Varian, VitalBeam, USA).Then, cells were fixed with 4% paraformaldehyde, and incubated with anti-nitrotyrosine (Sigma-Aldrich, 1:200) overnight at 4 °C followed by Alexa Fluor 647-labeled IgG (H+L) (Beyotime, 1:200) for 1 h at room temperature, and then counterstained with DAPI for visualization under CLSM.Moreover, the production of ONOO -was further detected and quantified using a BBoxiProbe ® O 58 fluorescent dye on a microplate reader (Ex 516nm, Em 606nm, Bio-Tek Instrument Inc., USA) To identify their impact on the lipid peroxidation induced by ONOO -, GL261 cells (1×10 5 cells per well) were seeded into a 6-well plate and cultured for 12 h.Then, they were respectively incubated with free Gem, LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations.After 4 h, cells were exposed to X-ray radiation at 2 Gy (6 MV, Varian, VitalBeam, USA) and incubated for a further 6 h.Cells were stained with C11-BODIPY 581/591   (5 μM) for 15 minutes at room temperature and then analyzed by flow cytometry (BD FACSVantage SE, USA).Meanwhile, the generation of lipid peroxidation was observed by CLSM.In brief, the GL261 cells (1×10 5 cells per well) were seeded into the confocal dish, cultured for 12 h, and performed with the same process as above.The production of lipid peroxides in the cell membrane was visualized by CLSM, wherein the oxidized lipid was denoted as green fluorescence signals.
DNA damage and repair.The impact of C-LNG mediated radiation treatment on DNA damage was performed using DNA Damage Assay Kit (γ-H2AX Immunofluorescence, Beyotime).S8 GL261 cells (1×10 5 cells per well) were seeded into the confocal dish and cultured overnight.
Then, cells were respectively treated with free Gem, LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations for 4 h, exposed to X-ray radiation at 2 Gy, and incubated for a further 20 h.Cells were treated with the Fixation/Permeabilization solution, then stained with primary γ-H2AX antibody following the manufacturer's protocol (DNA Damage Assay Kit, C2035S, Beyotime).Afterward, cells were stained with DAPI for visualization under CLSM.
Considering the essential role of RAD51 in DNA repair, we measured the RAD51 expression in cells from each treatment by immunofluorescence assays.In brief, GL261 cells (1×10 5 cells per well) were seeded into the confocal dish and followed the same treatment as above.Cells were treated with the Fixation/Permeabilization solution, stained with primary RAD51 antibody (Abcam, ab133534, 1:200) and Alexa Fluor 647-labeled IgG (H+L) (Beyotime, A0468, 1:200), then counterstained with DAPI for visualization under CLSM.
Then, the expression of the γ-H2AX and RAD51 in cells from each treatment was further investigated by western blot analysis.In brief, GL261 cells were seeded into the 6-well plate (5×10 5 cells per well) and cultured overnight.Then, cells were respectively treated with free Gem, LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations for 4 h, exposed to X-ray radiation at 2 Gy, and incubated for a further 20 h.Afterward, cells from each treatment were collected and treated with the pre-cooled RIPA lysis buffer (Beyotime, China) and centrifuged at 12,000 rpm at 4 °C for 20 minutes.The protein concentration in each sample was quantified with a BCA protein assay kit.The expression of γ-H2AX and RAD51 was measured using a primary γ-H2AX antibody (Abcam, ab81299, 1: 500) and a primary RAD51 antibody (Abcam, ab133534, 1:1000), followed by a secondary HRP-linked antibody (Abcam, ab97051, 1: 2000) according to the manufacturer's protocols for detections (Bio-Rad, USA).

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The cytotoxicity of C-LNG was measured in GL261 cells and Bend.3 cells by a cell counting kit (CCK-8).Both of them were seeded into the 96-well plate (1×10 4 cells per well) and cultured overnight, and then incubated with C-LNG at NG concentration ranging from 0 to 400 ng/mL for 20 h.The cell viability from each treatment was monitored using the CCK-8 kit for quantification.To evaluate the therapeutic efficacy of different treatment strategies in vitro, GL261 cells were seeded into the 96-well plate (1×10 4 cells per well) and cultured overnight.
Then, they were respectively treated with free Gem, LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations for 4 h, exposed to X-ray radiation at 2 Gy, and incubated for a further 20 h.The cell viability from each treatment was measured using the CCK-8 assay kit on a microplate reader.
Next, the therapeutic efficacy was further measured by colony formation assays.In brief, GL261 cells were seeded into the 6-well plate (5×10 3 cells per well) and cultured overnight.
Then, they were respectively treated with free Gem, LG, LNG, and C-LNG at 20 μg/mL of NG or comparable concentrations for 4 h, exposed to X-ray radiation at 2 Gy, and further cultured for 10 days to form clusters of cells.Afterward, the cell colonies were fixed with 4% paraformaldehyde and stained with Giemsa dye.
Targeting behavior to orthotopic GBM tumors.To evaluate their targeting to the GL261-lucinduced orthotopic GBM tumors, both LNG and C-LNG were fluorescently labeled with DiD for the imaging.The formation of the GL261-luc-induced orthotopic GBM tumor model was verified by bioluminescence assays prior to the experiments.At different time intervals after injection (1.0 mg/kg of DiD), the fluorescence signals from each group were monitored using an IVIS imaging system (IVIS, Perkin Elmer, U.K.), and analyzed by corresponding software.

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Compound NG: The 2.6 g of the compound, N, N-diisopropylethylamine 1.9 g, and HATU 3.2 g were added in 20 mL of N, N-dimethylaniline (DMA) and stirred at 25 o C for 0.5 h, and then 2.0 g of compound gemcitabine was added, stirred for another 10 h, and the reaction solution was concentrated under reduced pressure to yield a yellow solid.Afterward, the residual was extracted with ethyl acetate and water.And then, the combined organic layer was concentrated to give the desired product 0.9 g (yield, 20.7%), which was characterized by the NMR spectrum.

Figure S11
Synthesis procedure and characterization of C14-Gem.
Human tumor specimens: The tumor and peritumor tissues of GBM patients were obtained from the Department of Neurosurgery at the Second Affiliated Hospital of Chongqing Medical University.All specimens were obtained with written informed consent and standard research protocol approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (Approved No. YLS 2023-079).

Figure S12
Figure S12The particle size distribution of LG, LNG, and C-LNG upon their incubation in

Figure S13 Figure S14
Figure S13The mean diameter of LG, LNG, and C-LNG upon their incubation in PBS (pH

Figure S15
Figure S15 Degradation of NG in GSH-contain media.(A) ESI-MS analysis of NG, which was presented as [NG + H] + .(B) ESI-MS analysis of Gem, which was presented as [Gem + H] + .

Figure S18 Figure S19
Figure S18Flow cytometry analysis of lipid peroxidation in GL261 cells from each treatment.

Figure S20
Figure S20The in vivo pharmacokinetic profiles of DiD-labeled LNG and C-LNG in healthy

Figure S21
Figure S21The ex vivo fluorescence images of the heart, liver, spleen, lungs, kidney, and

Figure S23 Figure S24 Figure S25
Figure S23The quantified gemcitabine distribution in different major organs at 12 h after tail vein injection of LNG and C-LNG NPs (n = 3), *** P < 0.001.

Figure
Figure S27 H&E staining images of the whole brain from GL261-luc induced GBM tumor