1. Introduction
Tumor progression causes the generation of a very complex pathological environment called a tumor microenvironment (TME), which directly promotes tumor growth. The rapid metabolism and proliferation of tumor cells require an increased blood supply to meet their demand for nutrients and oxygen. This leads to excessive oxygen consumption, thus creating a large disorganized and leaky tumor-associated vasculature and the formation of a hypoxic region [
1]. This complex TME seriously interferes with the efficacy of common tumor therapies (e.g., causing chemotherapy resistance) [
2], but also creates new ideas and possibilities for tumor treatment options [
3,
4].
For instance, antiangiogenic therapy relying on targeting vascular endothelial growth factor (VEGF) is a strategy for tumor treatment through the avoidance of new blood vessel formation [
5]. However, a single antiangiogenic treatment strategy is ineffective in eliminating the tumor [
6]. Moreover, long-term employment of sorafenib (SF), a multi-kinase inhibitor that suppresses tumor neo-angiogenesis by targeting VEGF, exacerbates tumor hypoxia [
7]. And it has been found that clinically resistant hepatocellular carcinoma exhibited increased intratumoral hypoxia compared to pretreatment or sorafenib-sensitive hepatocellular carcinoma [
8]. Alternatively, alleviating hypoxia appears to be a more promising strategy. Malignant tumor cells constantly adapt to hypoxic environments, a process mediated by a class of transcriptional activators called hypoxia-inducible factors (HIFs) [
9]. One of the major isoforms, HIF-1α, is closely associated with tumorigenesis, progression, invasion, and metastasis, as up-regulated HIF-1α expression activates various downstream target genes [
10], including activation of glucose transporter proteins GLUT1 and GLUT3 to promote glycolysis and VEGF to facilitate angiogenesis [
11,
12]. Hence, inhibition of HIF-1α has been considered an efficient approach to relieve hypoxia [
13].
Given current challenges, we envision that integrating small interfering RNA (siRNA)-targeting HIF-1α (siHIF-1α), which silences the specific gene by RNA interfering, with SF can synergistically reconstruct TME by overcoming the hypoxia and angiogenesis to improve tumor treatment outcomes. However, due to their different properties, it requires a brilliant delivery system for co-delivering siRNA and small molecular drugs.
Poly-lysine and cell-penetrating peptides are common non-viral gene delivery vectors with a well-defined architecture, high density of surface charges, and good nucleic acid binding ability [
14,
15]. Nevertheless, cationic peptides have abundant positive charges that can cause severe cytotoxicity, and they are serum intolerant, easily forming large aggregates with proteins and other substances in the serum, resulting in reduced transfection efficiency [
16]. The optimization of cationic carriers by architecture changes (e.g., branching, dendritic, or stellate), surface modifications (introduction of hydrophobic, and hydrophilic, fluorinated units, etc.), and the introduction of responsive linkers (disulfide bonds, ester bonds, etc.) can improve gene transfection efficiency and reduce cytotoxicity [
17,
18,
19,
20]. Especially, as in our previous study, fluorination modification of the peptide was a facile strategy to improve serum tolerance and promote cellular uptake and endosome escape, thereby improving transfection efficiency [
21,
22].
Coincidentally, it has been reported that fluorine-containing drugs can be loaded into fluorinated materials via interactions between fluorine atoms [
23]. In this way, sorafenib-containing fluorine atoms can be loaded into the fluorinated peptide through F-F interactions. More intriguingly, perfluorocarbons (PFC) have unique oxygen storage and transport properties [
24]. For example, Liu et al. applied erythrocyte membranes wrapped with PFC and poly(lactic-co-glycolic acid) to form nanoscale artificial red blood cells that could effectively transport oxygen to the tumor site after intravenous injection [
25].
Herein, a perfluorocarbon was linked to a dendritic poly-lysine (DEN) conjugated cell-penetrating peptide (TAT) through a disulfide bond to form a fluorinated peptide (DEN-TAT-PFC), which was employed for co-delivering SF and siHIF-1α, aiming at both vascular normalization and alleviating hypoxia for TME reconstruction, consequently combined gene therapy and chemotherapy synergistically enhances anti-tumor efficacy. PFC not only served as a component to enhance the efficiency of the peptide-based gene delivery vector and had a high affinity for the fluorinated drug SF, but also potentially played a role in this system by delivering oxygen and synergistically regulating the tumor hypoxic microenvironment (
Scheme 1). The findings can provide references for constructing a co-delivery system for siRNA and fluorinated drugs and a targeting-hypoxia therapeutic strategy for tumor treatment.
2. Materials and Methods
2.1. Reagents and Materials
Bankpeptide Co., Ltd. (Hefei, China) custom-make the peptide (DEN-TAT) (sequence, K4K2KGRKKRRQRRRPPQC). 2-(2-Pyridyldithio)ethylamine hydrochloride (HY-101794-50) was purchased from MedChemExpress (Shanghai, China). Perfluorooctanoyl chloride and Cobalt chloride (CoCl2) were from Sigma-Aldrich (St. Louis, MO, USA). TrypLETM Express, Opti-MEM®, and HEPES buffer were from Gibco (Waltham, MA, USA). Lipo8000™, DTT, Agarose, TBE buffer, LysoTracker Green, 100 × Hoechst 33342, Calcein AM cell activity assay kit, and Propidium iodide were obtained from Beyotime (Shanghai, China). Triton X-100 was purchased from Solarbio (Beijing, China). Matrigel® matrix was from Corning (New York, NY, USA). Sorafenib was obtained from CSNpharm (Arlington Heights, IL, USA). Glutathione was purchased from Adamas-beta (Shanghai, China). DMOG was obtained from TCL. β-Actin antibody, HIF-1α antibody, and secondary antibody were purchased from Proteintech (Rosemont, IL, USA). siRNA-targeting VEGF (siVEGF) (anti-sense strand: 5′-GAUCUCAUCAGGGUACUCCdTdT-3′, sense strand: 5′-GGAGUACCCUGAUGAGAUCdTd-3′), siRNA-targeting HIF-1α (siHIF-1α) (sense strand: 5′-CGAUCAUGCAGCUAC UACAdT dT-3′; anti-sense strand: 5′-UGUAGUAGCUGCAUGAUCGdTdT-3′), Cyanine 5 labeled siRNA (Cy5-siRNA), and negative control scrambled siRNA (siNC) were all from Genepharma (Shanghai, China).
2.2. Synthesis of Fluorinated Peptide DEN-TAT-PFC
First, as in our previous study [
21], N-(2-(2-pyridyldithio)ethyl)perfluorooctanamide (PFC) was produced. Then, the peptide DEN-TAT (5 mg, 1.76 μmol) was dissolved in distilled water containing 0.1% TFA, and PFC (1.2 mg, 2.06 μmol) was dissolved in acetonitrile containing 0.1% TFA, then the two solutions were mixed in equal volumes and reacted at room temperature (RT) for 4 h. The product was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) (
Figure S1) (YMC, Kyoto, Japan—Triart C18, S-5 µm, 10 × 250 mm, solvents: acetonitrile–water mixtures (0.1% (
v/
v) TFA), flow rate: 2.0 mL/min,) and the detailed solvent gradient was shown in
Table S1. The separated DEN-TAT-PFC was freeze-dried with an ultra-low temperature freeze dryer for 48 h to obtain the desired product. Finally, the identity and purity of the obtained product were characterized by mass spectrometry ESI-MS and HPLC, respectively.
2.3. Gel Shift Assay
The siRNA compaction ability at various vectors/siRNA molar ratios (0, 2, 5, 10, 20, 50, and 100, 7.5 pmol siRNA) was evaluated by gel retardation assay. After dilution with 20 μL of the complexes in 20 mM HEPES buffer pH 7.2 (prepared with nuclease-free water), the vectors and siRNA were gently pipetted and incubated for 30 min at RT. The prepared 20 μL nanocomplexes were then gently mixed with 4 μL of 6 × DNA loading buffer, and the samples were loaded on a 1% agarose gel followed by electrophoresis at 100 V for 50 min. The gel was stained with ethidium bromide (EB) and observed by the iBright FL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
2.4. Serum Protection Assay
Scrambled siRNA-loaded DEN-TAT-PFC complexes prepared at a vector/siRNA molar ratio of 50 were selected for a serum protection assay. Complexes containing 7.5 pmol siRNA were incubated with 50% fetal bovine serum (FBS) for 0, 0.5, 2, 4, 6, and 24 h at 37 °C. A quick addition of 1 µL of 0.5 M EDTA was made to stop any nuclease activity. The siRNA remaining in the complexes was displaced with 1 µL of 50 μg/µL heparin to ensure the complete release of siRNA. Naked siRNA acted as a control and was treated in the same manner. As described above, the released siRNA was visualized by 1% agarose gel electrophoresis stained with EB.
2.5. siRNA Release Assay
To evaluate the siRNA release capacity, DEN-TAT-PFC/siRNA complexes formed with 7.5 pmol scrambled siRNA at a molar ratio of 50 were incubated at RT for 15 min with a 5 µL solution containing 0.5, 1, 2, 5, and 10 µg of heparin sodium. As a reference, 5 µL of nucleic acid-free pure water was added. In addition, the release profiles of siRNA from DEN-TAT-PFC/siRNA complexes (with 7.5 pmol scrambled siRNA at a molar ratio of 10) in reductive condition were further evaluated by gel electrophoresis assay after incubation with DTT (20 mM) for 4 h at 37 °C.
2.6. Assembly and Characterization of Vector/siRNA Complexes
DEN-TAT/siRNA and DEN-TAT-PFC/siRNA complexes were prepared by mixing siRNA and DEN-TAT or DEN-TAT-PFC gently at molar ratios of 50, 100, and 200 in HEPES buffer with 40 pmol siRNA to a final volume of 200 μL, and incubated for 30 min at RT before use. DEN-TAT-PFC/SF/siRNA complexes were prepared according to a DEN-TAT-PFC/siRNA molar ratio of 75, a siRNA content of 40 pmol, a final concentration of 25 μM of SF, and a final volume of 200 μL. First, the carrier and SF were gently pipetted and mixed well. Let it stand at RT for 30 min, then add siRNA and ultrapure water (nuclease-free, sterile) to the mixture, gently pipetted to mix evenly, and again let it stand for 30 min at RT to obtain DEN-TAT-PFC/SF/siRNA nanocomplexes. Next, the size distribution, as well as the zeta potential of complexes, was assessed by a dynamic light scattering (DLS) analyzer (Zetasizer, Malvern, UK).
2.7. Oxygen-Carrying Capacity Measurement
First, 3 mL of DEN-TAT and DEN-TAT-PFC solutions (2 mg/mL) was placed in a 5 mL centrifuge tube, and nitrogen was introduced into the solution to expel oxygen dissolved in the solution. The oxygen contents in these solutions were detected as a control. Next, oxygen was introduced into the solution until it was saturated with oxygen, and then ultrapure water, rapidly cooled after boiling, was added, and the change in dissolved oxygen contents in the solution was detected by a portable dissolved oxygen meter and monitored continuously for 10 min.
2.8. Transmission Electron Microscopy
The morphology of DEN-TAT-PFC/SF/siRNA complexes was observed by TEM (Jeol Ltd., Tokyo, Japan—Jem-f200). The sample solution was dropped on the TEM grid. After a few minutes, the excess solution was blotted away with filter paper. Then, 0.5% (w/v) of phosphotungstic acid was placed on the above grids. The grid was dried at RT for several minutes before observation.
2.9. Determination of Encapsulation Rate and Drug Load Efficiency of DEN-TAT-PFC/SF/siHIF-1α Nanocomplexes
HPLC was used to analyze the EE (%) and DLE (%) of nanocomplexes. The obtained nanocomplexes were centrifuged at 4 °C at 143,000×
g for 15 min to remove free SF. The free SF concentration was determined using HPLC. Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm, Dikma, Beijing, China); Mobile phase: acetonitrile: water = 75:25 (
v/v); Detection wavelength: 265 nm; Column temperature: 25 °C; Flow rate: 1.0 mL/min; Injection volume: 30 μL. The encapsulation efficiency (EE %) and drug load efficiency (DLE %) of nanocomplexes were calculated as follows:
2.10. In Vitro Release Profile of DEN-TAT-PFC/SF/siHIF-1α Nanoparticles
In vitro SF release from DEN-TAT-PFC/SF/siHIF-1α nanocomplexes was analyzed by dialysis. The prepared nanocomplexes were added to dialysis bags (molecular weight cut-off: 3500 Da) and then immersed in 11 mL PBS (pH 7.4) with or without 10 mM GSH containing 0.5%
w/v Tween 80. Different sets of dialysis bags were placed in a 37 °C thermostatic shaker and shaken at 90 g. Then, 300 μL of dialysate was removed at preset time points of 0 h, 1 h, 4 h, 10 h, 24 h, 48 h, and 72 h, respectively, and replenished with equal amounts of the fresh-release medium. After the sampling was completed at each point, the solution in the dialysis bag was added to the same volume of methanol, the demulsification was carried out by ultrasound for 10 min, and 300 μL of the same sample was taken to calculate the total drug content. HPLC analysis was performed on samples from the above sampling points and demulsification samples. The conditions of HPLC analysis were the same as that in
Section 2.9. The in vitro release curve of SF was plotted according to the sampling time point and the percentage of total drug content.
2.11. Hemolysis Assay
The hemolysis rate test was performed according to the following steps. First, 10 mL of fresh rat blood was taken into a vacuum blood collection tube coated with sodium heparin, mixed evenly by inverting slightly, centrifuged at 1000×
g for 10 min after mixing, and then the supernatant was discarded. The above red blood cells (RBCs) were resuspended with PBS (about 10 times the volume of RBC) and centrifuged at 888 g for 10 min, washed 3 times until the supernatant was clear, and diluted with PBS to make a 2% RBC suspension. Then, 20 μL of 50 μM, 100 μM, and 200 μM peptides were added to 180 μL of 2% RBC suspension, which was mixed gently and incubated in a 37 °C water bath for 1 h, then centrifuged at 1065×
g for 5 min. The optical density (OD) of the supernatant was read at 545 nm using enzyme labeling. The positive control (100% lysis) was a blood/Triton X-100 mixture, and the negative control (0% lysis) was a blood/PBS mixture. The hemolytic ratios of the samples were calculated as follows:
2.12. In Vitro Cytotoxicity
The cytotoxicity of vectors was evaluated against HepG2 and LO2 cells using MTT Assay. The cells were seeded in 96-well plates so that the cell density could reach around 60% after 24 h. Residual serum was washed away with sterile PBS, and 100 μL of Opti-MEM medium containing various formulations (different concentrations of vectors or different complexes) was added. After 4 h, 100 μL of 10% DMEM medium and 10 μL of FBS were added. Cells were continued to be cultured for 24 h or 48 h. Then, 10 μL of MTT solution (5 mg/mL) was added on a clean table protected from light and incubated in a cell incubator at 37 °C for 4 h, and blue-violet formazan crystals appeared at the bottom of the plate. The medium was carefully aspirated, then 150 μL of DMSO was added per well and incubated at 37 °C, 50 g for 10 min until the crystals were completely dissolved. The absorbance (A) of each well at 570 nm was measured with a microplate reader, and the cell viability was calculated as follows:
2.13. In Vitro Cell Uptake and Intracellular Tracking Study
The cells were seeded in 96-well plates to ensure the cell density could reach about 70–80% after 24 h. Complexes with various vectors/siRNA molar ratios were added to the cells in the Opti-MEM medium at the Cy5-siRNA concentration of 50 nM per well or in the 10% FBS medium at the Cy5-siRNA concentration of 100 nM per well. After incubation at 37 °C for 4 h, cells were washed three times with 200 μL FACS buffer (0.5 mg/mL heparin sodium, 2% (v/v) FBS in PBS) and once with PBS. Next, 25 µL TrypLETM was added to each sample and incubated for 5 min at 37 °C. Following that, 150 µL FACS Buffer was introduced to every well to obtain the cells, and then the cell uptake efficiency of Cy5-siRNA was assessed by flow cytometry (Accuri, Ann Arbor, MI, USA—C6, BD with FL4 detector (640/675 nm)).
The cell uptake was also evaluated by confocal laser scanning microscopy (CLSM). HepG2 cells were seeded into a confocal dish, and various Cy5-labeled siRNA-loaded nanocomplexes at the molar ratio of 100 (Cy5-siRNA 100 nM per well) were added. After incubation at 37 °C for 4 h, cells were washed with FACS buffer three times and once with PBS to remove the nanocomplexes that were not taken. The lysosomes and nucleus were stained with LysoTracker Green (200 nM) and Hoechst 33342 (1×), respectively, and then the cells were observed by CLSM.
2.14. In Vitro Gene Silencing
HepG2 cells were seeded in a 12-well (mRNA extraction) or 6-well (protein extraction) tissue culture plate and cultured overnight. Then, the complete medium was replaced by Opti-MEM medium or 10% FBS medium, followed by the addition of various complexes (with the siVEGF or siHIF-1α concentration of 100 nM per well at a molar ratio of 75 and SF concentration of 25 μM per well). Lipo8000TM was used following the protocol as a control group. After incubation for 4 h, CoCl2 (100 μM) was added (HIF-1α mRNA and protein extraction) and further cultured for 48 h in serum-containing DMEM. HepG2 cells without treatment were used as a negative control. The expression of VEGF and HIF-1α were determined at mRNA (expression of VEGF and HIF-1α) and protein (expression of HIF-1α) levels by quantitative real-time PCR and Western blot assay as described below.
2.15. Quantitative Real-Time PCR
A Steady Pure Quick RNA Extraction Kit (Code No. AG21023) was used to isolate and purify total RNA from samples. Reverse transcription was carried out to create complementary DNAs (cDNAs) employing a reverse transcription kit (Code No. AG11728, Evo M-MLV RT Mix Kit with gDNA Clean), according to manufacturer’s instructions. Subsequently, qPCR experiments were conducted using the SYBR
® Green Premix Pro Taq HS qPCR Kit (Rox Plus) (Code No. AG11718-S; Accurate Biotechnology Co., Ltd, Hunan, China) on Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA—Step One Plus
TM Real-Time PCR System) as directed by manufacturer’s instructions. A separate real-time PCR using primers for detecting GAPDH (expression of VEGF) and β-actin (expression of HIF-1α) was used as a control. All PCR experiments were performed in triplicate, and the expression levels of genes were calculated against the GAPDH or β-actin. Specific primer sequences for VEGF, HIF-1α, GAPDH, and β-actin are listed in
Table S2.
2.16. Western Blot Assay
Cells were washed once with cold PBS and then resuspended in RIPA lysis buffer (200 μL), freshly supplemented with complete protease inhibitor cocktail tablets and DMOG (1 mM). The lysates were sonicated until the lysate was clear and non-viscous. Total protein was separated on a 6% Bis-Tris-polyacrylamide gel and then transferred to PVDF membrane at 100 V. After incubation in 5% skimmed milk in 1 × TBST for 1 h, the membrane was incubated in 5% skimmed milk in 1 × TBST with a monoclonal antibody against HIF-1α (1:3000) overnight. The membrane was further incubated in 5% skimmed milk with goat anti-rabbit IgG (1:3000) for 1 h. Immunoreactive complexes were visualized using ECL reagents. β-actin (1:10,000) was used as a loading control.
2.17. In Vitro Angiogenesis Assay
A tube formation assay was employed to assess the effect of various formulations on human umbilical vein endothelial cells (HUVEC) vascularization. Liquefied Matrigel matrix was first placed into 48-well plates (150 µL per well), followed by 30 min incubation at 37 °C for solidifying. HUVECs were resuspended with the supernatants of HepG2 cells (condition medium) after transfection with different formulations (siRNA concentration in each well was 100 nM, the molar ratio of vectors to siRNA was 75), then the cells were added into the solidified Matrigel matrix, and incubated for a further 2~4 h. Images of tubule formation were observed by an inverted bright field microscope, and the total length (TL) was processed by Image J (2.3.0/1.53q) software. The rate of tubule structure formation was calculated using the following formula:
2.18. Statistical Analysis
Data were expressed as mean ± standard deviation (SD) for at least three independent experiments. The differences in each group were statistically analyzed by one-way or two-way analysis of variance (ANOVA) by GraphPad Prism 9. When p < 0.05, the results were regarded as statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
4. Conclusions
In conclusion, a well-designed and well-defined fluorinated cell-penetrating peptide DEN-TAT-PFC was synthesized to co-deliver siHIF-1α and sorafenib. Compared to non-fluorinated peptides, DEN-TAT-PFC exhibited an enhanced siRNA binding ability, GSH-responsive release, facilitated cellular uptake, endosome escape, serum-resistance ability, and oxygen-carrying capacity. Meanwhile, the biocompatible DEN-TAT-PFC was suitable for co-loading siHIF-1α and sorafenib to assemble cationic nanoscale complexes, which could effectively knockdown HIF-1α both at mRNA and protein levels and further suppress VEGF expression, which could realize TME reconstruction with both vascular normalization and hypoxia-relieving. Consequently, DEN-TAT-PFC/SF/siHIF-1α exhibited favorable anti-tumor cell proliferation and anti-angiogenesis via the synergetic effect. Altogether, DEN-TAT-PFC is a promising carrier for siRNA and fluorine-containing drugs for tumor treatment with targeting hypoxia.