Development and Efficacy of Enzyme-Responsive Squalene-Chidamide Nanoparticles for Pancreatic Cancer

Summary

This protocol details the synthesis and characterization of folic acid-modified squalene-chitosan nanoparticles, evaluates their enzyme-responsive drug release and cellular uptake in vitro, and applies them to enhance pancreatic cancer therapy via targeted delivery.

Abstract

Here, we present a protocol to address the limited intratumoral penetration of small-molecule drugs caused by the dense extracellular matrix (ECM) of pancreatic ductal adenocarcinoma (PDAC). Prodrugs offer great potential to overcome this challenge by enhancing drug penetration and tumor-killing efficacy. Squalene (SQ), a natural precursor for cholesterol biosynthesis with excellent biosafety and biocompatibility, can improve the membrane compatibility of hydrophilic drugs and enhance their cellular uptake when conjugated to chemotherapeutic agents or bioactive small molecules. In this study, we developed a novel lipophilic SQ-based prodrug system: the hydrophilic anticancer drug chidamide (CHI) was conjugated to SQ via an amide bond -- a linkage responsive to pancreatin and cathepsin B (key enzymes overexpressed in the PDAC microenvironment). This conjugation yielded an amphiphilic SQ-CHI prodrug, which was further self-assembled into folate (FA)-modified nanoparticles (FA-SQ-CHI NPs). The optimized NPs exhibited a uniform hydrodynamic diameter of 173.3 ± 1.5 nm, a polydispersity index (PDI) of 0.181 ± 0.18, a high drug loading capacity of 59.0% ± 0.77%, and a stable Zeta potential of -13.10 ± 0.86 mV. In vitro release studies showed that the NPs achieved 80.2% ± 4.22% cumulative drug release within 72 h in the presence of 0.25% pancreatin, while only 33%-38% release was observed in pH-adjusted buffers (pH 4.5 or 7.4) without enzymes. Cellular uptake assays confirmed that FA modification significantly enhanced intracellular delivery efficiency, with 1.8-2.3-fold higher fluorescence intensity in PDAC cells (PSN-1 and CFPAC-1) compared to non-targeted NPs at 12-24 h. The current protocol provides a comprehensive methodology for the synthesis and characterization of the prodrug, in vitro evaluation of enzyme-responsive release kinetics, and comparative analyses of therapeutic efficacy and tissue penetration, highlighting the nanocarrier's core advantages of targeted delivery, high drug loading, and enzyme-triggered controlled release.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor of the digestive tract, with an incidence rate nearly equal to its mortality rate, and a 5-year survival rate of only 8%-9%¹. A complex tumor microenvironment (TME) is the hallmark feature of PDAC². It is primarily composed of an abnormal extracellular matrix (ECM), activated cancer-associated fibroblasts (CAFs), and a profoundly immunosuppressive cellular milieu. The PDAC ECM is rich in type I collagen. Both its altered content and abnormal architecture promote tumor progression and are associated with poor prognosis³. CAFs are the most abundant stromal cells and consist of functionally distinct subtypes, such as matrix-producing myCAFs and inflammatory iCAFs, which exhibit phenotypic plasticity⁴.

Immunologically, the PDAC TME is dominated by pro-tumor myeloid cells. Anti-tumor lymphocytes are scarce or dysfunctional. Mature B lymphocytes also contribute to immunosuppression^3,4. Collectively, these components establish multiple therapeutic barriers. The dense stroma, characterized by high interstitial fluid pressure, creates a significant penetration barrier that severely impedes the delivery of therapeutic agents into the deep tumor regions, thereby substantially limiting the clinical efficacy of chemotherapeutic drugs⁵. Consequently, developing drug delivery systems capable of overcoming this stromal barrier and penetrating deeply into pancreatic tumor tissue is critically important.

In recent years, squalene (SQ), a natural precursor for cholesterol biosynthesis, has attracted considerable attention due to its excellent biosafety and biocompatibility⁶. Being highly hydrophobic, SQ can be conjugated with hydrophilic drugs to form amphiphilic prodrugs, thereby improving the biocompatibility of hydrophilic small-molecule drugs with cell membranes and enhancing their cellular uptake efficiency⁷. Previous studies have demonstrated that SQ can be covalently conjugated with various chemotherapeutic agents, such as cisplatin, paclitaxel, and gemcitabine, forming prodrug nanoparticles with high drug loading capacity, which improves drug delivery to solid tumors^8,9. These properties underscore the broad application potential of SQ in drug delivery system development.

To enhance tumor targeting, this study employs folic acid (FA) as a targeting ligand. FA is a water-soluble vitamin crucial for one-carbon metabolism, biosynthesis¹⁰, redox homeostasis¹¹, and methylation reactions¹². Conjugation of FA via its carboxyl groups to the nanocarrier enables specific recognition and binding to the folate receptor (FR), followed by receptor-mediated endocytosis¹³. By leveraging the enhanced permeability and retention effect of tumor cells, Zheng et al.¹⁴ evaluated folate receptor α (FRα) expression in pancreatic cancer, normal pancreas, adjacent tissues, and chronic pancreatitis using Western blotting and immunohistochemistry, and analyzed its correlation with clinicopathological features. FRα was expressed in 94.7% (72/76) of pancreatic cancer cases, correlating with metastasis, but was absent in normal pancreatic tissues. FA-modified nanoparticles can selectively accumulate in FR-positive tissues, enabling molecularly targeted therapy.

Chidamide (CHI) is a novel histone deacetylase inhibitor. Preclinical studies have demonstrated that it also exhibits potent antitumor activity against solid tumors such as pancreatic and breast cancer, and can reverse drug resistance in tumor cells^15,16. However, its strong hydrophilicity is associated with low penetration efficiency into the tumor microenvironment and poor distribution within solid tumor tissues, which severely limits its clinical therapeutic potential for pancreatic cancer⁵. Using chidamide (CHI) as a model drug, this study proposes the preparation of self-assembled nanoparticles via the conjugation of CHI with SQ and subsequent modification with FA. This strategy aims to enhance the antitumor efficacy of CHI against pancreatic cancer by overcoming its poor penetration into the tumor microenvironment and suboptimal distribution within solid tumor tissue. Furthermore, by evaluating the in vitro efficacy of CHI in pancreatic cancer cell lines, this approach is expected to significantly improve the penetration and cytotoxic effects of CHI -- a representative hydrophilic small-molecule drug -- within the pancreatic tumor microenvironment, thereby providing an experimental basis for guiding CHI-based therapy in pancreatic cancer.

Our study elaborates on the experimental procedures for preparing CHI-SQ-PEG-FA self-assembled prodrug nanoparticles for the treatment of pancreatic cancer. Specifically, we describe the optimization and characterization of the preparation process for CHI-SQ-PEG-FA nanoparticles, along with in vitro drug release studies. The process involves: first, optimizing the formulation using a single-factor experimental approach and determining the optimal particle size and drug loading; second, characterizing the nanoparticles using UPLC and MTS assays; and finally, establishing an in vitro co-cultured tumor spheroid model. Using coumarin-6 (C6) as a fluorescent probe in targeted nanoparticles, we further validate the penetration and distribution of the nanoparticles within three-dimensional tumor spheroids and obtain relevant data.

Protocol

1. Synthesis of SQ-CHI and SQ-PEG-FA

1. Synthesis of SQ-CHI: Weigh 120.4 mg (0.3 mmol) of 1,1',2-tris-norsqualenoic acid and dissolve it in 1 mL of N, N-Dimethylformamide (DMF). Sequentially add NHS (46 mg, 0.4 mmol) and EDCI (72 mg, 0.38 mmol) and allow the reaction to proceed for 90 min under nitrogen protection.
2. Dissolve chidamide (58.56 mg, 0.15 mmol) in 1 mL of DMF and add it dropwise to the reaction mixture. Stir the reaction at room temperature under a nitrogen atmosphere for 72 h.
3. After completion, evaporate the solvent under reduced pressure at 5-15 kPa to obtain the crude product. Purify the crude product by flash column chromatography using a gradient elution of DCM/MeOH (99:1, 99:5, 85:5).
4. Structural verification of SQ-CHI: Place the synthesized SQ-CHI product in a glass vial and dry it under vacuum in a desiccator for 24 h. Dissolve the product in approximately 1 mL of deuterated chloroform (CDCl₃) and transfer it into an NMR sample tube. Perform ¹H NMR analysis to confirm the chemical structure of the product¹⁷.
5. Synthesis of SQ-PEG-FA: Dissolve 41.12 mg of 1,1',2-tris-norsqualenoic acid in 35 µL of DMF. Sequentially add N-hydroxysuccinimide (NHS,15.71mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 24.59 mg) to this solution.
6. Allow the reaction to proceed for 90 min under a nitrogen atmosphere. Dissolve 20 mg of FA-PEG-NH₂ in 35 µL of DMF and add it dropwise to the reaction mixture.
7. Stir the resulting mixture at room temperature for 72 h under nitrogen. Purify the crude product with a 3 kDa MWCO ultrafiltration centrifuge tube at 5,200 x g for 30 min to remove unreacted free acid. Place the purified SQ-PEG-FA complex aqueous solution in a glass vial and pre-freeze it at -50 °C. Lyophilize the pre-frozen solution in a freeze dryer to obtain the SQ-PEG-FA complex as a lyophilized powder.

2. Structural verification of SQ-PEG-FA

1. Place the lyophilized SQ-PEG-FA complex in a 2 mL centrifuge tube and further dry it under vacuum in a desiccator for 24 h. Dissolve the product in approximately 1 mL of deuterated dimethyl sulfoxide (DMSO-d₆) and transfer it into an NMR sample tube. Perform ¹H NMR analysis to confirm the chemical structure of the complex.
2. Optimization of preparation and characterization of SQ-CHI-PEG-FA NPs
3. Weigh 6.64 mg of SQ-CHI and dissolve it in 2 mL of absolute ethanol to prepare a 3.32 mg/mL (4.3 µM) stock solution.
4. Add the SQ-CHI ethanolic stock solution (1, 0.2, 0.1, or 0.05 mL) dropwise to 1 mL of deionized water under stirring. Stir for 5 min, then remove ethanol by reduced-pressure evaporation. Sonicate the resulting mixture for 30 min to obtain a CHI-SQ nanoparticle suspension. Measure the hydrodynamic diameter and polydispersity index (PDI) of the nanoparticles with a Zetasizer to identify the optimal formulation¹⁸.
5. Add 0.1 mL of the SQ-CHI ethanolic stock solution dropwise to 1 mL of deionized water after different stirring durations (0, 5, 10, or 20 min). After the respective stirring period, remove ethanol by pressure-reducing evaporation, followed by sonication for 30 min to obtain CHI-SQ nanoparticle suspensions. Measure the hydrodynamic diameter and PDI to determine the optimal stirring time.
6. Add 0.1 mL of the SQ-CHI ethanolic stock solution dropwise to 1 mL of deionized water under stirring for 5 min. After stirring, remove ethanol by pressure-reducing evaporation. Sonicate the resulting mixture for different durations (0, 5, 20, or 30 min) to obtain CHI-SQ nanoparticle suspensions. Measure the hydrodynamic diameter and PDI to determine the optimal sonication time.
7. Confirm the optimal conditions based on the above results. Here, the obtained volume ratio of water to stock solution = 10:1, stirring time = 5 min, and sonication time = 30 min. Repeat all experiments independently in triplicate to ensure reliability.
8. Use the single-factor method while keeping other parameters constant. Sequentially vary the following factors to assess their impact on the particle size distribution and Zeta potential of the nanoparticles: mass ratio of components (SQ-CHI: SQ-PEG-FA = 1:0, 1:0.05, 1:0.5, 1:0.7), phase volume ratio (aqueous phase: organic phase = 1:1, 1:5, 1:10, 1:20), and sonication time (5, 15, 30, 45 min).
9. According to the optimized protocol, mix the SQ-CHI and SQ-PEG-FA stock solutions at the determined ratio. Add this mixture dropwise into deionized water under constant stirring. Continue stirring for 5 min to ensure thorough mixing. Remove ethanol by pressure-reducing evaporation, followed by probe sonication for 30 min to yield the final CHI-SQ-PEG-FA nanoparticle suspension.
10. Measure the hydrodynamic diameter, size distribution (PDI), and Zeta potential of the nanoparticles using dynamic light scattering with a laser particle size analyzer. Observe the morphology of the nanoparticles by transmission electron microscopy (TEM, 150,000x magnification) and scanning electron microscopy (SEM, 80,000x magnification).
11. Determination of drug loading content by ultra-performance liquid chromatography (UPLC)
12. Set the chromatographic conditions as follows: C18 column (2.1 mm x 50 mm, 1.7 µm); mobile phase: 0.1% formic acid aqueous solution - acetonitrile (75:25, v/v); detection wavelength: 258 nm; flow rate: 0.2 mL/min; column temperature: 23 °C; injection volume: 5 µL; run time: 10 min.
13. Reconstitute the CHI-SQ-PEG-FA NP suspension and centrifuge it at 7,500 x g for 30 min. Collect the supernatant and filter it through a 0.22 µm microporous membrane. Use the resulting filtrate as the test solution. Inject and analyze the test solution under the specified chromatographic conditions. Calculate the drug loading content of the nanoparticles based on the measured CHI concentration in the filtrate.

3. In vitro drug release study

1. Accurately pipette a 2 mL aliquot of either a freshly prepared CHI stock solution or the CHI-SQ-PEG-FA nanoparticle suspension into a dialysis bag with a molecular weight cutoff of 3 kDa.
2. Immerse the dialysis bag in the release medium containing different concentrations of pancreatin (0%, 0.05%, 0.1%, 0.25%). At predetermined time intervals (0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, 72 h), withdraw 1 mL samples from the release medium and immediately replace them with an equal volume of pre-warmed fresh medium to maintain sink conditions.
3. Determine the concentration of CHI in all samples using the UPLC method (chromatographic conditions as specified in section 3) and calculate the cumulative drug release.
4. Use ammonium acetate buffer (pH 4.5) as the release medium. Accurately pipette 2 mL of the nanoparticle suspension into a dialysis bag (3 kDa). Add different activity units (0, 0.7, 1.4, 2.8 U) of Cathepsin B directly into the dialysis bag. Immerse the bag in 40 mL of release medium and incubate it in a constant temperature shaker at 37 ± 0.5 °C and 75 rpm. At the predetermined time points (0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, 72 h), withdraw 1 mL samples and immediately replace them with an equal volume of fresh, pre-warmed medium at the same pH to maintain system equilibrium.
5. Filter the collected samples through a 0.22 µm microporous membrane. Analyze the filtrates by UPLC to determine the drug concentration. Calculate the cumulative release percentage of CHI and plot the drug release profiles.
6. Accurately pipette a 2 mL aliquot of either the CHI stock solution or the CHI-SQ-PEG-FA NP suspension into a dialysis bag (3 kDa MWCO). Immerse the dialysis bag in the release medium containing different concentrations of pancreatin (0%, 0.05%, 0.1%, 0.25%). At the predetermined time points (0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, 72 h), withdraw 1 mL samples and immediately replace them with an equal volume of fresh, pre-warmed release medium to maintain sink conditions.
7. Quantify the drug concentration in the samples by UPLC. Calculate the cumulative drug release and generate the release curves.
8. Evaluation of nanoparticle stability in PBS
9. Mix the CHI-SQ-PEG-FA NPs with PBS (pH 7.4) at a ratio of 1:20 (v/v). Incubate the mixture in a constant temperature shaker at 37 ± 0.5 °C and 100 rpm. Take samples at 1, 3, 6, 12, 18, 24, 30, 36, 42, 48, 72, 96 h to measure the hydrodynamic diameter and Zeta potential.
10. Cell proliferation assay by MTS method
11. PSN-1 pancreatic cancer cells were cultured in complete RPMI-1640 medium. Cells in the logarithmic growth phase were harvested by digestion with 0.25% trypsin at 37 °C and resuspended to a density of 4-6 x 10⁴ cells/mL in complete medium. The suspension was seeded into 96-well plates at 100 µL per well, and pre-culture them for 2 h in a 37 °C, 5% CO₂ incubator to allow for complete cell adhesion.
12. Aspirate the original culture medium. Set up a control group (blank culture medium) and multiple concentration gradients of chidamide groups (0.2, 2, 8, 10, 20, 80 pM), with three replicate wells per group. Add 100 µL of blank or drug-containing medium to each well, then transfer the cell plate to the incubator for continued culture. Treat the experimental groups with 100 µL of fresh medium containing varying concentrations (0.2, 2, 8, 10, 20, 80 pM) of SQ-PEG, free CHI, CHI-SQ-PEG NPs, or CHI-SQ-PEG-FA NPs, while the control group receives an equal volume of blank medium. After further incubation for 96 h, remove the drug-containing medium from each well.
13. Add 20 µL of MTS working solution to each well, followed by incubation for 4 h. Shake the 96-well plate for 15 s to ensure homogeneity, and immediately measure the absorbance (A value) of each well at a wavelength of 490 nm using a microplate reader. Calculate the cell proliferation rate by subtracting the OD value of the blank group from the OD value of the drug-treated group, dividing the result by the difference between the OD value of the blank medium group and the OD value of the blank group, and then multiplying by 100.
14. Establishment of an in vitro co-cultured tumor spheroid model
15. Mix PSN-1 cells in the logarithmic growth phase with HPSC cells at a 2:7 ratio and seed them into ultra-low attachment 96-well plates at a density of 5,000 cells per well. After tumor spheroid formation, treat the spheroids with different nano-formulations (Coumarin 6-labeled CHI-SQ-PEG/C6 NPs and CHI-SQ-PEG-FA/C6 NPs) or DMSO (as a control) and co-incubate them for 48, 96, and 144 h to evaluate drug efficacy.
16. Following incubation, add an equal volume of cell viability assay reagent to each well. Place the plate on an orbital shaker and shake for 15 min to ensure complete lysis, followed by incubation at room temperature in the dark for 30 min.
17. Transfer the cell lysate to a black 96-well plate. Measure the chemiluminescence signal at a wavelength of 562 nm using a multifunctional microplate reader to quantify cell viability.
18. Investigation of cellular uptake of nanoparticles
19. Prepare targeted nanoparticles (CHI-SQ-PEG-FA/C6 NPs) and non-targeted nanoparticles (CHI-SQ-PEG/C6 NPs) incorporating coumarin-6 (C6) as a fluorescent probe. Routinely culture PSN-1 cells in the logarithmic growth phase for 24 h to form a monolayer. Dissolve coumarin 6 in ethanol to prepare a 200 µg/mL stock solution. Mix 10 µL of the solution with SQ-CHI and SQ-PEG-FA (or SQ-PEG) at a 1:0.7 ratio, then inject dropwise into deionized water under stirring. Stir for 5 min, remove ethanol by evaporation, and sonicate for 30 min. Finally, purify via ultrafiltration (3 kDa, 12,000 rpm, 20 min) to obtain the items.
20. Treat the cells as follows: the experimental groups receive serum-free medium containing free C6, CHI-SQ-PEG/C6 NPs, or CHI-SQ-PEG-FA/C6 NPs (with a final CHI concentration of 5 µmol/L and a C6 concentration of 2.6 µmol/L), while the control group receives an equal volume of blank medium.
21. After incubation for 4 h, 12 h, and 24 h, aspirate the drug-containing medium. Stain cell nuclei using 4',6-diamidino-2-phenylindole (DAPI)¹⁹.
22. Conduct observations under a fluorescence microscope (40x, excitation wavelength Ex=360 nm, emission wavelength Em=477 nm). Assess the cellular uptake of nanoparticles by measuring the fluorescence intensity of coumarin-6.

4. Statistical analysis

1. Perform all experiments with a minimum of 3 replicates to obtain the data presented in this study. Analyze statistics using GraphPad Prism Software. Present experimental data as mean ± standard error of the mean (SD), and compare between groups using Student's t test, one-way ANOVA, or Wilcoxon test where appropriate. Set the threshold for statistical significance at P < 0.05.

Results

This study successfully designed and systematically evaluated a novel targeted nanodrug delivery system -- Chidamide Squalenylation Prodrug-based Self-assembled Nanoparticles (CHI-SQ-PEG-FA NPs; Figure 1). We synthesized CHI-SQ-PEG-FA NPs (Figure 2) and systematically evaluated the effects of various parameters, including the oil-to-water phase ratio (Figure 3A), stirring duration (Figure 3B), and sonic...

Discussion

This study provides a practical step-by-step guide for implementing a low-waist system based on nanoparticle self-assembly. The self-assembled nanoparticles obtained in this study exhibited a regular spherical shape, good dispersion, uniform particle size, high drug loading and encapsulation efficiency, and demonstrated satisfactory stability²¹. A 3D tumor spheroid co-culture model (PSN-1/HPSC) was employed to evaluate nanoparticle penetration, revealing that the folic acid-modified CHI-SQ-PEG-FA ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco (USA)	25200072
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)	Sigma-Aldrich (USA)	E6383
2 mL cryogenic vials	Corning (USA)	430488
96-well cell culture plates	Corning (USA)	3598
absolute ethanol	China National Pharmaceutical Group Corp. (Sinopharm)	100092680
acetone	China National Pharmaceutical Group Corp. (Sinopharm)	32201-2.5L
acetonitrile	Tedia (USA)	A998-4	HPLC grade
ammonium acetate	China National Pharmaceutical Group Corp. (Sinopharm)	10002861
anhydrous sodium sulfate	Sigma-Aldrich (USA)	31481
biological safety cabinet	Thermo Fisher Scientific, USA	1380, 1332, 1356
cell culture flasks	Corning (USA)	CLS430372
cell culture incubator	Thermo Fisher Scientific, USA	HERA51901126
chidamide(CHI)	Shenzhen Microcore Biotech Co., Ltd. (China)		purity >95%
Cytation 5 multimode microplate reader	BioTek, USA	CYT5MV
DF-101S thermostatic magnetic heating stirrer	Zhengzhou Kechuang Instrument Co., Ltd., China
dichloromethane	China National Pharmaceutical Group Corp. (Sinopharm)	80047318
dimethyl sulfoxide(DMSO)	Sigma-Aldrich (USA)	D2650
dioxane	Aladdin (China)	D116156-5ml
DMEM medium	Gibco (USA)	12100046
Dulbecco's phosphate-buffered saline (DPBS)	Gibco (USA)	14040141
ethyl acetate	China National Pharmaceutical Group Corp. (Sinopharm)	10009460
ethyl ether	China National Pharmaceutical Group Corp. (Sinopharm)
FA2004B electronic balance	Shanghai Tianmei Balance Instrument Co., Ltd., China	FA2004B
Fetal bovine serum(FBS)	Gibco (USA)	10099141C
folate-poly(ethylene glycol)-amine (FA-PEG-NH2)	Shanghai Pengshu Biological Technology Co., Ltd. (China)	PS2-NFA-2000
Formic acid	Aladdin (China)	F112034	HPLC grade
freeze dryer	Thermo Fisher Scientific, USA
human cathepsin B	Sigma-Aldrich (USA)	SRP0289
Human pancreatic cancer cell lines (PSN-1, CFPAC-1)	the American Type Culture Collection (ATCC)	CRL-3211, CRL-1918
human pancreatic stellate cells (HPaStec, HPSC)	the American Type Culture Collection (ATCC)
human trypsin	Sigma-Aldrich (USA)
hydrochloric acid	China National Pharmaceutical Group Corp. (Sinopharm)	CFSR-10011008
inverted microscope	Olympus, Japan	IX83
Iscove's Modified Dulbecco's Medium (IMDM)	Gibco (USA)	12440053
IX 1000 vortex mixer	Hangzhou Ruicheng Instrument Co., Ltd., China	IX 1000
methanol	China National Pharmaceutical Group Corp. (Sinopharm)	M433287-10L
methanol	Tedia (USA)	MS1922-001	HPLC grade
microcentrifuge tubes (1.5/2.0 mL)	Axygen (USA)	MCT-150-C
micropipettes (20, 100, and 1000 μL)	Thermo Fisher Scientific, USA	4642050, 4642070, 4642090
Mill-Q water purification system	Millipore, USA	ZR0Q00800
MTS assay kit (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)	Promega (USA)	G3582
N,N-Dimethylformamide (DMF)	Aladdin (China)
N-bromosuccinimide (NBS)	Sigma-Aldrich (USA)	B81255
N-Hydroxysuccinimide (NHS)	Sigma-Aldrich (USA)	56480
Optima Max ultracentrifuge	Beckman, USA	393490
penicillin/streptomycin solution	Gibco (USA)	15140-122
periodic acid	Sigma-Aldrich (USA)	P7875
petroleum ether	China National Pharmaceutical Group Corp. (Sinopharm)
phenazine methosulfate(PMS)	Sigma-Aldrich (USA)	3180806
phosphate-buffered saline (PBS)	Gibco (USA)	70011069
Poly(ethylene glycol)-amine (PEG-NH2)	Shanghai Pengshu Biological Technology Co., Ltd. (China)	PG2-AM-20k
RPMI-1640 medium	Gibco (USA)	31800022
silica gel powder	Sigma-Aldrich (USA)	53698	300 mesh
sodium bicarbonate	Sigma-Aldrich (USA)	401676
sodium chloride	China National Pharmaceutical Group Corp. (Sinopharm)	10019318
sodium dichromate dihydrate	Sigma-Aldrich (USA)	1.93689
Squalene	Sigma-Aldrich (USA)	Y0002131
sterile centrifuge tubes (15 and 50 mL)	Corning (USA)	430791, 430828
sulfuric acid	China National Pharmaceutical Group Corp. (Sinopharm)	10021618
Tetrahydrofuran	China National Pharmaceutical Group Corp. (Sinopharm)	34865-4X4L	analytical grade
ultrafiltration centrifugal tubes (molecular weight cut-off: 3.5 kDa)	Beckman, USA	344088