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 t....

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 ....

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