Fabrication of Dissolvable Microneedle Patches Loaded with α-Lactalbumin Nanomicelles for Transdermal Capsaicin Delivery and Adipose Tissue Reduction

Summary

This protocol describes the fabrication of dissolvable microneedle patches incorporating α-lactalbumin nanomicelles loaded with capsaicin. The method offers a reproducible, minimally invasive approach for targeted transdermal delivery of hydrophobic bioactives to adipose tissue, facilitating efficient local fat reduction and potentially expanding applications in obesity management.

Abstract

This protocol outlines the fabrication and application of dissolvable microneedle (MN) patches containing α-lactalbumin nanomicelles for the targeted transdermal delivery of capsaicin to adipose tissue. The process begins with the partial enzymatic hydrolysis of α-lactalbumin, resulting in amphiphilic peptides that self-assemble into nanomicelles encapsulating the hydrophobic compound capsaicin. The capsaicin-loaded nanomicelles are mixed with a hyaluronic acid (HA) and polyvinyl alcohol (PVA) matrix, cast into polydimethylsiloxane (PDMS) molds, and centrifuged to produce 10 × 10 MN arrays. After vacuum drying, the MNs exhibit adequate mechanical strength for skin penetration and dissolve within approximately 30 min of application. Representative results include scanning electron microscopy, which confirms uniform morphology of MNs, confocal microscopy, which verifies skin penetration and drug release, and in vivo experiments in high-fat diet (HFD) mice showing reduced fat mass, enhanced metabolic activity, and induction of adipose browning. This protocol provides a reproducible and minimally invasive method for the transdermal delivery of hydrophobic bioactives, offering a promising therapeutic approach for the management of obesity. This protocol is optimized for reproducibility and is suitable for adaptation to other hydrophobic bioactives.

Introduction

Obesity has become one of the most pressing global health challenges, with prevalence rising steadily in both developed and developing countries. According to the World Health Organization, more than 650 million adults were classified as obese in 2016, and in 2022, approximately 43% of adults aged ≥18 years were overweight, and 16% were living with obesity¹. Obesity is closely associated with metabolic syndrome, cardiovascular disease, type 2 diabetes, and several cancers, imposing substantial socioeconomic and healthcare burdens^2,3. Current therapeutic strategies, including dieta....

Protocol

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Science and Technology Ethics Review Committee of Ludong University (Approval No. LDU-IRB2025020011). The reagents and equipment used are listed in the Table of Materials.

1. Preparation of α-Lactalbumin nanomicelles encapsulating capsaicin ¹²

1. Dissolve α-lactalbumin in deionized water to a concentration of 6 mg/mL and adjust the pH to 7.4. Stir the solution using a magnetic stirrer at 8-28 × g until α-lactalbumin i....

Results

Morphology and characterization of nanomicelles
Transmission electron microscopy (TEM) showed that α-lactalbumin nanomicelles were uniformly dispersed, spherical particles with a narrow size distribution (Figure 1). After capsaicin encapsulation, the mean particle diameter increased from ~22 nm to ~30 nm, indicating successful drug loading into the micellar core. Consistently, the zeta potential shifted slightly from −16.8 mV to −20.3 mV, further confirming efficient enc.......

Discussion

This study demonstrates that dissolvable microneedle (MN) patches loaded with α-lactalbumin nanomicelles encapsulating capsaicin (M(Cap)) can achieve localized, minimally invasive anti-obesity effects. α-Lactalbumin nanomicelles efficiently encapsulated hydrophobic capsaicin, improved its aqueous dispersibility, and enhanced cellular uptake, leading to stronger inhibition of adipogenesis and more prominent mitochondrial biogenesis in 3T3-L1 adipocytes than free capsaicin. These results are consistent with previ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3T3-L1 preadipocytes	ATCC	CL-173	Cell line for adipogenesis studies
Bacillus licheniformis protease (BLP)	Sigma-Aldrich	P5380	Enzymatic hydrolysis of α-lactalbumin
Capsaicin (≥98%)	Sigma-Aldrich	M2028	Active compound to be encapsulated
Centrifuge (≥4000 rpm)	Eppendorf	5810R	To fill molds
Confocal laser scanning microscope (CLSM)	Zeiss	LSM 900	To visualize micelle distribution and penetration
Cy3 fluorescent dye	GE Healthcare	PA23001	For labeling nanomicelles
Cy5 or Cy7 fluorescent dyes	Fanbo Biochemical, Beijing, China	N/A	For biodistribution imaging
Dulbecco’s Modified Eagle’s Medium (DMEM)	Gibco (Thermo Fisher Scientific)	11965-092	For cell culture
Ethanol (≥99.7%)	Sinopharm Chemical Reagent	100092683	Solvent for capsaicin
Fetal bovine serum (FBS)	Gibco (Thermo Fisher Scientific)	16000-044	For cell culture
High-fat diet (60% kcal fat, D12492)	Research Diets, USA	D12492	To induce obesity in mice
Hyaluronic acid (HA, Mw 100–300 kDa)	Aladdin Biochemical Technology Co., China	H108124	Polymer for microneedle fabrication
Isoflurane	RWD, China	R510-22-10	Anesthetic
Metabolic cage system	Panlab, USA	N/A	To assess activity, oxygen consumption, and RQ
Milli-Q Deionized water	Millipore	N/A	Solvent
MRI system	NiuMag, China	MesoQMR23-060H-I	To measure fat/lean body composition
PDMS microneedle molds (10 × 10, 600 μm height)	Blueacre Technology or custom	N/A	Microneedle fabrication
Penicillin–streptomycin	Gibco (Thermo Fisher Scientific)	15140-122	For cell culture
Phosphate-buffered saline  (PBS, pH 7.4)	Thermo Fisher Scientific	10010023	Buffer for in vitro and in vivo studies
Polyvinyl alcohol (PVA, Mw 30–70 kDa)	Aladdin Biochemical Technology Co., China	P109389	Polymer for microneedle fabrication
Scanning electron microscope (SEM)	FEI Tecnai	G2 F30	To characterize microneedle morphology
Texture analyzer	BosinTech, Shanghai, China	TA.XTC-20	To test microneedle strength
Trypan blue solution (0.4%)	Thermo Fisher Scientific	15250061	To visualize microneedle insertion sites
UV–Vis spectrophotometer	PerkinElmer	Lambda 35	For drug release assays
Vacuum oven/desiccator	Thermo Fisher Scientific	N/A	To dry microneedles
Western blotting apparatus	Bio-Rad	Mini-PROTEAN Tetra	Protein analysis
α-Lactalbumin (≥95%)	Sigma-Aldrich	L6010	Protein source for nanomicelle formation