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 dietary restriction, exercise, pharmacotherapy, and bariatric surgery, provide only partial and often transient benefits. These approaches frequently suffer from poor patient adherence, limited long-term efficacy, high invasiveness, and undesirable adverse effects^4,5. Consequently, there is an urgent need for safe, effective, and patient-compliant localized therapies for obesity management.

Natural bioactive compounds have attracted increasing interest as anti-obesity agents due to their multi-targeted actions and generally favorable safety profiles. For example, rutin, a flavonoid glycoside, exhibits anti-lipogenic and metabolic regulatory effects by suppressing adipogenic transcription factors and promoting mitochondrial biogenesis⁶. Capsaicin, the pungent component of chili peppers, has been reported to enhance energy expenditure, induce browning of white adipose tissue, and inhibit lipid accumulation via activation of transient receptor potential vanilloid 1 (TRPV1) signaling^7,8. However, the clinical translation of these compounds is hindered by poor aqueous solubility, low oral bioavailability, rapid metabolism, and, in the case of capsaicin, gastrointestinal irritation upon ingestion⁹.

Nanotechnology-based drug delivery systems offer promising solutions to these limitations. Liposomes, polymeric nanoparticles, and protein-based nanocarriers can markedly improve drug solubility, stability, and cellular uptake¹⁰. In particular, α-lactalbumin, a food-derived milk protein, can be partially hydrolyzed into amphiphilic peptides that self-assemble into nanomicelles capable of encapsulating hydrophobic molecules such as capsaicin¹¹. These α-lactalbumin nanostructures have been shown to enhance the solubility, stability, and bioavailability of diverse hydrophobic bioactives in food and pharmaceutical matrices^12,13. Nevertheless, systemic administration of nanocarriers can still lead to off-target tissue distribution and potential systemic toxicity¹⁴.

Microneedle (MN) technology has emerged as a minimally invasive, patient-friendly platform for transdermal drug delivery. Dissolvable MNs prepared from biocompatible polymers such as hyaluronic acid (HA) and polyvinyl alcohol (PVA) can painlessly penetrate the stratum corneum, dissolve after insertion, and enable precise local drug deposition in targeted tissues^15,16. Combining nanocarriers with MNs further increases drug accumulation in subcutaneous adipose tissue while reducing systemic exposure and side effects¹⁷. Importantly, dissolving HA- and PVA-based MN patches are being advanced toward clinical use for transdermal delivery of vaccines and biopharmaceuticals, demonstrating favorable safety, patient acceptance, and manufacturability at scale¹⁸.

Building on these advances, the present study describes the fabrication of dissolvable MN patches loaded with α-lactalbumin nanomicelles encapsulating capsaicin. In this protocol, α-lactalbumin is dissolved at 6 mg/mL and enzymatically hydrolyzed to generate amphiphilic peptides that self-assemble into capsaicin-loaded nanomicelles. The nanomicelles are incorporated into an HA/PVA matrix (5% w/v each) and cast into polydimethylsiloxane (PDMS) molds to form 10 × 10 MN arrays with a needle height of 600 µm, a configuration that is compatible with standard micro-molding processes and readily scalable to larger, human-sized patches. This integrated design is expected to improve capsaicin solubility and stability, provide pH-responsive release within adipose tissue, and enhance local anti-obesity efficacy in vivo by promoting adipose tissue browning, suppressing lipogenesis, and improving systemic metabolic parameters. By leveraging food-grade α-lactalbumin and clinically advanced dissolving MN materials, this platform offers a translationally relevant, minimally invasive strategy that could be adapted to human-scale patches for self-administered, localized obesity therapy.

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 is completely dissolved (typically 20-30 min).
2. Add Bacillus licheniformis protease (BLP) at an enzyme-to-substrate ratio of 1:100 (w/w).
3. Incubate the mixture at 50 °C for 30 min to generate amphiphilic peptides.
4. Terminate the reaction by heating the solution to 95 °C for 5 min, then cool to room temperature and filter through a 0.22 µm membrane.
5. Dissolve capsaicin in ethanol to a concentration of 1 mg/mL. Add the capsaicin solution dropwise into the peptide solution under gentle stirring.
6. Continue stirring for 2 h to allow self-assembly of nanomicelles.
7. Remove free capsaicin by dialyzing the mixture (MWCO 3.5 kDa) against deionized water for 12 h, replacing the water every 4 h.
8. Freeze-dry (lyophilize) the prepared α-lactalbumin nanomicelles.
9. Characterize the nanomicelles using TEM, DLS, and zeta potential analysis.

2. Fabrication of dissolvable microneedle patches ¹¹

1. Prepare the polymer solution by dissolving HA (5% w/v) and PVA (5% w/v) in deionized water at 60 °C and stirring until the solution becomes clear.
2. Cool the polymer solution to room temperature and mix it with the capsaicin-loaded nanomicelles (M(Cap)) at a 4:1 ratio.
3. Degas the mixture under vacuum in a desiccator connected to a vacuum pump (approximately −80 kPa) for 10 min to remove air bubbles before casting into PDMS molds.
4. Load 200 µL of the mixture into PDMS molds (10 × 10 needle array, 600 µm height).
5. Centrifuge the molds at 1800 × g at RT for 10 min to ensure complete filling of the microneedle cavities.
6. Repeat the filling once, followed by a second centrifugation under the same conditions as in step 2.5, and then dry the molds under vacuum (approximately −80 kPa) at room temperature for 12 h.
7. Carefully peel the microneedle patches from the molds.
8. Examine microneedle morphology by SEM.

3. Mechanical testing of microneedles ¹⁹

1. Place the microneedle patch on the stage of a texture analyzer with the needles facing upward.
2. Compress the patch using a probe at a speed of 20 µm/s until a displacement of 0.7 mm is reached.
3. Record the fracture force and confirm that it is ≥0.15 N per needle to ensure skin penetration capability.

4. In vitro insertion and dissolution test ¹¹

1. Shave the abdominal skin of mice and excise the skin for ex vivo testing.
2. Place the excised skin on an agarose gel support.
3. Press the microneedle patch firmly onto the skin for 30 s.
4. Maintain contact between the patch and the skin for 30 min.
5. Observe needle dissolution.
6. Stain the skin with H&E staining and visualize insertion sites under CLSM.

5. In vitro release study ¹¹

1. Immerse microneedle patches in PBS buffer (pH 6.5 or 7.4) at 37 °C.
2. At defined intervals, withdraw buffer samples and immediately replenish with an equal volume of fresh buffer.
3. Determine the capsaicin concentration in the collected samples using HPLC.
4. Compare the release profiles at different pH conditions using HPLC.

6. In vivo application in HFD-induced obese mice ¹¹

1. Induce obesity in C57BL/6N mice by feeding a high-fat diet (HFD, 60% kcal from fat) for 8 weeks.
2. Randomly divide the mice into five groups: normal control, HFD control, blank micelle microneedle patch (MP-M), capsaicin-loaded nanomicelle injection (InJ-M(Cap)), and capsaicin-loaded nanomicelle microneedle patch (MP-M(Cap)) groups.
3. Depilate the inguinal region of each mouse.
4. Anesthetize mice with inhaled isoflurane using a multi-channel small-animal anesthesia machine equipped with an anesthetic gas scavenging and filtration system. Apply one microneedle patch to the depilated area every 2 days for 28 days, and maintain each patch in place for 30 min.
   NOTE: Before completion of the procedure, residual anesthetic gas in the induction chamber was rapidly cleared by oxygen flushing.
5. Record body weight and food intake every 2 days during the treatment period.
6. Analyze whole-body fat and lean tissue composition by MRI at the end of the treatment.
7. Assess physical activity, oxygen consumption, CO₂ production, and respiratory quotient (RQ) in metabolic cages according to the manufacturer's instructions.
8. Collect iWAT, sBAT, and major organs, fix, embed, section, and stain the tissues with H&E, and evaluate histological changes by light microscopy.
9. Perform Western blotting for TRPV1, PPARγ, C/EBPα, UCP1, and Cyt C using standard procedures.

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