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

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.

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Ethics Committee in Beijing. The assigned approval number of the laboratory/investigator is AW03019102-3. 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. Apply one microneedle patch to the depilated area every 2 days for 28 days and maintain the patch in place for 30 min.
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.

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 encapsulation and altered surface properties.

Cellular uptake and anti-lipogenic effects in 3T3-L1 adipocytes
Confocal laser scanning microscopy (CLSM) using Cy3-labeled micelles revealed rapid and efficient uptake of nanomicelles into 3T3-L1 preadipocytes within 2 h (Figure 2A). BODIPY staining showed that M(Cap) markedly reduced intracellular lipid droplet accumulation compared with untreated controls and free capsaicin (Figure 2B). Western blot and qPCR analyses demonstrated significant downregulation of the adipogenic transcription factors PPARγ and C/EBPα, together with upregulation of TRPV1, UCP1, and Cyt C (Figure 2C,D). TEM of adipocytes further confirmed an increased number of mitochondria and smaller lipid droplets in M(Cap)-treated cells (Figure 2E).

Fabrication and mechanical properties of microneedle patches
SEM imaging confirmed successful fabrication of microneedle patches with well-defined pyramidal needles (600 µm height, 300 µm base width) arranged in uniform arrays (Figure 3A). CLSM of Cy5-labeled M(Cap) demonstrated homogeneous distribution of nanomicelles throughout the microneedle matrix (Figure 3B). Texture analysis showed that each microneedle exhibited a fracture force >0.15 N, sufficient to penetrate murine skin.

Insertion, dissolution, and release of microneedle patches
In vivo insertion studies using murine abdominal skin were performed, and the microneedles dissolved completely within ~30 min after application. The results showed that the microneedles penetrated the stratum corneum effectively, creating distinct microchannels visualized by H&E staining (Figure 4A,B). In vitro release experiments revealed pH-responsive behavior, with cumulative capsaicin release reaching ~98% at pH 6.5 and ~78% at pH 7.4 after 50 min, mimicking adipose tissue versus physiological pH conditions (Figure 4C).

In vivo anti-obesity effects in HFD-induced obese mice
In HFD-fed obese mice, treatment with M(Cap)-loaded microneedle patches induced marked body weight reduction compared with HFD controls and free capsaicin groups (Figure 5A-C). MRI analysis showed a pronounced decrease in whole-body fat mass (4.7% vs. 15.7% in untreated HFD mice), accompanied by an increase in lean mass (Figure 5D,E). Histological examination of WATs and sBAT (E-WAT: epididymal white adipose tissue; P-WAT: perinephric WAT; iWAT: inguinal WAT; sBAT: shoulder-blade brown adipose tissue) revealed smaller adipocytes and features consistent with adipose tissue browning in the M(Cap) MN group (Figure 5F).

Physiological and molecular impacts of treatment
Metabolic cage studies demonstrated that M(Cap)-treated mice displayed higher physical activity and oxygen consumption, together with a lower respiratory quotient, indicative of enhanced fatty acid oxidation. Immunohistochemistry and Western blotting of iWAT confirmed significant downregulation of lipogenic proteins (PPARγ, C/EBPα) and upregulation of browning-related markers (UCP1, Cyt C, PGC1α) (Figure 6A-D). No evident toxicity was observed in major organs, and serum biochemical parameters remained within normal ranges after treatment.

Figure 1: Characterization of α-lactalbumin nanomicelles (SNS) and capsaicin-loaded micelles (SNS-Cap). (A) TEM images of SNS and SNS-Cap showing uniformly dispersed spherical micelles. Scale bar: 100 nm. (B) Size distribution histograms of SNS and SNS-Cap. This figure has been modified from Bao et al.11. Please click here to view a larger version of this figure.

Figure 2: Characterization of α-lactalbumin nanomicelles (SNS) and capsaicin-loaded micelles (SNS-Cap). (A) CLSM imaging shows efficient intracellular uptake of Cy3-labeled micelles (M(Cy3); nuclei stained with DAPI, scale bar: 20 µm). (B) Fluorescence lipid staining demonstrates reduced lipid accumulation following M(Cap) treatment (scale bar: 200 µm). (C,D) Western blot and qPCR analyzed adipogenic and browning-related genes. (E) TEM imaging shows mitochondrial content and lipid droplets in M(Cap)-treated adipocytes (scale bar: 200 nm). This figure has been modified from Bao et al.11. Please click here to view a larger version of this figure.

Figure 3: Structural characterization and micelle incorporation of dissolvable microneedle patches. (A) Optical and SEM images of the microneedle mold and resulting microneedle (MP) array, showing uniform pyramidal structures (~600 µm height). Scale bars: 600 µm and 1.00 mm. (B) CLSM images of Cy5-labeled nanomicelles distributed within the microneedles and showing microneedle dissolution at body temperature. This figure has been modified from Bao et al.11. Please click here to view a larger version of this figure.

Figure 4: Skin insertion, dissolution, and release behavior of dissolvable microneedle patches. (A) Histological images showing successful microneedle penetration into mouse skin. (B) Representative images of microneedle application, in situ dissolution, and post-removal skin appearance by H&E staining. (C) In vitro cumulative capsaicin release profiles of microneedle patches at pH 6.5 and 7.4. This figure has been modified from Bao et al.11. Please click here to view a larger version of this figure.

Figure 5: In vivo anti-obesity efficacy of capsaicin-loaded microneedle patches in HFD-induced obese mice. (A) Animal treatment protocol. (B,C) Body weight progression and representative mouse images. (D,E) MRI analyses of fat mass and lean mass in treated mice. (F) The representative adipose tissues of different treated mice. This figure has been modified from Bao et al.11. Please click here to view a larger version of this figure.

Figure 6: Physiological and molecular effects of capsaicin-loaded microneedle treatment in obese mice. (A) Physical activity and respiratory quotient (RQ) measurements used to assess energy expenditure following M(Cap) microneedle treatment. (B) Immunohistochemical staining of UCP1 and Cyt C in adipose tissues of the different treatment groups. (C,D) Western blot analysis of adipogenic and browning markers in treated mice (TRPV1, 95 kDa; PPARγ, 53 kDa; C/EBPα, 42 kDa; PGC1α, 130 kDa; UCP1, 30 kDa; Cyt C, 14 kDa). This figure has been modified from Bao et al.11. Please click here to view a larger version of this figure.

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 previous work showing that protein-based nanocarriers improve the bioavailability and cellular penetration of lipophilic nutraceuticals¹¹.

The dissolvable MNs provided robust transdermal delivery. The HA/PVA-based needles showed uniform pyramidal morphology and sufficient mechanical strength for skin penetration, then dissolved within ~30 min, fulfilling key requirements for patient-friendly application. A pH-responsive release profile, with faster capsaicin release at pH ~6.5 than at pH 7.4, favored drug liberation in the adipose tissue microenvironment and helped limit systemic leakage, in line with prior reports on dissolving MNs for controlled delivery^12,20.

In HFD-induced obese mice, M(Cap) MNs significantly reduced body weight, fat mass, and adipocyte size while preserving lean mass. Indirect calorimetry revealed increased oxygen consumption and decreased respiratory quotient, indicating enhanced fatty acid oxidation. Molecular analyses showed downregulation of adipogenic markers (PPARγ, C/EBPα) and upregulation of TRPV1 and browning markers (UCP1, PGC1α, Cyt C), supporting a dual mechanism involving inhibition of lipogenesis and promotion of beige adipogenesis. These improvements exceeded those obtained with free capsaicin or blank MNs, highlighting the benefit of combining nanomicelles with localized MN delivery.

No obvious organ toxicity or serum biochemical abnormalities were observed, underscoring the biocompatibility of the α-lactalbumin/HA/PVA system. Compared with systemic nanocarrier injections, MN delivery confines the drug mainly to subcutaneous adipose tissue, reducing off-target exposure and systemic side effects. This localized, minimally invasive strategy aligns well with current efforts to develop MN platforms for chronic, self-administered therapies^21,22.

Critical steps and key parameters
Several steps strongly influence protocol success. During nanomicelle preparation, α-lactalbumin concentration (6 mg/mL), the BLP enzyme-substrate ratio (1:100, w/w), and hydrolysis conditions (50 °C, 30 min) must be tightly controlled to generate amphiphilic peptides with suitable self-assembly properties. The capsaicin feeding concentration and slow, dropwise addition under gentle stirring are important for achieving high encapsulation efficiency and narrow size distributions. For MN fabrication, the HA/PVA concentrations (each 5% w/v), the M(Cap):polymer ratio (4:1), and centrifugation (1800 × g, 10 min, repeated once) are critical for complete mold filling, homogeneous micelle distribution, and adequate mechanical strength. The centrifugation conditions were selected based on preliminary optimization and our previous work with similar α-lactalbumin nanomicelle-loaded HA/PVA microneedles, where lower forces led to incomplete filling of needle cavities and higher forces or longer times caused polymer overflow and tip deformation^6,11, while 12 h vacuum drying ensures structural integrity and storage stability.

Troubleshooting and protocol modifications
If nanomicelles show aggregation or broad size distributions by TEM/DLS, hydrolysis time, pH, and ionic strength should be re-optimized, and 0.22 µm filtration and adjustment of the capsaicin-to-peptide ratio are recommended. Low loading efficiency can be addressed by increasing the capsaicin concentration within its solubility limit, extending self-assembly time, or applying more gradual solvent removal. During MN casting, bubbles or incomplete cavity filling can be mitigated by longer degassing, slightly higher centrifugation speed/time, or gentle vibration of the mold. Inadequate skin insertion may require increasing needle height or polymer concentration; excessive irritation can be reduced by shortening needles or lowering array density. Patch adhesion issues can often be resolved by better depilation and cleaning of the site, using an adhesive backing, and applying standardized pressure for a fixed period.

Practical usability and throughput
The protocol uses reusable PDMS molds (10 × 10 arrays, 600 µm height) and water-based HA/PVA formulations, which are compatible with batch and potentially semi-automated casting/drying processes. Multiple patches can be fabricated in parallel at relatively low cost, and the workflow could be adapted to higher-throughput production by scaling mold number and integrating temperature- and humidity-controlled drying. For end users, the patches require only a short application (~30 min), generate no sharps waste, and can, in principle, be supplied as pre-dosed, single-use devices with adhesive backing, which would facilitate routine or home-based use and improve treatment adherence.

Limitations and future directions
The present study is limited to murine models, so translation to human obesity requires caution. The 600 µm microneedles used here were optimized for murine abdominal skin and will likely require redesign for human application, where thicker skin and different anatomical sites demand adjusted needle length (e.g., ~700-900 µm), density, and patch size, supported by ex vivo human skin testing and larger-animal studies to confirm penetration depth, drug deposition, and tolerability. In parallel, long-term biocompatibility, immunogenicity, and practical aspects such as patient comfort and feasibility of self-administration should be systematically evaluated. The platform is also adaptable to other hydrophobic nutraceuticals or drugs (e.g., rutin, resveratrol, or anti-diabetic agents), alone or in combination, and comparative studies of cargos, dosing regimens, and application frequency will help define the therapeutic window and broaden indications. Overall, the α-lactalbumin nanomicelle-loaded dissolvable MN system provides a reproducible, tunable platform for localized delivery of hydrophobic bioactives to adipose tissue and holds promise for future development as a practical anti-obesity therapy in humans.