Carboxylated gelatin-based instant dissolvable microneedles with robust mechanical properties and biomolecule stabilization for biomedical applications

Abstract

Gelatin dissolvable microneedle (DMN) patches offer a promising, painless, and rapid transdermal delivery platform. However, conventional DMNs with <5% w/v gelatin exhibit poor mechanical strength and storage stability of biomolecules, while higher concentrations (>5% w/v) hinder dissolvability due to gelation. To address this, we introduced a tailored number of carboxylic groups into the gelatin backbone, generating Modified Gelatin (MG) with improved solubility and reduced viscosity by limiting intra- and intermolecular interactions. MG-DMNs fabricated from MG at a concentration of 10%–20% w/v and ≥5% w/v stabilizing molecules (e.g., trehalose) exhibited rapid dissolution (5 minutes), high mechanical strength (>95 N per patch), and excellent storage stability. Notably, MG-DMNs retained >80% of platelet-rich plasma (PRP) activity after one month of storage at 4 °C and 25 °C, and ∼60% at 40 °C under 75% relative humidity, as confirmed through an in vitro bioassay, an in ovo CAM assay, and in vivo diabetic wound healing studies. MG-DMNs enable the cold-chain-free and stable delivery of biomolecules for biomedical applications.

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1. Introduction

Microneedle (MN) patches represent an innovative and minimally invasive platform for transdermal drug delivery, utilizing micrometer-sized needles to efficiently penetrate the skin layers.^1–3 Depending on the material composition and structural design, MNs can be classified into various types, such as solid, coated, hollow, dissolving, and hydrogel-based microneedles. Among these, instant-dissolving microneedles (DMNs) have gained attention for their ability to rapidly release therapeutic agents in a pain-free manner. Their ease of application and rapid dissolution make them particularly advantageous for delivering biomolecules, such as proteins and nucleic acids, especially in vaccinations and emergency medical treatments.^4–7 Apart from that, they offer several advantages, including painless injections, improved patient compliance, reduced application time compared to hydrogel microneedles, potential for cold-chain independent storage, and the ability to operate without the need for trained healthcare personnel, making them an ideal choice for use in resource-limited settings.^8,9

Biopolymers, such as hyaluronic acid (HA) and proteins like gelatin, albumin, and silk, are commonly used for the fabrication of dissolvable microneedles (DMNs) due to their ability to maintain the stability of incorporated biomolecules from processing and storage-related stress.^10,11 Although HA-based DMNs have been widely studied, they have limited biomedical applications due to their high cost of raw materials and a drastic reduction in mechanical properties when stored at elevated temperatures, high relative humidity, and high drug loading.^12–14 In contrast, gelatin offers a cost-effective raw material and a promising alternative for the fabrication of affordable DMNs.^15–18 However, gelatin-based instant DMNs (without being a composite matrix) remain unexplored for biomolecule delivery mainly due to the inferior mechanical strength (<0.1 N per needle) when made from a <5% w/v concentration, with or without a stabilization matrix such as trehalose.¹⁹ Increasing the concentration of gelatin >5% w/v can improve the mechanical strength (>0.1 N per needle), but it compromises rapid dissolvability due to the possible concentration-driven intermolecular interaction and random coil to helix transformation of gelatin molecules to form a gel.

Most previous studies used high gelatin concentrations or incorporated additives or polymers to achieve sufficient mechanical properties. For instance, Chiu et al.,²⁰ Chen et al.,²¹ and Chen et al.²² used 50% w/v gelatin to fabricate microneedles with sufficient mechanical properties, but this resulted in delayed dissolution of more than 2 hours. In another approach by incorporating additives/polymers, Weijiang Yu et al. reported a gelatin microneedle using 50% w/v gelatin mixed with hydroxyapatite, which achieved a fracture force of 24 N; however, the dissolution time exceeded two hours.²³ While Lee et al. developed a gelatin microneedle by blending 10% w/v gelatin with 10% w/v carboxymethyl cellulose (CMC), which achieved a higher fracture force for effective skin penetration, dissolution was delayed by more than three hours.²⁴ Furthermore, incorporating stabilizing excipients such as trehalose, sucrose, or maltose (>5% w/v) into 5% w/v gelatin formulations drastically decreases the mechanical strength (<0.19 N per needle), necessitating higher gelatin concentrations (>5% w/v) to achieve sufficient strength.^25–28 For instance, Zhang et al. reported that a 5% w/v gelatin–starch DMN failed to penetrate the skin unless modified with gold nanomaterials.¹⁹

These challenges highlight a key unmet need: developing an instantly dissolvable gelatin-based DMN with more than 5% gelatin and stabilizing agents, with robust mechanical strength and superior biomolecule stability at both 4 °C and 25 °C. Achieving this requires minimizing intermolecular interactions and helix structure formation of gelatin at higher concentrations (>10% w/v), thereby maintaining low viscosity for fabrication and ensuring the instant dissolvability of the microneedle. In our previous publication, we demonstrated that introducing a carboxylic group into silk fibroin protein molecules limited β-sheet formation at higher concentrations and instant dissolubility after storage at different temperatures.²⁹ However, silk and gelatin possess fundamentally different protein structures, from primary to tertiary levels. Silk fibroin undergoes a random coil (silk I) to crystalline β-sheet (silk II) transition by hydrogen bonding and hydrophobic interaction between the repetitive units of glycine–alanine–glycine, which improves mechanical strength but reduces solubility. In contrast, gelatin, derived from collagen, typically exists as a random coil in a dilute solution but transitions into a triple helix at higher concentrations, upon cooling, or upon drying via hydrogen bonding between the amine group of glycine and the carbonyl group of proline or hydroxyproline.^17,18 We therefore hypothesised that controlled introduction of carboxylic groups into gelatin could reduce intermolecular interactions, suppress helix formation, and prevent gelation even at higher concentrations. This would yield a low-viscosity solution that is favorable for fabrication while maintaining the instant dissolvability of microneedles. Moreover, the abundant hydroxyl groups of biomolecule-stabilizing excipients may contribute to the robust mechanical strength of MNs by forming hydrogen bonds with the carboxyl groups of modified gelatin molecules.³⁰

In this study, we introduced a tailored number of carboxylic groups into gelatin molecules (modified gelatin (MG)) and validated our hypothesis by demonstrating the instant dissolvability of MNs fabricated with >10% w/v gelatin, without gelation. In this study, we prepared various modified gelatin microneedles with varying concentrations to investigate their mechanical strength. We also examined the stabilization properties of the modified gelatin microneedles by varying the concentrations of stabilizing excipients and by incorporating different molecules (SSD, HRP, and PRP). This MG-based microneedle (MG-DMN) retained predominantly a random coil structure (>50%), with minimal triple helix structure (<10%), by limiting intermolecular interactions.^31,32 As a result, MG enables the fabrication of DMNs with concentrations up to 25% w/v, which exhibited rapid dissolution and robust mechanical strength (>0.45 N per needle). Additionally, the robust mechanical properties of MG-DMNs were attributed to hydrogen bonding between the stabilizing excipients and the carboxyl groups introduced in the gelatin backbone. This strategy enables the use of more than 5% w/v of excipients to enhance the storage stability of biomolecules in microneedles, thereby significantly improving the mechanical properties from 6 N per array of UMG to 90 N per array of MG. We demonstrated the remarkable storage stability of this modified gelatin using model biomolecules, including platelet-rich plasma (PRP) and horseradish peroxidase (HRP). After one month of storage at 4 °C and 25 °C, MG-DMNs retained 80% of model biomolecule activity. Finally, an in vivo diabetic wound model was used to evaluate the storage stability of PRP in one-month stored PRP-loaded MG-DMNs.

2. Materials and methods

2.1. Materials

Gelatin (porcine type A, bloom strength 300), succinic anhydride (SA), and horseradish peroxidase (HRP; 250 U mg⁻¹, RZ >3.0) were obtained from Sigma-Aldrich (USA). o-Phenylenediamine dihydrochloride (OPD), silver sulfadiazine (SSD), and Rose Bengal were purchased from Sisco Research Laboratories Pvt. Ltd (SRL), India. Polydimethylsiloxane (PDMS) microneedle molds, featuring tip diameters of 60 μm, base widths of 300 μm, and varying heights of 400, 500, and 600 μm, were procured from Micropoint Technologies Pvt. Ltd, Singapore. All chemicals and solvents used were of analytical grade.

2.2. Modification of gelatin and preparation of microneedles

2.2.1. Carboxylic acid modification of gelatin. A 10% w/v gelatin solution was prepared in deionized water (DI) with the pH adjusted to 11.5. Succinic anhydride was then gradually added to the gelatin solution at different molar ratios of SA (R = 5 mmol and R = 10 mmol). The pH was maintained above 11 by using 1 M NaOH. The modified gelatin (MG) was dialyzed against deionized (DI) water to remove the unreacted molecules. The dialysed solution was then freeze-dried and stored at −80 °C for further experiments.

2.2.2. Fabrication of gelatin microneedles. To optimize the properties (mechanical strength, instant dissolvability, and stabilization of biomolecules) of the modified gelatin-based dissolvable microneedles (MG-DMNs), multiple microneedles were designed by varying key parameters.

1. Modified gelatin concentration: different concentrations of modified gelatin were employed to enhance the mechanical strength of the microneedles, as summarized in Table 1.

Table 1 Summary of the details of the fabricated, modified, and unmodified gelatin microneedles

Sample name	Composition	Gelatin concentration	Trehalose/sucrose concentration
UMG4.5-DMNs	Unmodified gelatin	4.5% w/v	—
UMG20-DMNs		20% w/v	—
MG20-DMNs	Modified gelatin	20% w/v	—
MG15-DMNs		15% w/v	—
MG10-DMNs		10% w/v	—
UMG4.5-T7.5-DMNs	Trehalose/sucrose incorporated Unmodified gelatin MNs	4.5% w/v	7.5% w/v
UMG4.5-T5-DMNs		4.5% w/v	5% w/v
UMG4.5-T2.5-DMNs		4.5% w/v	2.5% w/v
MG20-T7.5-DMNs	Trehalose/sucrose incorporated modified gelatin MNs	20% w/v	7.5% w/v
MG20-T5-DMNs		20% w/v	5% w/v
MG20-T2.5-DMNs		20% w/v	2.5% w/v

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2. Stabilizing agent concentration: various concentrations of stabilizing agents were incorporated into the MG-DMNs to evaluate their efficacy in preserving the bioactivity of encapsulated biomolecules, as presented in Table 1.

3. Model biomolecules: different molecules (SSD, PRP, and HRP) were loaded into the MG-DMNs to assess the instant release behaviour and the stabilizing capacity of the modified gelatin matrix; the details are provided in Tables 2–5.

Table 2 Summary of the details of the fabricated HRP-loaded modified and unmodified gelatin microneedles

Sample name	Composition	Gelatin concentration	Trehalose/sucrose concentration	HRP concentration
HRP-UMG-T7.5-DMNs	HRP-loaded unmodified gelatin MNs	20% w/v	7.5% w/v	2 μg
HRP-UMG-T5-DMNs		20% w/v	5% w/v	2 μg
HRP-UMG-T2.5-DMNs		20% w/v	2.5% w/v	2 μg
HRP-MG20-T7.5-DMNs	HRP-loaded modified gelatin MNs	20% w/v	7.5% w/v	2 μg
HRP-MG20-T5-DMNs		20% w/v	5% w/v	2 μg
HRP-MG20-T2.5-DMNs		20% w/v	2.5% w/v	2 μg

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Table 3 Summary of the details of the fabricated PRP-loaded modified and unmodified gelatin microneedles

Sample name	Composition	Gelatin concentration	Trehalose/sucrose concentration	PRP concentration
PRP-MG20-T5-DMNs	PRP-loaded unmodified gelatin MNs	4.5% w/v	5% w/v	8 × 10^6 cells
PRP-MG20-T10-DMNs	SSD-loaded modified gelatin MNs	20% w/v	10% w/v	8 × 10^6 cells

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Table 4 Summary of the details of the fabricated SSD-loaded modified and unmodified gelatin microneedles

Sample name	Composition	Gelatin concentration	Trehalose/sucrose concentration	SSD concentration
SSD2-UMG20-DMNs	SSD-loaded unmodified gelatin MNs	20% w/v	5% w/v	2 mg
SSD2-UMG20-DMNs		20% w/v	5% w/v	2 mg
SSD2-UMG20-DMNs		20% w/v	5% w/v	2 mg
SSD2-UMG4.5-DMNs	SSD-loaded unmodified gelatin MNs	4.5% w/v	5% w/v	2 mg
SSD2-UMG4.5-DMNs		4.5% w/v	5% w/v	2 mg
SSD2-UMG4.5-DMNs		4.5% w/v	5% w/v	2 mg
SSD2-MG420-DMNs	SSD-loaded modified gelatin MNs	20% w/v	5% w/v	2 mg
SSD2-MG15-DMNs		15% w/v	5% w/v	2 mg
SSD2-MG10-DMNs		10% w/v	5% w/v	2 mg

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Table 5 Summary of the details of the fabricated cocktail of molecules (PRP and SSD) loaded on modified and unmodified gelatin microneedles

Sample name	Composition	Gelatin concentration	Trehalose concentration	PRP	SSD
SSD-PRP-MG20-T5-DMNs	PRP-loaded unmodified gelatin MNs	4.5% w/v	5% w/v	8 × 10^6 cells	2 mg
SSD-PRP-MG20-T5-DMNs	SSD-loaded modified gelatin MNs	20% w/v	10% w/v	8 × 10^6 cells	2 mg

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Two types of dissolvable microneedles (DMNs) were fabricated using unmodified gelatin (UMG) at concentrations of 4.5% (w/v) and 20% (w/v), referred to as UMG4.5-DMNs and UMG20-DMNs, respectively. Similarly, microneedles based on modified gelatin (MG) were prepared at concentrations of 10%, 15%, and 20% (w/v), designated as MG10-DMNs, MG15-DMNs, and MG20-DMNs, respectively. The gelatin microneedles were fabricated with slight modifications to the previously reported procedure.³³ Briefly, unmodified gelatin (UMG) and modified gelatin (MG) were prepared in phosphate-buffered saline (PBS, pH 7.4) to form precursor solutions for DMN fabrication. The stabilizing agent trehalose (T) or sucrose (S) was added at varying concentrations (7.5%, 5%, and 2.5% w/v) to produce formulations such as UMG-T7.5, UMG-T5, and UMG-T2.5, and MG-T7.5, MG-T5, and MG-T2.5, respectively.

The prepared matrix solutions were cast onto negative PDMS molds with a 10 × 10 array, and a positive pressure of 1 bar was applied to ensure complete filling of the microneedle cavities. (Note: PDMS molds with different dimensions were used for specific microneedle designs.) To eliminate trapped air bubbles, the casting procedure was repeated three times. The molds were then allowed to dry at room temperature for 48 hours. After complete drying, the microneedles were gently demolded and stored in a desiccator at 4 °C and 25 °C until further use. Detailed formulations and microneedle specifications are provided in Table 1.

2.2.3. Fabrication of model biomolecules and drug-loaded gelatin microneedles. Biomolecules, including platelet-rich plasma (PRP) and horseradish peroxidase (HRP), were loaded into different gelatin-based microneedles (UMG-DMNs and MG-DMNs). The storage stability of model biomolecules with stabilizing agents at 4 °C and 25 °C was investigated.
HRP-loaded modified and unmodified microneedles (HRP-MG-DMNs and HRP-UMG-DMNs). Specifically, 1 μg of HRP was incorporated into MG-DMNs (HRP-MG-DMNs) and stored under various conditions (4 °C and 25 °C). Trehalose or sucrose was added at different concentrations (7.5%, 5%, and 2.5% w/v) during formulation. The patches prepared with MG were named HRP-MG-T7.5-DMNs, HRP-MG-T5-DMNs, and HRP-MG-T2.5-DMNs, while those made using UMG were designated HRP-UMG-T7.5-DMNs, HRP-UMG-T5-DMNs, and HRP-UMG-T2.5-DMNs.
PRP-loaded modified and unmodified microneedles (PRP-MG-DMNs and PRP-UMG-DMNs). PRP-loaded microneedles (PRP-MG-DMNs) were prepared by incorporating 8 × 10⁶ cells of activated PRP into the microneedle tip solution. The PRP was isolated from blood samples collected from healthy volunteers, following a published protocol.³³ All experiments were performed in accordance with the Guidelines of the Indian Institute of Technology, Hyderabad. Experiments were approved by the ethics committee at the Indian Institute of Technology, Hyderabad (approval number IITH/IEC/2018/12/13), with consent obtained from the human participants of this study. Trehalose was added at concentrations of 5% and 10% w/v for the preparation of PRP-MG-T5-DMNs and PRP-MG-T10-DMNs. The fabricated microneedles were stored under vacuum conditions in a desiccator at 4 °C and 25 °C, labelled as PRP-MG-T-DMN-0 for freshly prepared microneedles and PRP-MG-T-DMN-1 for those stored for one month.
SSD-loaded modified and unmodified microneedles (SSD-MG-DMNs and SSD-UMG-DMNs). The model antibacterial drug silver sulfadiazine (SSD) was incorporated into gelatin microneedles (with UMG and MG) to evaluate their instant release properties from microneedles. Modified gelatin microneedles with different concentrations (MG20-DMNs and MG10-DMNs) were loaded with SSD (2 mg), labelled as SSD2-MG20-DMNs and SSD2-MG10-DMNs, respectively, following the aforementioned fabrication protocol.
Cocktail of molecules (PRP and SSD) in the modified microneedles. Finally, the cocktail of biomolecules (PRP and SSD) containing microneedles was stored in a desiccator at 4 °C and 25 °C, and the samples were denoted as SSD-PRP-MG-DMN-0 (immediately prepared) and SSD-PRP-MG-DMN-1 (stored for 1 month at 4 °C and 25 °C).

2.3. Characterization of gelatin DMNs

2.3.1. Fourier-transform infrared (FTIR) spectroscopy. The secondary structures of MG and UMG were evaluated, and their influence on the dissolvability of different microneedle formulations was explored. Additionally, their interactions with stabilizing excipients, such as trehalose and sucrose, were examined.^34,35 The degree of modification in MG samples was analyzed using a Bruker FTIR Tensor 37 spectrometer in the wavelength range of 4000 to 400 cm⁻¹ and a resolution of 4 cm⁻¹.
Secondary structure analysis. To further assess the influence of modification on MG's secondary structure, Attenuated Total Reflectance (ATR)-FTIR measurements were performed on dried MG-DMN and MG-T-DMN samples using an Alpha Bruker FTIR instrument (Germany). All the spectra were acquired with 128 scans in the above-mentioned wavelength range and resolution. The amide I region (1590–1705 cm⁻¹) was deconvoluted using OriginPro 9.0 software to estimate the secondary structures of UMG and MG.³⁶ The amide I regions were curve-fitted employing Lorentzian line shape fitting, guided by peak positions identified through secondary derivative analysis. All measurements were conducted in triplicate for each microneedle formulation.

2.3.2. Nuclear magnetic resonance (NMR). ¹H NMR spectroscopy was performed to analyze the chemical structure and quantify the extent of carboxyl group modification in UMG and MG samples with R = 5 and R = 10. Measurements were carried out using a Bruker Avance III 400 MHz spectrometer, with samples dissolved in deuterated water (D₂O). Chemical shifts were referenced to tetramethylsilane (TMS) as an internal standard. 10 mg of the samples were dissolved in 1 mL of D₂O and thoroughly mixed before analysis. The degree of modification in MG samples was determined by comparing the integral areas corresponding to lysine residues in MG samples to those in the UMG spectrum.

2.3.3. Carboxylic acid determination in UMG and MG. The carboxyl group content in UMG and MG with varying degrees of modification was quantified using an electric conductivity titration method.³⁷ Briefly, 100 mg of each sample was dissolved in 60 mL of deionized (DI) water at 37 °C for 1 hour. The pH was initially kept at 3.0 using 0.1 M hydrochloric acid (HCl), and 0.05 M sodium hydroxide (NaOH) was added gradually at a constant flow rate of 100 μL min⁻¹ until the pH reached 12. The pH was recorded throughout the titration process, and a plot of pH versus the volume of NaOH added was generated to determine the carboxyl content of each sample.

2.3.4. Morphology characterization of DMNs. The morphology of the UMG and MG dissolvable microneedles (DMNs) was analysed by field emission scanning electron microscopy (FE-SEM, JEOL JSM-7600F, Tokyo, Japan). Before imaging, the microneedles were sputter-coated with gold for 60 seconds on the tip side to enhance conductivity. SEM analysis was performed at an accelerating voltage of 5 kV with the sample stage tilted at an angle of 30°.

2.3.5. Differential scanning calorimetry (DSC) analysis. DSC was used to analyze the glass transition temperatures (T_g) of UMG-DMNs and MG-DMNs. Approximately 10 mg of each sample, UMG, MG, and MG-T-DMNs, was sealed in Tzero aluminium pans, with an empty pan serving as the reference. Thermal analysis was performed using a DSC 204 system (Netzsch, Germany) over a temperature range of −10 °C to 120 °C at a heating rate of 20 °C min⁻¹ to determine T_g values.

2.3.6. Circular dichroism (CD) spectroscopy. The secondary structures, specifically coil-to-helix transitions in all microneedles, were analyzed using a JASCO J-1500 CD spectrometer, following an established protocol.³⁸ In brief, UMG4.5-DMNs, MG20-DMNs, and MG20-T5-DMNs were dissolved in deionized (DI) water to make a final concentration of 0.25 μg mL⁻¹. CD spectra were recorded in the 180–250 nm wavelength range at a scanning speed of 50 nm min⁻¹. Baseline correction was performed using DI water as the reference blank.

2.3.7. Water content analysis of UMG-DMNs and MG-DMNs. The percentage water content of UMG-DMNs and MG-DMNs was determined with a Q500 Thermo Gravimetric Analyser (TA Instruments, Elstree, Herts, UK). Samples of 2.0 mg were heated from ambient temperature to 300 °C at a heating rate of 10 °C min⁻¹. Nitrogen flow rates of 40 ml min⁻¹ (balance purge gas) and 60 ml min⁻¹ (sample purge gas) were maintained for all samples. The data from thermogravimetric analysis experiments were analyzed with TA Instruments Universal Analysis 2000 software, version 4.4A (TA Instruments, Elstree, Herts, UK).

2.3.8. Evaluation of instant dissolvability. To assess the rapid dissolution behaviour, both MG-DMNs and UMG-DMNs were inserted into a 20% w/v gelatin gel designed to mimic the hydration level of the skin.³⁹ The dissolution process was documented using a Canon PowerShot SX420 IS digital camera. For detailed observation of the microneedle tip dissolution, the patches were applied onto the gelatin gel, separated by a parafilm membrane, and left in place for 5 minutes. Afterwards, the microneedle morphology was examined with field emission scanning electron microscopy (FESEM; JOEL-JSM-7600F, JOEL, Tokyo, Japan) to verify the dissolution of the needle tips.

2.3.9. Ex vivo drug release study.
SSD release study. An ex vivo transdermal drug release study was performed using a Franz diffusion cell to compare the penetration efficiency of the model drug silver sulfadiazine (SSD) delivered via instant-dissolving MG-DMNs with that of UMG-DMNs. In brief, fresh porcine ear skin was isolated, and the fat layer was carefully removed. Then, 1 mg of SSD loaded with various concentrations of MG-DMNs and UMG-DMNs was inserted into the porcine skin until the microneedles completely dissolved. The microneedle-dissolved skin was placed into the Franz diffusion cell with the epidermis facing upward, and the receptor compartment was filled with 20 mL of PBS. The diffused SSD concentration in the released samples was determined using HPLC (Agilent Technologies 1260 Infinity II) by following the optimized method of SSD from our previously published procedure.⁴⁰ Note that the cumulative release is the complete release of SSD into the skin.
HRP release study. HRP-MG-DMNs and HRP-UMG-DMNs were dissolved in PBS, and the resulting supernatant was collected to quantify the HRP concentration. An OPD-based assay, with slight modifications, was performed to assess the residual horseradish peroxidase (HRP) activity in the HRP-MG-T-DMNs, following previously established protocols.⁴¹ 6.28 mM of o-phenylenediamine (OPD), 6 mM of hydrogen peroxide (H₂O₂), and the HRP standards (0 to 300 μg mL⁻¹) were prepared in sodium phosphate buffer (pH 7.2). For the enzymatic reaction, 100 μL of each sample was combined with 100 μL of OPD solution and 20 μL of hydrogen peroxide. After 30 minutes of incubation, the absorbance was recorded at 417 nm using a MultiMode Plate Reader (iD5, Molecular Devices, USA). The HRP content in the DMNs was quantified based on a standard HRP calibration curve, as illustrated in SI Fig. S13.
PRP release study. Sandwich ELISA was used to quantify the amount of active (capable of binding to antibodies) EGF in PRP-encapsulated microneedles. The released EGF from the PRP-MG-DMNs was determined by dissolving PRP-MG-T-DMNs and PRP-UMG-T-DMNs in 1 ml of PBS with PRP in PBS as a control. The manufacturer's ELISA protocol was followed to prepare the plate with primary and secondary EDGF antibodies. Finally, the dissolved PRP-MG-DMN and PRP-UMG-T-DMN samples were added, followed by the addition of 100 μL of ABTS liquid substrate. The resulting absorbance was recorded at 405 nm every 5 minutes for 25 minutes using the iD5 MultiMode Plate Reader (Molecular Devices, USA). The sample concentrations were determined using a standard PDGF plot.

2.3.10. Mechanical properties and insertion capability of DMNs. MG-DMNs and UMG-DMNs were evaluated for their mechanical properties using different concentrations of stabilizers, such as trehalose and sucrose, with a texture analyzer (TA-XT Plus, Stable Micro Systems), based on a previously established protocol with slight modifications.⁴² MG-DMN patches were kept on a platform with the needle tips facing upward, and a cylindrical probe (1 cm diameter) was used to compress the microneedles at a test speed of 0.25 mm s⁻¹. The pre- and post-test speeds were set to 1.0 mm s⁻¹, and the trigger force was fixed at 0.049 N. All measurements were performed in triplicate.
Parafilm insertion study. Parafilm M® was used as an alternative model of skin to check the insertion capacity of the microneedles.⁴³ Briefly, a single piece of Parafilm M was folded into eight layers, resulting in a total thickness of approximately 1 mm. The folded Parafilm was laid out on the mold, and microneedles of varying heights (500 μm and 600 μm) and interspacing distances (100 μm and 200 μm) were inserted into it using thumb pressure for 30 seconds. After the insertion, the microneedle was carefully peeled off, and the parafilm was unfolded. The number of holes in each layer was then counted, and the hole efficiency was calculated using the following formula:

Also, Rose Bengal dye was loaded onto MG20-T5-DMNs, which was then applied to freshly excised porcine ear skin. The microneedles were inserted for 5 minutes, and the dye penetration into the skin was visualized using an optical microscope to confirm successful delivery.

2.4. In vitro studies

2.4.1. Biocompatibility of gelatin microneedles. The biocompatibility of MG-DMNs, with a stabilizing agent (MG-T-DMNs), and SSD (SSD-MG-T-DMNs) was evaluated using MTT and Alamar Blue assays, with slight modifications to established protocols.⁴⁴ In the past studies, human embryonic kidney 293 (HEK293) cells were used to assess the biocompatibility of various microneedles.^45,46 Human embryonic kidney (HEK293) cells were seeded at a density of 3500 cells per well in 96-well plates and incubated overnight at 37 °C under a 5% CO₂ atmosphere to allow for proper adherence. All MG-DMN formulations were dissolved in 1 mL of PBS, and 100 μL of each dissolved microneedle sample was added to the respective wells containing the cells. The concentration details of the MG-DMNs added to the cells are provided in Table 6. Wells treated with culture medium served as the untreated control. After 48 hours of incubation, the culture medium was removed, and 100 μL of MTT solution (0.5 mg mL⁻¹) was added to each well. After 3 hours of incubation, 100 μL of DMSO was added to each well to dissolve the formazan crystals for 15 minutes. Absorbance readings were taken at 570 and 650 nm using a MultiMode Plate Reader (iD5, Molecular Devices, USA). Cell viability was then calculated using the following formula:

The metabolic activity of the cells was checked using the Alamar Blue assay. After 48 hours of incubation with the samples, the samples were removed and replaced with 100 μL of a 10% (v/v) Alamar Blue solution (HiMedia). Following 4 hours of incubation at 37 °C, the supernatant was added to 96-well plates, and fluorescence was measured using a MultiMode Plate Reader (iD5, Molecular Devices, USA) with excitation and emission wavelengths set at 570 nm and 600 nm, respectively.

Table 6 Concentration of all microneedle formulations used in the biocompatibility studies

Sample code	Concentration of gelatin	Concentration of trehalose	Concentration of SSD
The microneedles were initially dissolved in 1 ml of PBS, and 100 μL of the dissolved solution was added to the wells.
MG20-DMNs	2 mg	—	—
MG10-DMNs	1 mg	—	—
MG20-T5-DMNs	2 mg	0.5 mg	—
MG10-T5-DMNs	1 mg	0.5 mg	—
MG20-T5-DMNs	2 mg	0.5 mg	0.2 mg
MG10-T5-DMNs	1 mg	0.5 mg	0.2 mg

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Live-dead assay. The viability of HEK cells following incubation with various MG-DMNs has been further evaluated using the live/dead assay, as described in the previously described protocol with slight modifications. Following 48 hours of incubation with the dissolved microneedle samples, cells were stained with calcein AM and propidium iodide for 15 minutes. Fluorescent images were captured using an Olympus IX73 inverted fluorescence microscope.

2.5. Storage stability of biomolecules in gelatin-based DMNs

2.5.1. HRP enzyme bioactivity. Horseradish peroxidase (HRP) was used as a model enzyme to assess the impact of fabrication and storage-related stress on biomolecular stability within the MG-DMNs. HRP-loaded MG-T-DMNs were stored under vacuum in a desiccator at 4 °C and 25 °C for stability assessment. To assess the remaining HRP activity in HRP-MG-T-DMNs, we collected the HRP-MG-T-DMNs at predetermined time intervals and dissolved them in 1 mL of PBS. HRP activity assay was performed based on the protocol mentioned above.

2.5.2. In vitro bioactivity assay of PRP. The activity of platelet-rich plasma (PRP) encapsulated in PRP-MG-T-DMNs, both fresh and after storage, was tested in vitro by measuring the proliferation of umbilical mesenchymal stem cells (UMSCs). Previous studies have demonstrated that PRP significantly enhances the proliferation of UMSCs even under serum-free conditions.^47–49 These findings motivated the selection of UMSCs as a suitable cell model to investigate the retained bioactivity of PRP following storage within the microneedles. To assess the influence of the stabilizing agent concentration (5% and 10% w/v) on the long-term stability of PRP within the microneedle matrix, an accelerated storage stability test was conducted at 40 °C and 75% relative humidity (RH). The in vitro PRP activity of the storage samples was determined using a proliferation assay of UMSC cells.

2.6. Chorioallantoic membrane (CAM) assay

The bioactivity of platelet-rich plasma (PRP) encapsulated within MG20-T5-DMNs and stored at 25 °C for one month was assessed using the chorioallantoic membrane (CAM) assay. The in ovo CAM assay was done by applying dissolved microneedle samples (PRP-MG20-T5-DMN-0 and PRP-MG20-T5-DMN-1) onto live chicken embryos, using a modified version of a previously described protocol.⁵⁰ Fertilized chicken eggs were rinsed with a 20% saline solution and incubated at 37 °C in a humidified incubator. On the third day of incubation, the eggshells were carefully opened, and the embryos were maintained in the incubator at 37.5 °C with 60% relative humidity. UV-sterilized DMNs were placed on the embryo on the 7^th day. Angiogenesis in the embryo on day 14 was recorded by capturing digital images using a Canon PowerShot SX420 IS camera.

2.7. In vivo bioactivity evaluation of PRP-loaded MG-DMNs using diabetic wound healing

The therapeutic efficiency of PRP-loaded MG-DMNs was assessed in a diabetic wound-healing model, with modifications based on previously reported methodologies.⁴⁴ Male Sprague Dawley rats (8 weeks old) were established with type 1 diabetes.

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Indian Institute of Technology Hyderabad and were approved by the Animal Ethics Committee of the Institutional Animal Ethics Committee, with approval number IITH_IAEC 2023_M2_P09. Animals were fasted overnight before receiving an intraperitoneal injection of alloxan monohydrate (120 mg kg⁻¹) the following morning. Diabetes was confirmed by plasma glucose levels exceeding 16.7 mM, along with typical clinical symptoms including polyphagia, polydipsia, polyuria, and weight loss. Diabetic rats were divided into three groups (n = 3 per group): an untreated control, blank MG20-T5-DMNs (without PRP), and SSD-PRP-MG20-T5-DMN-1. Before surgery, anaesthesia was induced using ketamine (100 mg kg⁻¹) and xylazine (10 mg kg⁻¹). The dorsal area was shaved and disinfected with 70% ethanol. Two full-thickness excisional wounds (10 mm in diameter) were created bilaterally, 1.5 cm lateral to the vertebral midline, using sterile surgical blades.⁵¹ One day post-wounding, treatment groups of PBS, blank MG20-T5-DMNs, or SSD-PRP-MG20-T5-DMN-1 were applied to the wounds via gentle thumb pressure for 10 minutes. All microneedles were sterilized by UV exposure before use. Wound healing progression was documented using digital photography (Canon PowerShot SX420 IS) on days 0, 3, 7, and 14. At the study endpoint, animals were euthanized, and peri-wound tissue samples were collected for histological examination. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to assess tissue morphology and evaluate the healing stages in accordance with established protocols.⁵²

2.8. Statistical analysis

All quantitative results are expressed as mean ± standard deviation (SD). Statistical evaluations were conducted using OriginPro 9 and GraphPad Prism 8. For comparisons among groups, one-way or two-way analysis of variance (ANOVA) was used, followed by Tukey's post hoc test for multiple comparisons. Statistical significance is denoted in the figures with asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, while “n.s.” indicates no significant difference.

3. Results and discussion

3.1. Modification of gelatin

Gelatin-based dissolvable microneedles (UMG-DMNs) exhibit poor mechanical properties due to the random coil to triple helix formation, which is further reduced with the incorporation of stabilizing agents, even at lower concentrations (<5% w/v), leading to slow dissolution rates.^11,13 To overcome these limitations, gelatin was chemically modified by introducing the tailored carboxylic groups to limit inter- and intra-molecular interactions, particularly the random coil to triple helix transition, using a similar chemical modification strategy as reported in our previous work (Fig. 1A).²⁹ This tailored carboxylic group introduction enhances solubility, reduces solution viscosity, and restricts coil-to-helix transitions and gelation of MG during DMN fabrication at higher concentrations, while simultaneously improving mechanical properties.

Fig. 1 (A) Schematic representation of gelatin modification with succinic anhydride (SA). (B) ¹H NMR spectra of unmodified gelatin (UMG) and modified gelatin (MG) samples, demonstrating the incorporation of carboxylic groups. (C) Quantification of the carboxylic acid content in MG samples synthesized using varying SA ratios, as determined by conductivity titration, compared with UMG. (D) FTIR spectra of UMG, the UMG gel, and MG, showing that carboxylic modification disrupts coil-to-helix transition by reducing intermolecular interactions. (E) Secondary structure content in UMG-DMNs and MG-DMNs derived from deconvoluted amide I FTIR spectra. (F) Solubility of MG10, MG15, MG20, and UMG20 at different concentrations; UMG20 undergoes gelation due to coil-helix transition, whereas MG20 retains a liquid state.

¹H NMR spectra shown in Fig. 1B show unmodified gelatin (UMG) and modified gelatin with different degrees of modification, MG (R = 5 and R = 10). The degree of carboxylic group modification was quantified by using the phenylalanine aromatic peak at 7.1–7.4 ppm as an internal reference and assessing the relative increase in the vinyl proton signals around 5.8–6.2 ppm (–OCH₂–CH=CH₂).³⁸ The decrease in the native amine group in gelatin, observed around 2.8–3 ppm, occurs due to nucleophilic substitution with SA, resulting in carboxylic acid-rich modified gelatin. Titration analysis (Fig. 1C and SI Fig. S1A) determined the carboxylic acid group content to be 0.35 mmol g⁻¹ for UMG. It increased to 0.78 mmol g⁻¹ and 1.3 mmol g⁻¹ for MG-R5 and MG-R10, respectively, consistent with the ¹H NMR data shown in Fig. 1A. This linear increase correlates with the rise in succinic anhydride used in the modification reaction, 5 mmol (R = 5) and 10 mmol (R = 10), following the Le Chatelier principle, which states that the reactant concentration governs the substitution rate.

FTIR spectra (SI Fig. S1B) further validated this modification with the characteristic peaks corresponding to C–C bonds appearing at 1618, 1516, and 1456 cm⁻¹, while a distinct C–O stretch was observed around 1232 cm⁻¹, indicating the incorporation of succinic anhydride into the gelatin backbone.⁵³ Moreover, the increasing intensity of N–H stretching vibrations near 3223 cm⁻¹ and the C=O stretching associated with hydrogen-bonded carboxyl groups (COO⁻) in the 1635–1632 cm⁻¹ range with higher SA molar ratios (R = 5 and R = 10) further supports the increasing degree of modification.

The secondary structures of UMG gel, UMG, and MG were analyzed using ATR-FTIR by focusing on the amide I region (1705–1595 cm⁻¹), which was deconvoluted to identify characteristic peaks corresponding to different structural motifs (Fig. 1C). Specific secondary derivative peaks were identified at 1630 cm⁻¹ (β-sheet), 1645 cm⁻¹ (random coil), 1660 cm⁻¹ (triple helix), 1683 cm⁻¹ (β-turn/sheets), and 1692 cm⁻¹ (β-turn), as reported previously.^54,55 Deconvolution of the amide I region (SI Fig. S1D), followed by quantitative analysis (Fig. 1E), revealed that the modified gelatin (MG) gel consisted of approximately 18% β-sheet, 37% β-turn, and 38% triple-helix structures, with random coils accounting for less than 10% of the total composition. In contrast, the unmodified gelatin (UMG) exhibited a higher β-sheet content of around 40%, a triple-helix content of approximately 20%, and a random coil content of about 25%.

The tailored introduction of the carboxylic group in MG limited the hydrogen bonding between glycine and proline/hydroxyproline by electrostatic repulsion. This modification resulted in 58% of random coil and less than ∼10% of triple helix, surpassing our previous silk modification, which showed <50% random coil.²⁹ Even a moderate modification (R = 5 to R = 10) significantly enhanced solubility, even at higher concentrations, without resulting in gelation. These structural changes correlate with a reduction in viscosity and an improvement in solubility of MG across different concentrations, as demonstrated in Fig. 1F, thereby confirming our rationale for using MG in high concentrations for preparing instant-dissolving microneedles.

3.2 Molecular interaction between MG-stabilizing molecules

To enhance the stability of biomolecules within DMNs, MG was combined with stabilizing excipients such as trehalose or sucrose to form MG-T microneedles (MG-T-DMNs). The hydroxyl groups of these excipients form intermolecular hydrogen bonds with the hydroxyl and carboxyl groups of MG, forming a protective matrix around the biomolecules by limiting their mobility. ATR-FTIR analysis (Fig. 2A, SI Fig. S2A) confirmed these interactions: MG-T-DMNs exhibited characteristic amide C=O stretching at ∼1650 cm⁻¹ and N–H bending at ∼1560 cm⁻¹, along with broad O–H stretching bands (∼3300–3650 cm⁻¹), indicating hydrogen bonding between MG and the sugars. The absence of a distinct free O–H peak and the appearance of a broad, single band at ∼3300 cm⁻¹ further suggest active hydrogen bonding. Analysis of the amide I region (SI Fig. S2B) revealed structural transitions in MG-T-DMNs, with quantitative analysis (Fig. 2B) showing that increasing trehalose concentration reduced triple helix and β-sheet content, while increasing the random coil fraction from 48% in MG20-DMNs to 58% in MG20-T5-DMNs.

Fig. 2 (A) FTIR spectra of MG combined with different concentrations of the stabilizing agent trehalose. (B) Secondary structure contents obtained by deconvoluting the amide I band (1600–1700 cm⁻¹) from the respective FTIR spectra. (C) The CD spectra of MG20-DMNs, with a 5% w/v stabilizing agent incorporated, compared with UMG4.5-DMNs, show a decrease in triple helix intensity in MG-DMNs and MG-T-DMNs compared to UMG-DMNs. (D) DSC graphs of MG20-DMNs, 5% w/v stabilizing agent incorporated in MG20-T5-DMNs, show the reduced T_g values of MG20-DMNs and MG20-T5-DMNs in comparison with UMG20-DMNs.

CD spectra (Fig. 2C) further validated this hypothesis that stabilizing agents prevent gelatin's coil-to-helix transition. UMG-DMNs exhibited a strong positive peak at 222 nm, indicating a higher triple helix content. In contrast, MG-DMNs and MG-T-DMNs showed reduced intensity, confirming that carboxylation effectively inhibits helix formation. Adding stabilizing excipients, such as trehalose, to MG-T-DMNs further suppressed this transition, as reflected by the lowest 222 nm intensity. In contrast, absorbance at 198 nm, indicative of the random coil content, was significantly higher in MG-DMNs and MG-T-DMNs compared to UMG-DMNs, with MG-T-DMNs showing the highest values. The unmodified gelatin, with a water content of approximately 15%, was found to have a T_g value of around 60 °C, as reported by Liu et al. and Mosleh et al.^56,57 As the plasticizer content increased in the gelatin, the T_g value was found to decrease drastically below 40 °C.⁵⁸ DSC analysis (Fig. 2D) revealed that the T_g value of the MG-T-DMNs decreased as the stabilizing agent's concentration increased, indicating that stabilizing agents can act as plasticizers in the matrix by intercalating into the protein matrix, thereby hindering intermolecular interactions between protein chains.³⁹ These results are consistent with our FTIR data and previous findings on carboxyl-modified silk, which show that, when paired with excipients, higher concentrations enhance solubility, a key feature in fabricating instant dissolving gelatin microneedles.^38,59,60

3.3. Morphology and dissolution behaviour of MG-DMNs

3.3.1 Morphology of the gelatin microneedles. MG-based DMNs were fabricated using the solvent casting method in PDMS to ensure precise shape, reproducibility, and dissolution behaviour (Fig. 3A). MG-based microneedles MG20-DMNs, MG15-DMNs, and MG10-DMNs exhibited rapid tip dissolution, while UMG4.5-DMNs and UMG20-DMNs remained largely insoluble (Fig. 3A and SI Fig. S3C), due to strong intermolecular interactions driving triple helix (40% in UMG gel, 20% in UMG) and β-sheet formation (18% in UMG gel, 42% in UMG), as confirmed by FTIR and CD analyses. In contrast, the incorporation of stabilizing agents in MG formulations acts as a solubility enhancer by limiting such interactions. SEM analysis (Fig. 3A) further confirmed the rapid dissolution of MG20-DMNs, MG10-DMNs, and even UMG-DMNs upon insertion into a 20% gelatin gel. Storage stability tests (SI Fig. S4A) revealed that MG-DMNs retained structural integrity and exhibited instant dissolution within 5 minutes (SI Fig. S4B) even after one month at 25 °C, whereas UMG-DMNs became brittle and fractured under identical conditions. Compared to previous reports by Weijiang Yu et al.^61,62 and Lee et al.,²⁴ wherein gelatin-based DMNs required over 2 hours to dissolve, our MG-DMNs achieve rapid dissolution while accommodating higher gelatin concentrations (up to 25%), attributed to reduced inter- and intra-molecular interactions via carboxylation.

Fig. 3 (A) SEM images showing the dissolution behaviour of MG-DMNs compared with UMG-DMNs. Scale bar: 200 μm. (B) Optical images showing the dissolution of MG-DMNs compared with UMG-DMNs. Scale bar: 5 mm. The instant (5 min) dissolvability of different microneedles and the resulting release of the model molecules from respective microneedle variants, such as the modified gelatin microneedle and the unmodified gelatin microneedle. (C) SSD, (D) HRP, and (E) PRP quantified using a calorimetric assay for HRP and FGF-2 concentrations, as determined by ELISA, among the PRP-associated growth factors.

3.3.2 Dissolution and release study of gelatin microneedles. The ex vivo model assessed the release of SSD from MG-based DMNs and compared it to that of unmodified gelatin-based DMNs (UMG-DMNs). Each MG-DMN patch contained approximately 480 μg of the drug, whereas the UMG20-DMN patch contained about 222.3 μg. The lower drug loading in the UMG20-DMNs is attributed to gelation, which resulted in incomplete filling of the microneedle mold. As shown in Fig. 3B, the different concentrations of MG-DMNs were dissolved within 5 minutes after insertion onto the skin, in contrast to UMG-DMNs. The instantaneous dissolution behavior of MG-DMNs ensures the complete release of SSD in the skin, and 100% diffused SSD was detected in the receiver compartment (Fig. 3C). However, those of UMG20-DMNs, UMG10-DMNs, and UMG4.5-DMNs were less than 10% within 5 minutes, indicating that UMG-DMN gelation causes slow drug release from the DMN (Fig. 3C). A similar pattern was observed for the other molecules, such as HRP (Fig. 3D) and PRP (Fig. 3E), with MG-DMNs exhibiting an instant release of molecules within 5 minutes, whereas UMG-DMNs showed a slower release. Although UMG-DMNs visually exhibited gelation (SI Fig. S3C), the release profiles revealed that HRP and PRP achieved approximately 50% release, whereas SSD showed less than 20% release. This difference can be attributed to the distinct hydrophilic (HRP, PRP) and hydrophobic (SSD) nature of the molecules and their interaction with the microneedle matrix.

3.4. Mechanical strength and insertion capacity of DMNs

3.4.1 Water content analysis. For effective skin penetration, microneedles (MNs) must possess sufficient mechanical strength.⁶³ Without adequate insertion, the tips fail to dissolve efficiently, limiting payload delivery.^64,65 Moisture content within the MN matrix plays a critical role; excess water can disrupt protein structure, reduce mechanical stability, and delay dissolution. Yuquan Chi et al.³⁰ reported that hyaluronic acid (HA)-based MNs, composed of a highly carboxylated biopolymer structurally similar to our modified gelatin (MG), displayed poor mechanical integrity (e.g., tip bending) after incubation at 80% relative humidity (RH) for 24 h due to their hygroscopic nature. Quantitatively, HA-MNs showed relatively stable water absorption (–5% to 5%) under 20–60% RH, but the absorption increased sharply to ∼25% at 80% RH, underscoring the moisture sensitivity of highly carboxylated biopolymers. In our system, the moisture content remained below 7% in the MG-DMN and MG-T-DMN formulations, whereas UMG-DMNs exhibited nearly 15% moisture, correlating with their reduced mechanical stability (Fig. 4A).

Fig. 4 (A) TGA graph of the UMG and MG-DMNs will be used to determine the water content after drying, and the representative water loss percentage at 110 °C. (B) Mechanical strength comparison of microneedles fabricated from varying concentrations of unmodified gelatin (UMG) and modified gelatin (MG). (C) Fracture force measurements of microneedles containing 5% w/v trehalose across different MG and UMG concentrations, showing a reduction in mechanical strength at lower MG concentrations. (D) The fracture force measurement of the PRP-loaded modified gelatin with varying concentrations of trehalose, (E) SEM and (F) parafilm insertion images depicting the morphology of UMG and MG microneedles before and after application into a parafilm. Scale bars: 200 μm.

3.4.2 Fracture force analysis. Mechanical performance was evaluated using a texture analyzer. UMG4.5-DMNs and UMG20-DMNs exhibited significantly lower fracture forces (6 N and 16 N per array, respectively). Conventional gelatin MNs typically require gelatin concentrations above 10% w/v^20,21,24 or blending with polymers and additives^61,62,66 to improve strength, but these strategies often reduce solubility. In UMG-DMNs, increasing trehalose from 0% to 5% w/v induced a plasticizer effect, reducing the strength from 16 N to 10 N (SI Fig. S6A).

In contrast, MG20-DMNs exhibited the highest force of 95 N per 10 × 10 array (∼0.9 N per needle), surpassing the 0.19 N per needle threshold required for skin penetration. This superior mechanical strength is attributed to MG's improved solubility and compatibility at high concentrations, similar to our previously developed silk microneedles, which maintained strong mechanical properties and rapid dissolvability at higher protein concentration. MG formulations maintained structural integrity even at higher trehalose levels, likely due to hydrogen bonding between the hydroxyl groups of the stabilizers and the carboxyl/hydroxyl groups of MG (Fig. 4A and B). As expected, increasing trehalose from 0%, 5%, to 10% w/v in MG20-DMNs caused a gradual decrease in the fracture force (95 N, 80 N, and 65 N, respectively; Fig. 4C), but values remained well above the insertion threshold. Beyond 10% w/v, the MNs became brittle and difficult to demold. The incorporation of model molecules (PRP and SSD) did not affect the mechanical properties of SSD-PRP-MG-DMNs. However, varying the trehalose concentration (5%, 10%) slightly decreased the fracture force from 90 N to 60 N, as depicted in Fig. 4D.

3.4.3 Insertion studies. Finally, insertion tests into parafilm confirmed the superior mechanical performance of MG-DMNs. SEM images (Fig. 4E and F) showed that MG-DMN tips remained intact post-insertion, while UMG4.5-DMN tips bent severely (Fig. 4C and SI Fig. S7B inset). Additionally, the insertion of Rose Bengal-loaded MG20-T5-DMNs into porcine skin resulted in a visible microchannel imprint (SI Fig. S5E), further validating its mechanical strength and skin insertion capability. All the concentrations of MG-DMNs successfully penetrated 3 layers of parafilm (∼375 μm) with a hole efficiency of 100%. In comparison, UMG-DMNs could penetrate only the first layer of parafilm (∼125 μm) with a hole efficiency of less than 80% for UMG20-DMNs and 40% for UMG4.5-DMNs, as shown in SI Fig. S6. Additionally, the insertion of Rose Bengal-loaded MG20-T5-DMNs into porcine skin resulted in a visible microchannel imprint (SI Fig. S5E), further validating its mechanical strength and skin insertion capability.

3.5. Assessment of biocompatibility and systemic safety of MG-DMNs

Fig. 5 shows the cytocompatibility of various gelatin microneedles. Initially, we assessed the biocompatibility of the MG with HEK293 cells. We found the cell viability to be more than 80% even at a concentration of more than 2 mg, as shown in the SI Fig. S7. Furthermore, all the MG-DMNs demonstrated cell viability exceeding 80%, as evaluated by the MTT assay. Previous studies have reported that even commonly used sugars, such as trehalose and sucrose, can markedly reduce cell viability when used at high concentrations.^67–69 In light of this, we evaluated the cytotoxicity of excipient-loaded microneedles (MG-T-DMNs). However, the addition of stabilizing excipients (trehalose) and SSD to the DMNs did not adversely affect cell viability, maintaining levels above 80% across all formulations. However, higher concentrations of trehalose significantly reduced HEK293 cell proliferation, indicating cytotoxic effects. Consequently, 5% trehalose was chosen as the optimal stabilizer concentration, as it strikes a balance between biocompatibility and enhanced mechanical integrity, facilitating efficient skin penetration. The fluorescence imaging of HEK293 incubated with MG20/15/10-T5-DMNs showed better cell proliferation and density than the MG-DMNs, as shown in Fig. 5A, corroborating the MTT (Fig. 5B) and Alamar Blue assay results (SI Fig. S8). These results showed that cells incubated with the different MG-DMNs had a better cell density, with no change in cellular morphology.

Fig. 5 (A) Live/dead staining of HEK293 cells treated with various MG-DMN formulations for 24 and 48 hours showed that cell viability remained above 80% in all experimental groups. Scale bar: 100 μm. (B) Quantitative evaluation of cytocompatibility of MG-DMNs using the MTT assay demonstrates high cell viability.

3.6. Storage stability of biomolecules in MG-DMNs

Preserving the stability of biomolecules is crucial to maintaining their therapeutic efficacy, as they are highly susceptible to degradation during processing and storage.⁷⁰ This study aimed to enhance the long-term stability of model proteins HRP and PRP within MN formulations. The retained enzymatic activity under various storage conditions is summarized in Fig. 6A. After one month at 4 °C, the PRP-MG20-T5-DMN formulation retained over 80% of HRP activity (SI Fig. S9), while storage at 25 °C resulted in a moderate reduction to ∼70% activity (Fig. 6A). Among formulations without stabilizing excipients, microneedles fabricated with higher concentrations of modified gelatin (MG20-DMNs) exhibited better protein retention than those manufactured with MG15-DMNs and MG10-DMNs. This enhanced stability is likely due to stronger hydrogen bonding and intermolecular interactions provided by the carboxyl groups in MG, as previously demonstrated through FTIR and CD analyses (Fig. 1 and 2).

Fig. 6 Effect of processing and storage on biomolecule activity in MG-DMNs. (A) Enzymatic assay assessing the preserved activity of HRP in HRP-MG-DMN formulations after one month of storage at 25 °C. (B) In vitro assessment of PRP bioactivity in PRP-MG-DMNs and PRP-MG-T-DMNs. (C) Live/dead fluorescence images of stored PRP-MG-DMNs and PRP-MG-T-DMNs after 48 hours of incubation with UMSC cells, stained with calcein AM and propidium iodide (PI). Scale bar = 100 μm. (D) Optical images of embryos incubated with MG20-T5-DMNs, PRP-MG20-DMNs (day 7), and PRP-MG20-T5-DMNs (day 14) using the CAM assay, showing the angiogenesis activity of PRP-MG-DMNs using the in ovo CAM assay.

Conversely, the presence of stabilizing agents, such as trehalose, results in the storage stabilization of HRP being observed to increase further due to increased hydrogen bonding and the formation of a glassy matrix.^71–74 A similar stabilization effect of our microneedle platform was observed for PRP in different DMN samples, as evaluated by cell proliferation and CAM assays. The cell (UMSCs) proliferation assay (Fig. 6B and C) was performed with the PRP extracted from the dissolved PRP-MG20-T5-MN and PRP-MG15-T5-MN samples stored for one month at 25 °C, which showed no significant activity loss compared to their prepared PRP-microneedle counterpart (without storage). An in vitro bioactivity assay was conducted to evaluate the retained activity of PRP in the PRP-MG20-T5-DMNs stored at 4 °C, revealing that its bioactivity remained stable even after 30 days of storage (SI Fig. S10). The microneedle platform is used for mass application storage stability at RT (25 °C) or 4 °C, which is needed to protect biomolecules from deactivation during transport, storage, and distribution.

3.7. In ovo CAM assay

The CAM assay provides a versatile and straightforward animal model as a screening tool for angiogenesis and metastasis among the various functional assays.⁷⁵ Several research studies have examined the biomaterial structures of the CAM, not only to check the angiogenic response but also to study the biomaterial–tissue compatibility. A broad spectrum of materials has been examined, comprising collagen, silk, and natural and synthetic polymer-based materials. We used the in ovo CAM assay to assess the storage stability of PRP and its bioactivity after DMN preparation and storage at various temperatures for a month, as shown in Fig. 6D.

The results demonstrate that PRP-loaded microneedles (PRP-MG20-T5-DMNs) significantly enhanced angiogenesis, inducing a 72% increase in blood vessel area and generating a total vessel length of 408 μm with an average length of 31 μm by embryonic day 7. In contrast, the unloaded MG20-T5-DMN group produced only 49% vessel area and a total vessel length of 260 μm (average 3 μm), as shown in SI Fig. S11. Quantitative analysis of vessel junctions further supports these findings: the MG20-T5-DMN group formed 101 new junctions, comparable to the control group (110). At the same time, the PRP-loaded formulations retained significantly higher angiogenic activity, with PRP-MG20-T5-DMN-0 and PRP-MG20-T5-DMN-1 forming 203 and 190 junctions, respectively. These results indicate that PRP retains its angiogenic potential after one month of storage at 4 °C and 25 °C. This preservation is likely due to the presence of trehalose, which limits protein mobility during the drying process. Higher concentrations of trehalose further enhance this effect, ensuring structural integrity and functional retention of PRP during microneedle fabrication and storage. The demonstrated long-term stability and bioactivity of PRP-MG-T-DMNs underscore their potential for clinical applications that require extended storage or transport without dependence on a cold chain.

3.8. Accelerated storage stability study

Typically, overcoming biomolecule instability involves incorporating stabilizing agents at concentrations above 5% w/v to reduce biomolecule mobility during the drying or dehydration process.⁷⁴ However, adding stabilizing agents in high concentrations to the microneedles is impossible due to the reduced mechanical properties.⁷⁶ Here, MG-DMNs could incorporate stabilizing agents up to 10% w/v without hindering the mechanical properties (Fig. 4C). Additionally, the MG20-DMNs formulated with 10% w/v trehalose (PRP-MG20-T10-DMNs) maintained over 80% of PRP bioactivity after 14 days at 40 °C, whereas the PRP-MG20-T5-DMNs retained only 40% under the same conditions. This proves our hypothesis that the stabilizing agent trehalose, when used at more than 10% w/v, retains PRP activity more than 5% w/v trehalose, even under harsh storage conditions (40 °C and 75% RH) (Fig. 7). As discussed in the following section, we have further performed a functional assay, specifically angiogenesis in the CAM assay of PRP in microneedles, which is stored at various temperatures and times.

Fig. 7 Accelerated bioactivity evaluation showing the impact of trehalose concentration (0%, 5%, and 10% w/v) on PRP stability in PRP-MG20-T-DMNs stored at 40 °C and 75% RH. The PRP-MG20-T10-DMNs retained approximately 80% of PRP activity after storage under accelerated conditions, while PRP-MG20-T5-DMNs retained only 40%.

3.9. In vivo studies

The therapeutic efficacy of PRP following processing and one-month storage in the SSD-PRP-MG20-T5-DMN formulation was assessed using a diabetic wound healing model. SSD-PRP-MG20-T5-DMN and MG20-T5-DMN (without PRP) patches were applied to full-thickness wounds in diabetic mice. By day 14, the control and blank DMN groups showed 73.6% and 80.8% wound closure rates, respectively (Fig. 8A and SI Fig. S12). In contrast, the SSD-PRP-MG20-T5-DMN-treated group exhibited significantly accelerated healing, achieving approximately 95% wound closure, indicating the retained bioactivity of PRP within the formulation. To further evaluate the biocompatibility of the microneedles and the functional efficacy of PRP in promoting tissue regeneration, histological analysis of the wound sites was performed across all treatment groups.

Fig. 8 (A) Assessment of PRP bioactivity in PRP-MG20-T5-DMNs stored at room temperature for one month using a diabetic wound rat model. Representative images show wound healing progression in diabetic rats treated with PBS (control), MG20-T5-DMNs, and stored PRP-MG20-T5-DMNs (PRP-MG20-T5-DMN-1). Scale bar: 2 mm. (B) Histological evaluation of wound tissues collected on day 14 post-treatment with PBS, MG-T5-DMNs, and SSD-PRP-MG-T5-DMN-1. (i) Low magnification (4×) and (ii) high magnification (200×). Scale bar: 100 μm.

Fig. 8B illustrates that the MG20-T5-DMN and SSD-PRP-MG20-T5-DMN groups exhibited minimal infiltration of inflammatory cells in the dermal region (highlighted by green arrows), comparable to the untreated control, indicating a low inflammatory response. By day 14, the SSD-PRP-MG20-T5-DMN group demonstrated notably enhanced granulation tissue formation, increased angiogenesis, better fibroblast infiltration, and clear regeneration of both dermal and epidermal layers (indicated by green arrows). These regenerative features were markedly more pronounced than those of the MG20-T5-DMN group (lacking PRP) and the untreated control, as shown in Fig. 8B(ii). Taken together, the PRP-microneedle group (SSD-PRP-MG20-T5-DMNs) exhibits the maximum wound healing efficacy due to the presence of both SSD (an antibiotic and antioxidant) and PRP (a cocktail of growth factors), which may contribute to enhanced wound healing.^48,77

4. Conclusions

Currently, gelatin microneedles fabricated from <5% w/v gelatin solutions are used to achieve instant dissolvability, as higher concentrations typically result in gel formation due to random coil-to-triple helix transitions. However, low-concentration formulations exhibit poor mechanical strength and storage stability, particularly when using stabilizing excipients at concentrations below 5% w/v. A possible solution to improve mechanical strength and biomolecule stability is to use higher concentrations of gelatin (>5% w/v) and stabilizing excipients (>5% w/v). However, this approach promotes a coil-to-triple helix transition, leading to gelation and significantly increased viscosity, which poses significant challenges during microneedle fabrication. Here, we address these challenges by introducing a tailored number of carboxylic groups into the gelatin backbone to limit their intra- and intermolecular interactions and aggregation, resulting in superior solubility. This allows for the preparation of MG-DMNs at higher concentrations without fabrication issues. Furthermore, MG-DMNs exhibited better mechanical properties, specifically 45 N, 80 N, and 95 N for MG10-DMNs, MG15-DMNs, and MG20-DMNs, respectively, whereas UMG-DMNs showed a fracture force of less than 19 N. Further incorporation of stabilizing agents reduced the mechanical strength to below 10 N in unmodified gelatin microneedles (UMG-DMNs). In contrast, modified gelatin microneedles (MG-DMNs) maintained significantly higher mechanical strength (>70 N), due to the interactions between the stabilizing agents and the carboxyl-modified gelatin matrix. The synergistic interaction between modified gelatin (MG) and stabilizing agents effectively preserves biomolecule activity, enabling the MG20-T5-DMN formulation to retain approximately 80% of PRP and HRP activity after one month of storage at 4 °C and 25 °C, and about 60% even under accelerated conditions (40 °C and 75% RH). This retained bioactivity contributed to enhanced wound closure rates in the diabetic wound healing model. The present study provides an essential development of mechanically robust and effective biomolecule stabilization of gelatin-based DMNs for biomedical applications. Though we have reported the storage stability of the biomolecule (PRP) in the microneedle for up to one month, stabilizing various biomolecules longer than six months is required for any practical application. This study is being conducted to validate our system for superior stabilization of biomolecules for one year or more.

Author contributions

Jayakumar Rajendran: conceptualization, methodology, validation, formal analysis, investigation, data curation, writing – original draft, writing – review & editing, and visualization for all the experiments. Jeyashree K.: methodology and investigation for cell culture studies. Sujith M.S.: methodology and investigation for the CAM assay. Lalitha Devi Alluri: resources. Jyotsnendu Giri: conceptualization, methodology, validation, formal analysis, writing – review & editing, supervision, project administration, and funding acquisition.

Conflicts of interest

The authors declare no conflicts of interest related to this work.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. Table of detailed comparison of existing conventional gelatin microneedles. pH vs. NaOH titration curve of the UMG and MGDMN for carboxylic group detection. FTIR spectra and deconvoluted amide I region of stabilizing agents incorporated into unmodified and modified gelatin microneedles. Optical image of the dissolution of MG-DMN after one month of storage. SEM images of one-month stored UMG-DMN and MG-DMN. Mechanical properties (Force vs. displacement) of UMG-DMN with varying concentration of trehalose and its corresponding bright field, fluorescence, and skin insertion study. Alamar assay of MG-DMN. In-vitro bioactivity of PRP-MG-DMN stored at 4 °C for a month. Quantitative analysis of the vascular area of the CAM assay and the wound closure rate. The standard curve of various HRP concentrations was analysed using an HRP enzymatic assay. See DOI: https://doi.org/10.1039/d5bm01184a.

Acknowledgements

The authors sincerely acknowledge the financial support received from the Department of Biotechnology, Ministry of Science and Technology, Government of India (Grant No. BT/PR36505/NNT/28/1818/2022), and the Department of Science and Technology (DST), Government of India (Grant No. SR/NM/-NS-1364/2014(G)). Further support from the DST-Science and Engineering Research Board (Grants SB/S3/CE/048/2015 and CRG/2020/005244) and the IMPRINT initiative (Grant DST/IMP/2018/000687) is gratefully appreciated. The authors also thank the Indian National Academy of Engineering (INAE) for the Abdul Kalam Technology Innovation National Fellowship (INAE/121/AKF/37). Jayakumar Rajendran acknowledges the Ministry of Education, Government of India, for the Prime Minister's Research Fellowship (PMRF) award.

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