Microneedle Patch-Based Transnasal Delivery of Naloxone

Abstract

This study evaluates dissolving microneedle (MN) patches for naloxone (NAL) delivery via the transnasal route, addressing limitations seen with transdermal application of same and the limitations of conventional NAL intranasal sprays, which often require frequent redosing, particularly for long-acting opioids like fentanyl. MN patches composed of polyvinylpyrrolidone (PVP) and PVP/Chitosan were tested on porcine nasal mucosa. PVP patches achieved significantly higher 1-h cumulative permeation (7295.12 ± 2585.17 μg/cm²) compared to transdermal application (103 ± 15.18 μg/cm², p<0.05). Over 24 h, cumulative permeation reached 13,113.20 ± 597.39 μg/cm² transnasally versus 4112.89 ± 773.40 μg/cm² transdermally (p<0.05). Chitosan-PVP MN patches improved bioadhesion and demonstrated high 1-h cumulative permeation (3800.19 ± 940.51 μg/cm²). PVP MN patches with drug-loaded tips (MN/TO, where TO implies “tip only”) delivered 933.90 ± 161.60 μg/cm² in 1 h that was also a remarkable increase over transdermal permeation (p<0.05) but had lower 24 h permeation. Similar observation was seen with the PVP/Chitosan variant with drug loaded in just MN tips indicating that sustained delivery requires drug in both the tips and base. To further refine patch designs, a mathematical modeling framework was employed to simulate drug dissolution, permeation dynamics, and plasma concentration kinetics. Simulations demonstrated that optimized patches could achieve plasma profiles comparable to intranasal and intramuscular administration, while minimizing drug dose and patch size. Increasing drug concentration from 50 to 60 mg/ml decreased permeation, likely due to drug crystallization. Overall, MN patches showed consistent, sustained NAL delivery, providing an alternative option for efficient opioid overdose treatment.

Graphical Abstract

1. Introduction

Transnasal route is advantageous for both local and systemic drug delivery due to its high permeability and vascularity, allowing drugs to bypass the gastrointestinal system, avoid first-pass metabolism and digestive enzyme degradation. Transnasal administration is also being explored for delivering drugs directly to the brain, showing potential for improved efficacy by increasing the maximum concentration (Cmax) and enhancing drug distribution within the brain [1–3]. This is achieved through the olfactory bulb or trigeminal nerve pathways, which provide direct access to the brain. Additionally, drug absorption through the nasal epithelium enters the bloodstream and can reach the brain, for drugs capable of crossing the blood-brain barrier (BBB) [4,5]. Nose to brain delivery for NAL has been established as a clinical option in opioid overdose emergencies. Intranasal NAL sprays are in the market and intranasal NAL delivery via aerosolized sprays have allowed for easier drug administration, over the counter access, and the evasion of the need for specialized intervention and needle related risks with parenteral NAL injections. However, the short half-life of NAL still presents challenges relating to the need for highly concentrated and repeated intranasal doses. This challenge is of utmost significance with long-acting opioids including fentanyl and its analogue with a relatively long half-life of up to 9 h [6]. Poorly sustained effect due to the short half-life of NAL persists with this drug delivery form and therefore places resuscitating overdose patients at risk of re-narcotization as opioids are in circulation in the blood a period far longer than the half-life of NAL. Similar to IV dosing, nasal naloxone sprays may require repeat dosing every 2–3 minutes which typically warrants continuous monitoring of patients by specialized care givers. Further, variability in dosing is typical with sprays, which could be due to surface area of absorption variation and spray device design/ inhalation technique. Nevertheless, a promising alternative to aerosolized sprays for sustained delivery and precise/uniform, and easy dosing are achievable with MNs [4,8,9].

Dissolving polymeric MNs are a non- invasive approach to drug delivery allowing for the creation of micron-sized channels on mucosa to facilitate the diffusion or absorption of drug at a faster rate. They promise controlled release kinetics and better permeability allowing for better therapeutic dose delivery in comparison to what is achievable with single doses of NAL from sprays. The use of this drug delivery type has been showcased in a couple of studies to be valuable in facilitating nose to brain delivery of small molecules including ergotamine and caffeine [10]. In this study, we evaluated our previously [11] fabricated fast dissolving polymeric MNs for transnasal application of NAL. The main objective of this study was to investigate the applicability of the dissolving MN patches for the transnasal delivery of NAL in opioid emergencies; investigate drug delivery parameters crucial to the clinical application of the NAL loaded MNs for use transnasally including flux, permeation rate and pharmacokinetic profile; and assess the feasibility for sustained delivery of NAL through this route with the MNs. The hypothesis is that the presence of high number of capillaries in the nasal mucosa will allow for rapid drug uptake from the nose to the bloodstream and the dissolution of MN tips in microchannels created in the nasal mucosa could allow for sustained delivery of NAL through the polymer matrix used, forming a depot for continuous drug delivery.

2. Materials

NAL, PVP (MW=40000), chitosan 85% deacetylated (1526.464 g/mol), 10X phosphate buffered saline, pH 7.4 (PBS), trifluoroacetic acid (TFA), citric acid monohydrate, potassium chloride, calcium chloride dihydrate, and potassium monophosphate were purchased from Fisher Scientific (Fair lawn, NJ, USA). Methanol was purchased from Concord Technology (Beichen, Tianjin, China). Fresh porcine nasal mucosa was procured from Animal Technologies (Tyler, Texas, USA). Silver/silver chloride electrodes (2 mm ×4 mm) were obtained from A-M systems (Sequim, WA, USA). Methylene blue was obtained from Electron Microscopy Sciences (Hatfield, PA, USA) and polydimethylsiloxane (PDMS) molds from MicroPoint Technologies (Pioneer Junction, Singapore). ScotchPak^™ 1022 was gifted by 3M (St. Paul, MN, USA) and silicone adhesive (BIO-PSA 7–4301) was also provided as a gift sample by Dow Corning Corporation (Washington, DC, USA).

3. Methods

3.1. Fabrication of microneedles

3.1.1. Fabrication of PVP MNs

The mold casting procedure was conducted using PDMS molds (225 array, MN height 600 μm, needle pitch 500 um, patch size 8×8 mm). To make the casting solution, a 7.5% w/v PVP solution was prepared. NAL solution (50 mg/ml) was prepared in the PVP solution as vehicle. We poured 170 μL of this drug in PVP solution into the molds and subjected this to vacuum at room temperature, applying a constant pressure of about −0.6 bar for 5 minutes with repeated degassing steps. Once all air bubbles were removed, an additional 130 μL of the drug in PVP solution was added. Degassing was completed, and the vacuum was maintained under the same conditions for at least an hour before allowing the solution to dry. After 24 hours, the molds were filled to the top with another 130 μL of the drug solution to reinforce and thicken the base of the needles. The needles were then left to dry in a desiccator at room temperature for two more days. Following this, the needles were carefully extracted from the molds and placed onto an adhesive tape backing for skin application.

3.1.2. Fabrication of PVP/Chitosan Microneedles with drug loaded in tips only

For muco-adhesivity in nasal mucosa, Chitosan (2% w/v) was prepared by dissolving in 0.1 N glacial acetic acid. The pH of chitosan solution was adjusted to 6 before use with 0.5 M sodium hydroxide to remove excess acid. To fabricate a PVP/chitosan patch, a 7.5% w/v PVP solution was prepared. This was used to prepare NAL in PVP solution (50 mg/ml). To form MNs with drug loaded in tip only, 170 μL of the NAL in PVP solution prepared was poured into the molds and subjected to vacuum at room temperature, applying a constant pressure of about −0.6 bar for 5 minutes with repeated degassing steps. Once all air bubbles were removed, this was allowed to sit for 24 hours, followed by the addition of 130 μL of blank 2% w/v chitosan solution twice to form base of adequate thickness (~0.1 cm).

3.1.3. Fabrication of PVP/Chitosan Microneedles with drug loaded in whole patch

As described above in Section 3.1.1, all steps were similar up until the addition of 170 μL of the NAL in PVP solution. The base in PVP/Chitosan patch fully loaded with drug was made with a 50 mg/ml NAL solution in chitosan 2% w/v. For this step, 130 μL of the prepared NAL in chitosan solution was added in steps twice and patches left to dry for 24 h. Following this the patches were moved into a desiccator for continuous drying up until use.

3.2. Characterization of PVP/Chitosan microneedles

3.2.1. Digital and Optical Microscope Imaging

MNs were imaged using an Omax brightfield microscope (OMAX MD82EZ10, China) with a 4X scanning objective lens. The MNs were mounted on the microscope stub using a lab-made sample holder, which included double-sided tape, a parafilm bed, and microscope glass slides to position the MNs for sideways viewing. This setup allowed for the evaluation of qualitative properties such as MN morphology, needle-tip integrity, and uniformity. Images were captured using ToupView imaging software.

3.2.2. Dye binding study

Using the well-established MN characterization method, the mechanical integrity and penetration efficiency of the patches were assessed through methylene blue dye staining. Thawed porcine nasal mucosa pieces were gently dabbed with Kim wipes to remove moisture and MNs were applied via thumb pressure. A 1% w/v aqueous solution of methylene blue dye in DI water was applied to nasal mucosa. After 1 min, excess dye was removed with Kim wipes and alcohol swabs, and the treated nasal mucosa was examined under an optical microscope (Omax brightfield microscope (OMAX MD82EZ10, China) with a 10X scanning objective lens.

3.3. Nasal Tissue preparation

Fresh porcine nasal mucosa coupled to olfactory lobes were supplied by Animal Technologies (Tyler, Texas, USA). This was stored in a −20°C freezer and used within one week of receipt. On the day of the experiment, nasal mucosa was thawed in saline solution (0.9% w/v NaCl in water) and sectioned to appropriate thickness of between 0.5–0.7 mm. This was further cut into sizes able to cover the diffusion orifice on the Franz diffusion cells.

3.4. In vitro permeation evaluation

A simulated nasal fluid (SNF) of pH ~6 was prepared (8.77 mg/ml NaCl, 2.98 mg/ml KCl, 0.78 mg/ml CaCl₂.H₂0) as previously described 203. The receptor of Franz cells was filled with 5 ml of the SNF. The prepared nasal mucosa cut to sizes were mounted on Franz cells and the donor of Franz cells were mounted on them. The donor sections of the Franz cells were filled with 300 μL of the SNF. Equilibration was allowed for 15 min. The epithelial barrier integrity of the nasal mucosa was tested by determining electrical resistance across the nasal mucosa. A load resistor R_L (AgCl, 100 kΩ) was placed in the donor solution without allowing contact with the nasal membrane while a silver electrode (5 cm long) was placed in the receptor compartment (300 μL SNF solution). An arbitrary 2 channel waveform generator (RIGOL DG822, 25 MHZ, TEquipment, NJ, USA) and a digital multimeter (RIGOL DM 3068 61/2 digit, TEquipment, Long Branch, NJ, USA) were used for measuring voltage drop across the entire circuit (V_S) and was recorded as seen on the display screen of the multimeter. Resistance was then calculated using the formula:

$$R_S=V_S\cdot \frac{R_L}{V_O-V_S}$$

Where V_S is the voltage drop across the entire circuit, R_L is load resistance and V_O is voltage drop across nasal mucosa 13. V_O and R_L were 100 mV and 100 kΩ, respectively. Nasal pieces with resistance of 4.0 kΩ were retained for the studies.

After measuring the resistance, the donor solution was removed and the area was dried with Kim wipes. The different fabricated MN patches, varying with patch type and drug loading as detailed in Table 1 were applied to the nasal mucosa. We investigated nasal delivery from PVP only MN patches (50 mg/ml) applied with thumb pressure for 2 minutes to ensure microporation (MN-Transnasal). Investigation of delivery from just the needle tips following patch application for 3 minutes on nasal mucosa with removal of patch base thereafter was also explored (MN/TO-Transnasal). This guided further studies to explore bioadhesive MN patches taking into consideration practical application of patches in nasal mucosa. For this the PVP/Chitosan MNs with either drugs in tips only (PVP layer) (MN1/TO-Transnasal) or drug in entire patch both (PVP tips and chitosan base) (MN1/WP-Transnasal) were applied with thumb pressure on nasal mucosa for 2 minutes. The nasal samples with the adhered patches were then mounted on the diffusion orifice of Franz cells for 24 hours. The details on all patches evaluated in this study are summarized in Table 1 below.

Table 1.

Summary details of MN patches fabricated and evaluated for transnasal permeation

Patch Description	Polymer content	Drug Strength	MN Height	Drug Dose	Application Route	Application Time
MN-Transdermal	PVP	50 mg/ml	600 μm	21.5 mg	Transdermal	2 min, patch base adhered on skin
MN-Transnasal	PVP	50 mg/ml	600 μm	21.5 mg	Transnasal	2 min, patch base adhered on nasal mucosa
MN/TO- Transnasal	PVP	50 mg/ml	600 μm	21. 5 mg	Transnasal	3 min, patch base withdrawn from nasal mucosa
MN1/TO	PVP/Chitosan	50 mg/ml	600 μm	8.5 mg	Transnasal	2 min, patch base adhered on nasal mucosa
MN1/WP	PVP/Chitosan	50 mg/ml	600 μm	21.5 mg	Transnasal	2 min, patch base adhered on nasal mucosa
MN2/WP	PVP/Chitosan	60 mg/ml	600 μm	25.8 mg	Transnasal	2 min, patch base adhered on nasal mucosa

In all investigations, receptor samples (300 μL) were collected at various time points: 0, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 5, 6, 8, 22, and 24 hours. These samples were filtered into vials via a 0.22 μm, 13 mm diameter nylon syringe filter, and an equal volume of fresh SNF was added to the receptor chamber after each sample collection. The concentration of the drug in the samples was determined using HPLC. Results for each group were reported as mean ± SE. The cumulative amount of NAL that permeated through the nasal mucosa per unit area was plotted over time. The flux of NAL from the patches was derived from the slope of the linear sections of the plots for both the initial hour and the full 24-hour period.

3.5. Quantitative analysis

HPLC analysis for NAL was done using the Agilent 1100 module (Agilent technologies, CA, USA) as previously described 13. The validated method used was an isocratic elution that involved the use of Kinetex^® biphenyl 100 A◦ 250 × 4.6 mm, 5 μm C18 column (Phenomenex, CA, USA). The mobile phase and volume ratios included 30:60:10 v/v methanol with 0.1% TFA, water, and 0.2 M phosphate buffer pH 4.0, respectively set to flow at the rate of 1.05 mL/min. Sample run time was 8 min with retention seen at about 4.7 min. Drug peaks eluted at wavelength 211 nm. A calibration curve was obtained over a drug concentration range of 0.1–50 μg/mL with 1X PBS used as diluent. Linearity was obtained with fitted plot (R² = 0.9999). The limits of quantification (LOQ) and detection (LOD) were determined as 0.13 and 0.04 μg/mL, respectively.

3.6. Flux and lag time calculation

The lag time to delivery of drug and the flux values were obtained by plotting a graph of the average cumulative drug permeated/ cm² against time. The slope of the linear portion of these graphs were used to predict the flux. Lag time was obtained from the x-intercept of the linear portion of the permeation graphs. Given that interests majorly lie in significant drug release as soon as drug is administered, the permeation and flux profile of drugs between 0–1 h and 1–24 h, upon nasal mucosa treatment with either of these techniques were determined.

3.7. Mathematical model development

In this study, we used our previously published mathematical model for drug delivery via dissolvable microneedle (MN)-based patches [13]. This model has been applied to predict systemic pharmacokinetics of drugs like naloxone and levonorgestrel, and to optimize patch designs for both rapid and sustained release applications to achieve therapeutic levels [13]. It has also been used to study iontophoretically-enhanced dissolvable MN patches for naloxone delivery [14]. Here, we apply the same model to investigate the systemic delivery of naloxone through the transmucosal route using dissolvable MN patches. For brevity, we present four key equations governing MN dissolution, backing material (or baseplate) dissolution, drug concentration in the mucosa, and systemic drug concentration. Readers are encouraged to refer to our earlier work for detailed derivations [13].

3.7.1. Microneedle dissolution

The dissolution process of MNs begins at the tip and progresses toward the base, changing the geometry of the undissolved portion over time. The available surface area ($A\left(t\right)$) for MN dissolution at time $t$ is given by:

$$A(t)=\pi \cdot \left[\frac{\text{tan}\theta }{\text{cos}\theta }\cdot \left(h^2-{\Delta z(t)}^2\right)+{\text{tan}}^2\theta \cdot {\Delta z(t)}^2\right]$$ (1)

Here, $h$ is the height of the MN, $\theta$ is the half-angle at the apex of the cone, and $\Delta z(t)$ is the height of the dissolved portion.

The rate of drug release from the dissolving MN matrix is modeled as:

$$\frac{d{M}_d(t)}{dt}=\beta \cdot k_D\cdot A(t)\cdot \left(c_s-\frac{1-\beta }{\beta }\cdot c(t)\right)\cdot H\left(\frac{\text{Dose}\cdot V_n}{N\cdot V_n+V_{\text{BP}}}-M_d(t)\right),M_d\left(0\right)=0$$ (2)

Here, $\beta$ is the drug loading fraction, $k_D$ is the dissolution constant, $c_s$ is the solubility of the matrix material (i.e., polymer out of which the MN is made), and $c(t)$ is the drug concentration in the mucosa. The Heaviside function $H$ ensures that dissolution occurs only while the released drug mass ($M_d(t)$) remains below the fraction of the total dose contained in a single needle. Here, $V_n$ is the volume of a single MN, $N$ is the total number of MNs in the patch, and $V_{\text{BP}}$ is the volume of the baseplate.

3.7.2. Baseplate dissolution

For patches with a backing material or baseplate, the drug diffuses into the mucosa through the pores created by the MNs. The rate of drug release from the baseplate is given by:

$$\frac{d{M}_{d,d,\text{BP}}(t)}{dt}=\beta \cdot k_D\cdot A_{\text{BP}}(t)\cdot \left(c_s-\frac{1-\beta }{\beta }\cdot c(t)\right)\cdot H\left(\text{Dose}\cdot \left(1-\frac{N\cdot V_n}{N\cdot V_n+V_{\text{BP}}}\right)-M_{d,d,\text{BP}}(t)\right),M_{d,d,\text{BP}}\left(0\right)=0$$ (3)

Here, $A_{\text{BP}}(t)$ represents the available surface area of the dissolving baseplate, calculated as the sum of the lateral and bottom surface areas of the cuboid. The remaining parameters are analogous to those in the MN dissolution equation.

3.7.3. Drug concentration in the mucosa

The total drug concentration in the mucosa is modeled as a balance of drug influx from the MNs and baseplate and drug absorption into the plasma. The rate of change of drug concentration in the mucosa is given by:

$$\frac{dc(t)}{dt}=\stackrel{\mathit{\text{from MN}}}{\overbrace{\frac{N}{V_s}\cdot \frac{d{M}_d(t)}{dt}}}+\stackrel{\mathit{\text{from baseplate}}}{\overbrace{\frac{1}{V_s}\frac{d{M}_{i,BP}(t)}{dt}}}-\stackrel{\mathit{\text{into plasma}}}{\overbrace{\gamma \cdot c(t)}},c\left(0\right)=0$$ (4)

Here, $\gamma$ represents the rate of drug absorption into the bloodstream, $V_s$ is the mucosa volume, and $\frac{d{M}_{i,BP}(t)}{dt}$ is the rate of drug influx from the baseplate governed by Fick’s law.

3.7.4. Drug concentration in the systemic circulation

The drug concentration in the systemic circulation is modeled using a two-compartment pharmacokinetic model. For naloxone, which follows a two-compartment model, the central compartment equation is given by:

$$\frac{d{c}_b(t)}{dt}=\frac{V_s}{V_b}\cdot \gamma \cdot c(t)-\frac{1}{V_b}\cdot \left({\text{Cl}}_b\cdot c_b(t)+Q_{\text{per}}\cdot c_b(t)-Q_{\text{per}}\cdot c_{\text{per}}(t)\right),c_b\left(0\right)=0$$ (5)

Here, $c_b(t)$ and $c_{\text{per}}(t)$ represent drug concentrations in the plasma and peripheral compartments, respectively; $V_b$ is the volume of distribution of plasma compartment, ${\text{Cl}}_b$ is the systemic clearance rate, and $Q_{\text{per}}$ is the intercompartmental mass transfer rate.

This model enables the prediction of naloxone pharmacokinetics following transmucosal delivery and provides a framework for optimizing MN patch designs. For additional context and a detailed derivation of equations, readers are referred to our prior work [13].

3.8. Data analysis

Data analysis was done using GraphPad Prism 9.5.0 and Microsoft Excel. Student t test and its non-parametric alternative, Ordinary one-way ANOVA, one-way ANOVA with Brown-Forsythe and Welch’s correction tests, were applied for statistical analysis depending on the satisfaction of statistical criterions including test for normality and equal variance. A significant level was established with a P-value of less than or equal to 0.05.

4. Results and Discussion

Nasal delivery via MNs is an emerging approach shown to hold potential for the delivery of drugs. It represents another feasible route for the application of MNs beyond transdermal, intraocular, and oral applications for which MNs have been explored in numerous studies. Few research works have demonstrated the reliability of MNs for transnasal application. These have been shown for drugs including rivastigmine and huperzine A[8,9]. Rapid MN delivery via the nose in these studies suggest the possibility of transnasal MNs being able to address key limitations associated with conventional naloxone sprays. While nasal sprays for NAL are user-friendly and readily accessible, the variability seen with spray distribution and inhalation techniques may lead to drug wastage and inconsistent dosing. Further, there is the need for frequent redosing due to the non-continuous delivery profile of aerosolized NAL formulations, hence a major pitfall with NAL use, particularly for opioid overdoses induced by long-acting opioids [6]. This warrants the need for frequent redosing and continuous monitoring even after initial NAL doses via nasal sprays. Therefore, there is the need for a transnasal NAL drug formulation obviating these challenges while also allowing for precise dosing, sustained drug release, lesser drug wastage and noninvasive delivery. MNs, depending on the design type may minimize drug degradation and mucociliary clearance issues, leading to better retention and effectiveness of naloxone. Additionally, they provide a precise and user-friendly administration method that ensures accurate dosing and potentially improves patient comfort and adherence, making them a viable alternative for consideration for emergency naloxone delivery.

The data presented in this study demonstrates for the first time the potential of MN patches as an innovative approach for delivering NAL transnasally in opioid overdose emergencies. The results show several key benefits of MN patches for nasal delivery, including enhanced drug delivery, improved bioadhesivity, and the potential for sustained release. Here, we discuss our findings on MN based transnasally delivered NAL in detail, exploring the implications for patch design and future development.

4.1. Characterization of MNs for nasal application

4.1.1. Optical imaging of MNs

Optical imaging revealed that the fabricated PVP/Chitosan MNs retained a pyramidal shape consistent with the PDMS mold design, with an average needle height of 587.82 ± 6.64 μm, closely matching the manufacturer’s 600 μm specification (Fig. 1). The sharp needle tips and uniform geometry confirmed successful mold replication, which is critical for penetration into nasal mucosa [11].

Fig. 1.

Optical image of PVP/Chitosan MN under a brightfield microscope. (Omax brightfield microscope (OMAX MD82EZ10, China) 10X scanning objective lens.

4.1.2. Dye binding study

As shown in Fig. 2, uniform pores were seen following the dye binding staining process, indicating the MNs possessed adequate strength and mechanical integrity for porating the nasal mucosa. Prior studies reported in the literature have explored PVP NAL loaded MNs for transdermal application and the ability of the MNs to breach the skin barrier and create channels was established [11]. While the nasal mucosa and human skin are distinct in terms of tissue organization which is anticipated to impact drug delivery differently, understanding the structure of the nasal mucosa sheds more clarity to drug delivery findings with MNs through this route. The nasal mucosa is a thin, moist, and highly vascularized membrane with a relatively delicate epithelial layer, allowing for easier drug absorption but may require MNs to be sharp and gentle to avoid tissue damage. On the contrary, the skin has a thicker, more robust stratum corneum that resists penetration, necessitating MNs to be more durable and stronger to penetrate effectively. Therefore, MNs designed for nasal delivery must balance sharpness and bioadhesivity for effective drug delivery and minimized mucociliary clearance, while those for skin need to ensure robustness and durability to overcome the skin’s barrier. These design considerations will ensure that MNs perform optimally in these respective routes of administration, delivering drugs efficiently while maintaining patient comfort and minimizing tissue damage. The PVP/Chitosan MNs in this study are considered viable considering they are dissolving type MNs, sharp enough to penetrate the nasal epithelium creating channels for quick uptake of NAL.

Fig. 2.

Dye binding study showing pores created in nasal epithelium with PVP/Chitosan MN under a brightfield microscope. (Omax brightfield microscope (OMAX MD82EZ10, China) 10X scanning objective lens.

4.2. In Vitro Permeation from MNs.

4.2.1. Nasal permeation from PVP MN patches

Interest in evaluating the transnasal application of our previously fabricated PVP microneedles (MNs) originally developed for transdermal delivery stemmed from the need to further enhance naloxone (NAL) flux to achieve a pharmacokinetic profile suitable for rapid onset opioid overdose reversal. In vitro permeation studies were conducted using the best-performing patch “P3” from prior work [11], referred to in this report as “MN-Transdermal” to directly compare its performance on nasal versus dermal tissue.[11] Transnasal permeation was observed to be greater with the transnasally applied patch “MN-Transnasal” achieving statistically greater (P < 0.05) average cumulative 1 h and 24 h permeation, 7295.12 ± 2585.17 and 13113.20 ± 597.39 μg/cm², respectively over 103.00 ± 15.18 and 4112.89 ±773.40 μg/cm² seen with the same patch when applied transdermally (Fig. 3a and b). This implies a 70- and 3.2-fold change in average cumulative permeation over 1 h and 24 h respectively. Similarly, the flux of NAL in 1 h was statistically higher (P< 0.05) when the dissolving PVP MN was applied transnasally with a value of 3866.95 ± 718.74 μg/cm²/h compared to 102.98 ± 16.03 μg/cm²/h seen with same patch transdermally (Fig. 3c). However, the drug flux of 447.17 ± 114.61 μg/cm²/h seen with the transnasal application in 24 h though higher wasn’t statistically different (P > 0.05) than the flux observed with transdermal application which was 178.827 ± 38.13 μg/cm²/h (Fig .3d). These findings corroborate the hypothesis of this study, with expectations being that the moistness and greater perfusion of the nasal mucosa will aid rapid uptake of NAL following the application of the MNs on the nasal mucosa. Furthermore, PVP, as a rapidly dissolving polymer, dissolved and released drug content more quickly in the nasal mucosa compared to its relatively slower release profile in transdermal delivery. This slower transdermal release may be due to the dissolution rate acting as a limiting step, influenced by the minimal hydration level of the skin’s outermost stratum corneum (SC) layer compared to the more hydrated nasal epithelium. When MNs penetrate the nasal mucosa, the drug is exposed to this moist environment, facilitating quicker dissolution and subsequent absorption into the systemic circulation. The moisture helps dissolve the drug that is embedded within the MNs, allowing it to be readily absorbed through the epithelial layers and into the underlying blood vessels. This rapid dissolution and absorption are key factors in the faster delivery profile observed with transnasal NAL MN delivery. The flux seen with the transnasally applied MNs was also significantly greater than with transdermal NAL MN application. There was a 30-fold difference observed in 1 h (P <0.05). This implied a faster rate of diffusion or absorption.

Fig. 3.

Permeation profile of PVP NAL MN patches applied transnasally compared to transdermal (a) average cumulative permeation in 1 h, (b) average cumulative permeation over 24 h (c) flux in 1 h, (d) flux in 24 h. (** Represents statistically higher than comparison group)

4.2.2. Nasal permeation from PVP MN patches with tips only applied for three minutes

Given the remarkable delivery profile for NAL seen with the transnasally applied NAL PVP MNs, further investigations were done to inform the design of suitable patch size and types specifically for transnasal NAL application. We investigated whether applying the MN tips alone for a duration sufficient to dissolve them (3 minutes) could enable significant and sustained NAL delivery. To test this, MN/TO-Transnasal (details in Table 1) was applied on nasal mucosa in an in vitro study for three minutes until the needle tips dissolved. The patch base was removed, and we assessed the amount of NAL that permeated over a 24-hour period. Compared to MN-Transdermal, MN/TO-Transnasal was observed to have also achieved greater (P < 0.05) permeation in 1 h across porcine nasal mucosa with average cumulative permeation value of 933.90 ± 161.60 μg/cm² (Fig. 3a). However, this significant difference wasn’t sustained up until 24 h (Fig. 3b). The average cumulative permeation with this patch in 24 h was 1597.26 ± 235.83 μg/cm². It was not significantly different (P > 0.05) from the cumulative permeation delivered transdermally. Similarly, while drug flux in 1 h with this patch was significantly greater (P < 0.05) than that of MN-Transdermal (Fig. 3c), and this was 871.65 ± 187.63 μg/cm²/h, the flux over 24 h (Fig. 3d) wasn’t any statistically different (P> 0.05) with a value of 116.77 ± 29.61 μg/cm²/h. As shown in this study, though greater average cumulative permeation and flux were observed with the tip-only applied PVP patch compared to transdermal delivery, this was still significantly lower than what was seen with the full patch. The relevance of maintaining a high concentration gradient, achievable with the whole patch containing 21.5 mg of NAL over a long period was evident. The withdrawal of the patch base reduced the concentration gradient across the nasal epithelium, which resulted in a significantly lower drug delivery profile with MN patches applied for just a few minutes before removing the base. This also accounts for the overall decline in permeation observed from the 8 to 24 h time point (Fig. 3b.). The reduced permeation over time suggests that while tip-only MN patches are effective for achieving immediate drug delivery, they may not sustain NAL release as effectively as patches with drug in both the tips and base. This indicates a potential need to be considered in the refinement of the PVP MN patch design to achieve both rapid and sustained drug delivery.

4.2.3. Nasal permeation from PVP/Chitosan patches

For practical applications, it is crucial for the MN patch for transnasal applications to have bioadhesivity as the use of independent adhesive systems and the need to remove patch post application needs to be bypassed. Further, bioadhesive nasal formulas are known to minimize quick mucociliary clearance in the nasal mucosa [15]. For this reason, a chitosan/PVP patch was formulated with patch MN tips made with PVP polymer whereas the base comprised chitosan. In this study, both PVP/Chitosan (MN1) with drug in tip only (MN1/TO-Transnasal) to explore the prospects of minimizing drug loading in patch and another variant with the drug in the entire patch, both tips and patch base (MN1/WP-Transnasal) were investigated. The feasibility of MN1 adherence to nasal mucosa was seen as shown in Fig. 4. Similar to the PVP only patch, MN-Transnasal as discussed above, the average cumulative permeation and flux in 1 h and 24 h with the PVP-Chitosan patch “MN1/WP-Transnasal”, (Table 1) was greater than that with MN-Transdermal (P < 0.05). Average cumulative permeation values in 1 h and 24 h recorded for these were 103.00 ± 15.18 and 4112.89 ±773.40 μg/cm², respectively. As shown in Fig. 5. (a & b), compared to MN-Transnasal, MN1/WP-Transnasal achieved no significant difference (P>0.05) in 1 h and 24 h for the average cumulative permeation which were 3800.19 ± 940.51 and 17740.66 ± 612.64 μg/cm², respectively. The drug flux in 1 h for MN1/WP-Transnasal (3846.13 ± 733.81 μg/cm²/h) wasn’t statistically different either (P > 0.05) from that of MN-Transnasal (Fig. 5c). However, the 24 h flux with MN1/WP-Transnasal (1244.58 ± 120.83 μg/cm²/h) (Fig. 5d) was statistically higher (P < 0.05) comparing these two. Further, there was no significant difference (P > 0.05) in average cumulative permeation in 1 h with the PVP-Chitosan drug in whole patch “MN1/WP-Transnasal” and the tip only patch “MN1/TO-Transnasal”(1858.32 ± 422.18 μg/cm²). However, over 24 h, the average cumulative permeation with MN1/WP-Transnasal was statistically greater (P < 0.05) than that with MN1/TO-Transnasal (5719.25 ± 159.18 μg/cm²). Similar findings were seen with drug flux for MN1/TO-Transnasal with its 1 h and 24 h flux being 1835.32 ± 416.15 μg/cm²/h (P > 0.05) and 576. 57 ± 35.87 μg/cm²/h (P < 0.05) respectively.

Fig. 4.

Image of Chitosan/PVP patch showing bioadhesive property to nasal mucosa as evident with patch not falling off with vertical fixation of the mucosa mounted MN patch for 1 min.

Fig. 5.

Permeation profile of Chitosan/PVP bioadhesive NAL MN patches transnasally (a) average cumulative permeation in 1 h, (b) average cumulative permeation over 24 h, (c) flux in 1 h, (d) flux in 24 h. (** Represents statistically higher than comparison group)

Overall, the incorporation of chitosan in MN patches improved bioadhesivity (Fig. 4), which reinforces the practical applicability of the patch transnasally. While the average cumulative permeation from PVP/ Chitosan patch wasn’t statistically different from that seen with PVP only patch in the first 1 h, the average cumulative values with the PVP/Chitosan patches were lower (Fig. 5a). This observed decline in PVP/Chitosan patches could be attributed to the slower dissolution of chitosan and its greater sustained delivery potential. However, the cumulative amount delivered and flux through 24 h from this patch was greater than what was obtained with the PVP patch transdermally. Further, limiting drug load in the PVP/Chitosan patches to just the MN tips (Table 1; MN1/TO) impacted delivery negatively. This is attributable to a change in concentration gradient across the nasal epithelium which had significantly reduced drug flux.

4.2.4. Nasal permeation from PVP/Chitosan patches with higher drug loading

To further optimize delivery from the PVP/Chitosan patch (MN1/WP), increase in drug loading was considered to increase drug concentration gradient and flux upon patch dissolution on nasal mucosa. While the mechanical integrity of PVP/Chitosan patches with drug loading as high as 70 mg/ml were compromised and not viable for mucosal penetration and in vitro permeation evaluation, a patch with drug load of 60 mg/ml was fabricated and evaluated in vitro. As shown in Fig. 6 a and b, the 1 h and 24 h permeation was statistically lesser (P<0.05) from the patch with higher drug load 60 mg/ml (MN2/WP) compared to MN1/WP (50 mg/ml). The 1 h and 24 h cumulative permeation for MN2/WP were 1005.07 ± 177.43 μg/cm² and 4396.15 ± 182.03 μg/cm², respectively. Similar to the findings with average cumulative permeation, as shown in Fig 6 c and d, the drug flux with MN2/WP was also statistically lesser (P<0.05) in 1h and 24 h. These were also 1154.45 ± 236.17 and 334.12 ± 31.71 μg/cm²/h, respectively. The impact seen with increasing NAL drug load in PVP/Chitosan patches on average cumulative drug permeation and flux was contrary to expectations. Specifically, MN2/WP, with a drug load of 25.8 mg (60 mg/ml), resulted in significantly lower permeation and flux compared to the original PVP/Chitosan patch (MN1/WP; 21.5 mg (50 mg/ml)). This unexpected outcome may be due to drug crystallization or polymorphism occurring beyond a specific concentration threshold of 50 mg/ml, which negatively affects drug release and dissolution when applied to the nasal mucosa. Observations had been seen during attempts to increase the drug loading to 70 mg/ml in preliminary fabrications that patches with such high drug load compromise the mechanical integrity of the patches, resulting in non-viable formulations. Though MN2/WP patches were physically viable, the high drug content could have reduced its mucosal penetration ability which explains the observed decline in delivery compared to MN1/WP.

Fig. 6.

Effect of increasing drug concentration on (a) average cumulative permeation in 1 h, (b) average cumulative permeation in 24 h, (c) average drug flux in 1h (d) average drug flux in 24 h. (** indicates statistically higher).

Similar crystallization issues were noted in prior reports with PVP-only patches at concentrations above 60 mg/ml [11]. The differing solubility of NAL in PVP and chitosan may affect the drug loading limits for these polymers in patch fabrication. The observed reduction in drug permeation with increased drug loading also aligns with established concerns regarding crystallization in drug formulations; as drug concentrations rise, the likelihood of crystallization also increases. This phenomenon can compromise drug release profiles, leading to diminished formulation performance. Previous studies have shown that crystallization not only affects drug solubility but also significantly impacts permeation rates through biological membranes.

4.3. Model calibration and patch design optimization

The model was calibrated using in vitro cumulative drug release kinetics data (Fig. 7a), with the dissolution rate constant ($k_D$) as the sole adjustable parameter. Least-squares fitting performed in MATLAB yielded $k_D$ = 0.0585 cm/h, consistent with previously reported values [11,13,14]. The goodness of fit was strong, with a Pearson correlation coefficient R = 0.98, confirming the model’s ability to accurately capture the observed dissolution and release dynamics. All model parameters are summarized in Table 2.

Fig. 7. Model calibration and optimization for naloxone delivery.

(a) Model fitting of predicted cumulative drug release to in vitro data from microneedle-based patches. (b) Predicted plasma concentration kinetics of naloxone following transmucosal patch delivery, compared to the clinical gold standards of intranasal (IN) and intramuscular (IM) delivery devices. The goal is to match the plasma concentration profiles achieved with these clinical devices using the patch. (c) A contour plot of the squared error between model predictions and ideal plasma concentration kinetics (achieved with IN and IM devices), showing the non-linear relationship between needle height and total needle count. The color bar represents the magnitude of the squared error, with dark blue indicating the lowest error, identifying the optimal parameter combinations for achieving the desired plasma profile. (d) Optimized plasma concentration profiles of naloxone using three updated patch designs selected from the optimal region of the contour plot, compared to the profiles achieved with IN and IM delivery devices. The inset extends the simulation to 3 hours, highlighting differences in peak concentrations and decay rates

Table 2.

List of model parameters.

Parameter	Description	Units	Value				Reference
			Experimental Design	Optimal Design 1	Optimal Design 2	Optimal Design 3
Patch design parameters
$\rho$	Density of the MN polymer material	$\mu \text{g}\cdot {\text{mL}}^{-1}$	1.5e+6				[13]
$c_s$	Solubility of the MN polymer material	$\mu \text{g}\cdot {\text{mL}}^{-1}$	1.6e+5				[13]
$\text{Dose}$	Total drug mass in the patch	mg	21.5*	30.7	39.3	40.8	Calculated
$\beta$	Drug loading fraction	–	0.219*				–
$\theta$	Half-angle at the apex of the MN cone	°	9.46	8.68	9.46	10.78	Calculated
$r$	MN base radius	cm	0.01*				Fixed
$h$	Needle height	$\mu m$	600*	655	600	525	Optimized
$p_w$	Pitch (center-to-center distance between adjacent MNs)	cm	0.05*	0.025			Fixed
$N$	Number of needles in the patch	–	225*	1156	1521	1600	Optimized
$V_n$	Volume of a single MN	mL	6.3e-6	6.9e-6	6.3e-6	5.5e-6	Calculated
$h_{\text{BP}}$	Baseplate height	cm	0.1*				Fixed
$b_{\text{BP}}$	Baseplate side length	cm	0.8*	0.92	1.05	1.07	Calculated
$V_{\text{BP}}$	Baseplate volume	mL	0.064	0.085	0.110	0.115	Calculated
$k_D$	Dissolution rate constant	cm⋅h−1	0.0585				Fitted
Mucosal tissue parameters
$h_{\text{muc}}$	Thickness of the mucosal tissue	cm	0.07				[16]
$V_s$	Volume of the mucosa	mL	0.0448	0.060	0.077	0.080	Calculated
Drug-related Parameters
$D$	Drug diffusivity in mucosa	cm2⋅h−1	0.003				[11,13,14]
$\gamma$	Drug absorption rate into systemic circulation	h−1	2.44				Calculated
${\text{Cl}}_b$	Systemic clearance rate	mL⋅h−1	2.07e+5				[11,13,14]
$V_b$	Volume of distribution in plasma compartment	mL	1.21e+4				[11,13,14]
$V_{\text{per}}$	Volume of distribution in peripheral compartment	mL	1.02e+5				[11,13,14]
$Q_{\text{per}}$	Intercompartment mass transfer rate	mL⋅h−1	2.84e+5				[11,13,14]

Following model calibration, we predicted the plasma concentration kinetics of naloxone for patch-based delivery and compared these profiles to the clinical gold standards of intranasal (IN) and intramuscular (IM) delivery devices [17] (Fig. 7b). There is a noticeable discrepancy between the predicted patch-based pharmacokinetics and the profiles achieved with IN and IM devices, particularly in the magnitude of the peak of the plasma concentration. This discrepancy underscores the need for design optimization to better match the performance of MN patches to existing clinical delivery devices.

To address this, we systematically optimized the MN patch design by varying two critical parameters: MN height ($h$) and total number of MNs in the patch ($N$). Needle height was sampled between 200 μm and 660 μm, while the number of needles ranged from 0 to 2756, generating a total of 10,000 simulations. The objective was to minimize the squared error between the model-predicted plasma concentration profiles of the patch and the IN/IM data [17].

The optimization results, visualized in the contour plot (Fig. 7c), reveal a non-linear relationship between $h$ and $N$. The dark blue region identifies the optimal parameter combinations where the patch achieves plasma concentrations closest to the IN/IM delivery profiles. The contour plot further revealed a design trade-off, i.e., as the number of needles increases, the required needle height can decrease without compromising performance. This relationship provides design flexibility, enabling the use of shorter needles while maintaining drug delivery efficiency. The higher number of needles increases tissue porosity, creating more pathways for drug permeation and enhancing overall delivery performance, albeit at the cost of a larger drug dose. While the contour plot analysis suggested that increasing the number of microneedles allows for a reduction in needle height without compromising performance, this finding warrants further validation through comparative ex vivo and in vivo studies to confirm its translational relevance.

To explore this trade-off, we selected three representative designs from the optimal region of the contour plot. These include a configuration with few but long needles ($N$ = 1156, $h$ = 655 μm), an intermediate configuration with a moderate count and height ($N$ = 1521, $h$ = 600 μm), and a configuration with many but short needles ($N$ = 1600, $h$ = 525 μm).

As shown in Fig. 7d, all three designs closely replicated the plasma concentration profiles achieved with IN and IM delivery devices, demonstrating the ability of the optimized MN patch to deliver naloxone effectively via the transmucosal route. Design 1 achieved these profiles while reducing the total drug dose by 43% compared to earlier designs required for transdermal delivery [13], and using comparable drug dose to iontophoresis delivery but without the need for applied voltage [14]. This efficiency can be attributed to the combination of optimized patch geometry and the thinner transmucosal barrier (about 700 μm) [16], which enhances drug permeation.

While designs 2 and 3 also achieved the target plasma profile and produced a slightly higher peak concentration (inset, Fig. 7d), they required approximately 30% more total drug dose compared to design 1 (Dose = 30.7 mg), making them less efficient in terms of drug usage (Table 2). Additionally, the larger patch areas associated with designs 2 and 3 (1.1 cm² and 1.15 cm², respectively) further emphasize the trade-off, as these designs would require more extensive skin coverage. Since the patch is being adapted for intranasal application, design 1, with its smaller patch area (0.85 cm²), is better suited for efficient drug delivery, minimizing the total drug dose while still achieving the desired plasma profile. This underscores the balance between achieving higher peak plasma levels and minimizing the total drug dose and patch size. Overall, these results demonstrate the clinical potential of MN-based naloxone delivery via the transmucosal route, offering a rapid, efficient, and minimally invasive alternative to existing IN and IM methods.

Overall, these studies have demonstrated key considerations to be considered in the refinement of an MN patch for transnasal application of NAL for both immediate and sustained delivery and factors to consider for practical designs. The results from this study highlights several implications for the design of MN patches for NAL delivery transnasally and important factors for consideration. Effective MN patches for NAL should incorporate both the mechanical advantages of MNs for enhanced drug delivery and the bioadhesive properties of materials like chitosan to ensure sustained contact with the nasal mucosa. Optimizing the MN design to balance these factors is crucial for achieving both rapid and sustained drug release. Future research shall explore variations in MN designs, including different tip geometries and materials, to further enhance drug delivery performance.

Translating these findings into clinical practice requires further investigation into the safety, efficacy, and practical usability of MN patches transnasally in real-world settings. Further studies will consider histological and inflammatory response profile to comprehensively assess the irritation potential of these transnasal MNs. Future clinical trials will be necessary to evaluate the effectiveness of MN patches in emergency settings, including their usability by first responders and their impact on critical patient outcomes.

5. Conclusion

This study demonstrates that MN patches, particularly those with enhanced bioadhesivity and sustained-release properties hold significant promise for transnasal NAL delivery in opioid overdose emergencies. The improved permeation and flux rates observed with transnasally applied MN patches underscore their potential to enhance the efficacy of NAL, thereby offering another valuable tool in combating the opioid crisis.

Abbreviations:

FDA

Food and Drug Administration

HPLC

High performance liquid chromatography

NAL

Naloxone

MN

Microneedle

IE

Insertion efficiency

IN

Intranasal

IM

Intramuscular

IV

Intravenous

PBS

Phosphate buffered saline

PDMS

Polydimethylsiloxane

PK

Pharmacokinetics

PVP

Polyvinylpyrrolidone

SC

Stratum corneum

TFA

Trifluoroacetic acid

TO

Tip Only

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.