Development and Optimization of PNIPAAm Based Ensifentrine Loaded In-situ Nasal Gel for Improved Bioavailability

doi:10.21203/rs.3.rs-7774769/v1

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Development and Optimization of PNIPAAm Based Ensifentrine Loaded In-situ Nasal Gel for Improved Bioavailability

https://doi.org/10.21203/rs.3.rs-7774769/v1

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Objectives: The primary goal of this research was to develop and optimise PNIPAAm-based in-situ nasal gels for ensifentrine delivery, aiming to enhance drug bioavailability, retention time, and therapeutic outcomes for the treatment of COPD and asthma.

Methods: The formulation was optimized using a 3² full factorial design, with the independent variables being the concentrations of PNIPAAm (1%, 3%, 5% w/v) and HPMC K4M (0.2%, 0.4%, 0.6% w/v). The dependent variables included gelation temperature, mucoadhesive strength, and drug release. Various characterization techniques including solubility studies, DSC, FTIR, viscosity, and ex-vivo permeation were employed to assess the performance of the nasal gel.

Results: Research showed that ensifentrine achieved its maximum solubility value in phosphate buffer solution at pH 6.4 (3.02 ± 0.05 mg/mL). Between 27.1°C and 34.2°C stood as the gelation temperature range and the mucoadhesive strength extended from 0.98 N/cm² to 2.72 N/cm². Family MF7 demonstrated the highest drug permeation rate achieving 91.7 ± 3.1% of cumulative drug delivery throughout an 8-hour ex-vivo permeation experiment. The formulation stability test showed no considerable modifications during three months of accelerated storage.

Conclusion: The PNIPAAm-based nasal gel formulation shows outstanding potential for maintaining drug release duration together with increased ensifentrine bioavailability. The favorable mucoadhesive and gelation properties of formulation MF7 provide evidence that it can serve as a potential alternative for improving drug systemic absorption through nasal administration for future clinical applications in chronic respiratory disease treatment.

PNIPAAm

in-situ nasal gel

ensifentrine

factorial design

mucoadhesion

drug permeation

bioavailability

chronic respiratory diseases

The global prevalence of COPD along with asthma, leads to substantial healthcare expenses and numerous deaths yearly among global citizens [1]. The respiratory disease COPD presents as a condition which progressively worsens while being mostly irreversible since it affects airflow completely and causes the combination of emphysema and chronic bronchitis and develops primarily through extended contact with toxic environmental hazards such as cigarette smoke and air pollutants, alongside occupational risks [2]. The disease produces permanent changes to airways together with bronchial wall expansion and loss of connections between alveoli, while causing excessive mucus secretion [3]. The episodic features of asthma differ from those of COPD because asthma is a chronic inflammatory disease that displays airway hyperresponsiveness, along with reversible bronchoconstriction, after exposure to allergens, cold air, exercise, or pollutants [4]. Both conditions share pathophysiological parallels through increased oxidative stress and cellular inflammatory infiltration, which cause symptom worsening through excessive inflammatory mediator release [5]. The available therapeutic agents, such as bronchodilators, along with corticosteroids and leukotriene receptor antagonists, often fail to achieve optimal clinical results because patients exhibit low drug retention, suboptimal delivery, and systemic side effects, as well as poor inhaler technique and adherence to treatment. Treatment effectiveness requires advanced as well as patient-friendly drug delivery systems that target specific areas and minimize side effects [6]. The chemical structure of ensifentrine is shown in Fig. 1.

The first-in-class medicine ensifentrine functions as a dual PDE 3 and 4 inhibitor which shows great potential to treat COPD along with asthma [7]. The PDE3 inhibitory action of ensifentrine enhances cAMP levels within airway smooth muscle cells, causing smooth muscle relaxation and resulting in bronchodilatory effects that relieve airway constriction [8]. When PDE4 inhibitors are combined with PDE3 inhibitors, they shut off inflammatory cell activation and prevent the release of IL-6, TNF-α, and GM-CSF cytokines and chemokines, resulting in a notable anti-inflammatory effect [9]. The dual benefits of ensifentrine represent substantial medicinal value, as it treats obstructive ventilatory disorders, respiratory diseases, and inflammatory processes [10]. The limited application of ensifentrine in medical practice results from several clinical barriers, which include extensive first-pass hepatic metabolism, low oral availability, speed of systemic elimination, and poor drug uptake. The logical delivery path for respiratory conditions has limitations, including complex device requirements and inconsistent drug deposition in the lungs, as well as substandard patient adherence, particularly among elderly and pediatric patients. Better systemic uptake and therapeutic effectiveness of ensifentrine require an improved delivery route that eliminates the current bottlenecks in accessibility [11].

An appealing solution for nasal medicine delivery consists of thermoresponsive polymer-based in-situ gels as they offer non-invasive drug delivery systems with excellent patient compliance [12]. The nasal cavity exposes medications to abundant microvascular tissue, allowing drugs to easily reach systemic circulation without encountering hepatic first-pass effects, as the epithelial membrane remains extremely thin. The absorption efficiency of traditional nasal formulation drugs becomes restricted because they experience quick mucociliary clearance [13]. The sol-to-gel transition of in-situ gels occurs when these liquid systems transform into a gel form following application due to environmental triggers such as temperature, pH, or ionic variations. The gelation process restricts drug contact with the nasal mucosa surface for extended periods while boosting drug adhesion and creating controlled delivery of medication [14]. The exhaustive scientific investigation of thermosensitive polymers selects Poly(N-isopropylacrylamide) (PNIPAAm) since it demonstrates a lower critical solution temperature (LCST) at 32°C, which perfectly suits nasal applications requiring gelation near room temperature [15]. The biocompatible PNIPAAm-based systems demonstrate both high stability and mechanical properties, as well as temperature reversibility, which makes them excellent matrices for drug delivery applications. The use of PNIPAAm in in-situ manufactured nasal gels enhances drug retention, as well as absorption and bioavailability of ensifentrine, while ensuring efficient systemic drug delivery [16].

2.1. Materials

Ensifentrine (analytical grade, ≥ 98% purity) was obtained from Sciquaint Innovations Pvt. Ltd. (Pune, India). Poly(N-isopropylacrylamide) – PNIPAAm (analytical grade, molecular weight ~ 40 kDa) was procured from Sigma-Aldrich (St. Louis, USA). Hydroxypropyl Methylcellulose K4M (pharmaceutical grade, viscosity 4000 cps) was purchased from Colorcon Asia Pvt. Ltd. (Goa, India). Mannitol (analytical grade, USP specification) was obtained from Loba Chemie Pvt. Ltd. (Mumbai, India). Methyl paraben (analytical grade, ≥ 99% purity) was sourced from S.D. OK Chemicals Ltd. (Mumbai, India). Potassium bromide (spectroscopic grade, > 99% purity), used for FTIR analysis, was obtained from Merck Specialities Pvt. Ltd. (Mumbai, India). Phosphate buffer salts and all other chemicals and reagents used were of analytical grade and were procured from reputable commercial sources. All materials were used as received without further purification.

2.2. Methods

2.2.1. Calibration curve determination

A solution containing 100 µg/mL ensifentrine was prepared by dissolving 10 mg of the drug in 100 mL of phosphate buffer at pH 6.4. Proper aliquots were extracted from the stock solution, which was then diluted with phosphate buffer to prepare standard solutions at concentrations of 5, 10, 15, 20, 25, and 30 µg/mL. The Shimadzu UV-1900 double-beam UV-visible spectrophotometer was used to measure absorbance readings, utilising phosphate buffer at pH 6.4 as the blank solution. A calibration curve was constructed by plotting absorbance measurements alongside drug concentration values to determine both the linear regression equation and correlation coefficient value (R²) for method linearity. The established calibration data enabled researchers to determine the drug content in their formulations during testing [17].

2.2.2. Determination of solubility in different solvents

The solvent saturation method was used to determine ensifentrine's solubility in distilled water, ethanol, methanol, phosphate buffer (pH 6.4), and simulated nasal fluid. Each glass vial received an excessive amount of ensifentrine and contained 10 mL of individual solvent solutions to achieve saturation. The vials underwent continuous agitation at 25 ± 2°C for 24 hours on a mechanical shaker to establish equilibrium. The equilibrated suspensions were filtered through Whatman filter paper No. 1 to collect solutions free from drug particles. Subsequent drug analysis with a Shimadzu UV-1900 double-beam UV-Visible spectrophotometer required proper dilution of the clear supernatant samples. New measurements determined the solubility of ensifentrine while following the standard measurement unit of milligrams per millilitre (mg/mL) [18].

2.2.3. Differential scanning colourimetry (DSC)

A DSC analysis was conducted to examine the thermal behaviour of the pure drug and various physical mixtures with excipients, evaluating possible drug-excipient interactions. Aluminium pans contained accurately weighed drug samples and physical mixture samples in a 1:1 ratio to undergo thermal analysis using a DSC instrument. The measurements were taken using nitrogen gas at a flow rate of 50 mL/min during both the temperature rise from 30°C to 300°C at a rate of 10°C/min. An analysis of the thermograms searched for any modifications in melting point behaviour or shifts between endothermic and exothermic transitions, together with peak appearance and disappearance patterns as markers for potential incompatibilities or interactions between the drug and excipients [19].

2.2.4. Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared Spectroscopy (FTIR) provides a method for studying how the drug interacts with its formulation components, as it detects changes in chemical bonds. The research evaluated both individual drug specimens and mixed drug and excipient components at a 1:1 weight ratio. The preparation method involved combining samples with dry potassium bromide (KBr) and then pelletizing them through a hydraulic press. Sample analysis was performed using an FTIR spectrophotometer, with spectra ranging from 4000 to 400 cm⁻¹. The analysis of the drug peaks and those from the physical mixture triggered alarms for peak modifications and the emergence or disappearance of new peaks, as these phenomena could signify drug-excipient binding interactions. [20].

2.2.5. Experimental design

A 3² complete factorial design with two factors and three levels was utilised to optimise the in-situ nasal gel formulation. The independent variables were the concentrations of PNIPAAm (A) and HPMC K4M (B), each evaluated at three levels: low (-1), medium (0), and high (+ 1). PNIPAAm (A) was tested at 1%, 3%, and 5% w/v, while HPMC K4M (B) was tested at 0.2%, 0.4%, and 0.6% w/v. The dependent variables measured included gelling temperature (R1), Mucoadhesive strength (R2), and percentage drug release (R3). Nine formulations were prepared according to the factorial design, and the experimental design was created and analysed using Design-Expert software (version 13.0, Stat-Ease Inc.), as shown in Tables 1 and 2 [20, 21]. The experimental layout of independent variables and levels is presented in Table 1.

Table 1
3² Factorial Design showing independent factors and Levels.
Independent Variables
Label	Factors	Level (% w/v)
Label	Factors	Low (-)	Medium	High (+)
A	PNIPAAm	1	3	5
B	HPMC K4 M	0.2	0.4	0.6
Dependant Variables
R1	Gelling temperature (◦C)
R2	Mucoadhesive strength
R3	% Drug diffused (after eight h)

The detailed composition of each formulation batch is listed in Table 2.

Table 2
Preparation of Hydrogel batches using 3² factorial designs
Ingredients	MF1	MF2	MF3	MF4	MF5	MF6	MF7	MF8	MF9
Ensifentrine (% w/v)	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3
PNIPAAm (% w/v)	1	3	5	1	3	5	1	3	5
HPMC K4M (% w/v)	0.2	0.2	0.2	0.4	0.4	0.4	0.6	0.6	0.6
Mannitol (% w/v)	5	5	5	5	5	5	5	5	5
Methyl Paraben (% w/v)	0.018	0.018	0.018	0.018	0.018	0.018	0.018	0.018	0.018
DD water (q.s)	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s

2.3. Formulation of PNIPAAm based ensifentrine loaded In-situ nasal

According to the experimental design, a cold method was employed to prepare in situ nasal gel formulations. The procedure for preparing formulations F1–F9 began by slowly adding PNIPAAm (1–5% w/v) to cold, distilled water at 4°C, while continuously stirring, until complete hydration was achieved. HPMC K4M (0.2–0.6% w/v), along with mannitol (5% w/v), was diluted with a small volume of cold distilled water. Both solutions were then combined with gentle stirring until minimal air bubbles remained. The preservative Methyl paraben (0.018% w/v) was received in a small quantity of warm water, followed by its addition to the solution. The drug ensifentrine received accurate measurements for weight before being dissolved in a small solution of phosphate buffer at pH 6.4. This solution was then added under continuous stirring to achieve a homogeneous distribution of the drug throughout the polymer solution. The disused water filled the remaining solution volume before the cold storage (4°C) of the formulated products at the laboratory. The formulations were prepared under aseptic conditions, employing sterile techniques throughout [22, 23].

2.4. Characterization of hydrogel

2.4.1. Appearance and clarity

Visual inspection of the prepared in-situ nasal gel formulations revealed the appearance and clarity. Under adequate lighting, each formulation was observed against a black and white background to determine colour, turbidity, particles and phase separation. Homogeneity and transparency were also examined for the formulations. Any precipitation, cloudiness or inhomogeneity in the gel structure was observed. Only those formulations with a clear, uniform, and transparent appearance without visible particles were regarded as acceptable for further studies [24].

2.4.2. pH determination

A digital pH meter measured the in-situ nasal gel formulation pH after it had received previous calibration using standard buffer solutions that spanned the pH range of 4.0, 7.0, and 9.0. The pH measurements required placing enough sample in a clean beaker, followed by immersion of the pH electrode directly into each sample. The system recorded the pH measurement only after the reading became stable. Three trials of the measurement were administered for each formulation, during which the average reading value was determined. The pH range objective for nasal formulations was set between 4.5 and 6.5 since these values deliver both mucosa suitability and reduce nasal irritancy [25, 26].

2.4.3. Drug content uniformity

Nasal gel formulation drug content uniformity tests determined the consistent distribution of ensifentrine content throughout the formulations. A precise weight of 1 mL from each formulation was added to a 10 mL volumetric flask for individual treatment before the addition of pH 6.4 phosphate buffer. A 15–20 minute stirring step followed the extraction process to achieve full drug recovery before performing filtration through Whatman filter paper No. 1. The tests used the Shimadzu UV-1900 double-beam UV-Visible spectrophotometer to examine the diluted filtrate. The drug content analysis was performed by measuring the absorbance against a blank reference and then determining the drug composition using the established calibration curve. Three replicated measures of each formulation underwent testing, with all results showing the standard deviation from the mean [27, 28].

2.4.4. Gelation temperature

A test tube inversion system was used to measure the gelation temperature values of in-situ generated nasal gel formulations. Each route was administered 2 mL of solution, which was placed in an individual, clean, dry test tube and maintained within the water bath. The phases of the water bath underwent a temperature increase of one degree Celsius per minute, starting from 25°C and reaching 45°C, while testers made continuous observations. The test tube tilt method allowed monitoring the flow pattern of each formulation during the observation timeframe. Characteristic of gel formation occurred when the test tube was reversed, resulting in the cessation of formulation flow at the gelation temperature point. The experiment required three repeated trials and utilised the mean data for each formulation [29, 30].

2.4.5. Gelation time

The gelation time of the in-situ nasal gel formulations was determined using the test tube inversion method. Approximately 2 mL of the formulation was transferred into a test tube and placed in a water bath maintained at a constant temperature of 37 ± 0.5°C, simulating the conditions of the nasal cavity. The test tube was observed continuously, and the time required for the formulation to transform from a liquid to a non-flowing gel upon inverting the test tube was recorded as the gelation time. The test was performed in triplicate for each formulation, and the average value was calculated and reported [31].

2.4.6. Viscosity

The measurement of in-situ nasal gel formulation viscosity occurred on a Brookfield digital viscometer (Model: Brookfield DV2T) equipped with a suitable spindle (Spindle No. 62 or 63) selection determined by sample viscosity range. Each formulation received approximately 10 mL of solution for clean beaker testing, while viscosity measurements were conducted at room temperature (25 ± 1°C) and physiological temperature (37 ± 0.5°C) to determine sol-to-gel transition properties. The spindle ran at a predetermined speed (10 or 20 rpm) until stabilisation, at which point the viscosity records in centipoise (cP) were recorded. The experiment was performed in three duplicate tests, followed by averaging the recorded values [32].

2.4.7. Spreadability

The researchers tested the spreadability of their in-situ nasal gel formulations through the glass slide method. The glass slide received 1 gram of gel positioned precisely at its centre. A second glass slide, matching the weight and dimensions of the ones placed under the gel, was gently set on top to produce a homogeneous thin layer. The phase lasted 5 minutes as researchers applied a 500-g weight to the upper glass surface. Two perpendicular measurements of diameter were recorded after removing the weight, using either a ruler or calliper, to determine the diameter of the spread gel in centimetres. We calculated the spreadability value by averaging both measurement results. The exam was executed three times for each drug composition, with average calculation and standard deviation presentation [33].

2.4.8. Mucoadhesive strength

The mucoadhesive strength assessment of in-situ nasal gel formulations was conducted using a modified physical balance system. Scientists received goat nasal mucosa upon fresh extraction from a local slaughterhouse, then cleaned them with a pH 6.4 phosphate buffer solution for subsequent mounting on the balance platform through cyanoacrylate application. Approximately 0.5 g of gel material was placed on a fixed mucosal section, which rested on the upper platform. A firm contact between the mucosal tissues lasted for two minutes to achieve proper adherence. The balance required a gradual addition of weight to its opposite pan until both mucosal surfaces became slightly separated. The measurement scale recorded the weight necessary to break the adhesive bond between the surfaces, which became the mucoadhesive strength expressed in grams. The entire procedure was performed three times per formulation to generate an average reading [34].

2.4.9. Ex vivo permeation study using nasal mucosa

The ex vivo permeation assessment utilised recently collected goat nasal mucosa, which was obtained from a local slaughterhouse and used prior to a 2-hour cut-off. A Franz diffusion cell received a mucosal tissue after the researcher cleaned it with pH 6.4 phosphate buffer solution. A phosphate buffer solution at pH 6.4, measuring 20 mL, was filled into the receptor compartment while maintaining a temperature of 37 ± 0.5°C under continuous stirring at 50 rpm using a magnetic stirrer. The nasal gel formulation was administered in a measured amount of 1 mL before being added to the donor compartment. The study sampled 1 mL of solution from the receptor compartment at each time point (0, 1, 2, 4, 6, 8, 10, and 12 hours) while simultaneously replacing it with 1 mL of fresh buffer solution to maintain the drug in a sink condition. The evaluation process incorporated filter-based dilutions of the samples, which were analysed using a Shimadzu UV-1900 double-beam UV-Visible spectrophotometer. The evaluation of drug permeation profile occurred through the determination of the drug amount permeated per unit area (µg/cm²) versus time measurement [35, 36].

2.4.10. Stability studies under ICH conditions

Using the ICH Q1A(R2) guidelines, stability tests were applied to the optimised PNIPAAm-based ensifentrine in-situ nasal gel to assess formulation stability across various storage environments. The formulation was stored in closed, light-protecting containers under two storage environments: 40 ± 2°C / 75 ± 5% RH (accelerated condition) and 4 ± 2°C (refrigerated condition). The experiment collected samples at periodic stages from time zero through 1 month, 2 months, and 3 months to evaluate appearance, clarity, pH value, gelation temperature, viscosity level, and drug concentration, as well as in vitro drug release performance. The physical stability analyses relied on visual inspection for any modifications that could affect colour appearance or create precipitate or phase separation. In contrast, chemical stability measurements relied on determining the drug content using a Shimadzu UV-1900 UV-Visible spectrophotometer. The formulation maintained its initial parameters while researchers compared these values with the impacts of storage conditions on product stability [37].

3.1. Calibration curve

The phosphate buffer solution at pH 6.4 exhibited a linear association between ensifentrine absorbance and drug concentration, with a substantial R² value. The methodology demonstrates reliable performance for measuring ensifentrine concentration levels in nasal gels, as it exhibits linear behaviour. The drug content evaluation for formulation samples exploited the absorbance results collected from six ensifentrine concentrations ranging from 5 to 30 µg/mL. The analytical method demonstrates superior effectiveness due to its precise and reproducible calibration curve (Fig. 2).

3.2. Results of solubility studies

The solubility data for ensifentrine in various solvents revealed maximum solubility levels of 2.35 ± 0.04 mg/mL in methanol and 1.92 ± 0.03 mg/mL in ethanol (Table 3). Additionally, solubility was observed in phosphate buffer at pH 6.4 and simulated nasal fluid, with solubility levels of 3.02 ± 0.05 mg/mL and 1.05 ± 0.03 mg/mL, respectively. Drug tests indicate that phosphate buffer at pH 6.4 functions as the best solution carrier for ensifentrine, which means it can be effectively incorporated into nasal gels for improved bioavailability. Based on its 0.48 ± 0.02 mg/mL water solubility value, it becomes necessary to consider adding solubilising elements or reformulating the drug to enhance drug release during aqueous settings.

Table 3
Solubility Profile of Pure Ensifentrine in Various Solvents Using Solvent Saturation Method
Solvent	Solubility of Ensifentrine (mg/mL)
Distilled Water	0.48 ± 0.02
Ethanol	1.92 ± 0.03
Methanol	2.35 ± 0.04
Phosphate Buffer (pH 6.4)	3.02 ± 0.05
Simulated Nasal Fluid	1.05 ± 0.03
values are expressed as mean ± SD (n = 3)

3.3. Differential scanning colourimetry

DSC analysis (Figs. 3 and 4) demonstrated that pure ensifentrine has a melting point of -177.73°C, which corresponds to the known thermal properties of the drug substance. The DSC thermogram of the drug-excipient physical mixture showed two separate endothermic peaks, appearing at 176.38°C and 213.42°C, indicating no substantial drug-excipient interactions that would impact stability or release behaviour. The peak shifts in the measurements confirmed that the drug crystals maintained their primary structure throughout gel preparation, indicating good formulation stability.

3.4. Fourier Transform Infrared Spectroscopy

The FTIR spectra analysis included pure ensifentrine (Fig. 5A) and its physical mixture with excipients (Fig. 5B) to determine their compatibility. Following analysis of the spectra for both samples, no substantial chemical reactions were detected between the drug substance and excipients. The fact that both drug-excipient combinations showed similar spectral readings demonstrates that the compound remained compatible for future release in a nasal gel product.

3.5. Evaluation of In situ nasal gel

The prepared in-situ nasal gel formulations were evaluated for their appearance and clarity through visual examination, as shown in Table 4. The prepared formulations (MF1-MF9) displayed a uniform appearance, as most were transparent or colourless to the eye. The gel formulations displayed stable characteristics without phase separation or particulate matter; therefore, they meet the criteria for clinical and further characterisation purposes. Multi-factorial tests established that formulations with uniform, stable and precise characteristics qualify for the following analysis.

Table 4
Visual Assessment of Appearance and Clarity of PNIPAAm-Based Ensifentrine In-Situ Nasal Gel Formulations
Formulation Code	Appearance	Clarity	Observations
MF1	Colorless	Clear	Uniform and stable
MF2	Colorless	Clear	Uniform and stable
MF3	Slightly translucent	Clear	Uniform and stable
MF4	Colorless	Clear	Uniform and stable
MF5	Slightly translucent	Clear	Uniform and stable
MF6	Colorless	Clear	Uniform and stable
MF7	Slightly translucent	Clear	Uniform and stable
MF8	Colorless	Clear	Uniform and stable
MF9	Slightly translucent	Clear	Uniform and stable

3.6. Results of physicochemical evaluation

The assessment of PNIPAAm-based ensifentrine nasal gels (Table 5) involved testing their physicochemical properties, including pH level, gelation time, viscosity, and drug content measurement. The pH measurements, between 6.1 ± 0.1 and 6.6 ± 0.1, satisfied the nasal application requirements (pH 4.5–6.5), which ensures compatibility with the nasal mucosa and avoids potential irritation. The onset of drug release following nasal administration may be faster for MF9 due to its short gelation time of 25 ± 1.2 seconds. All drug contents measured in the different formulations exhibited consistent results, ranging from 97.2% to 99.5%, leading to equal ensifentrine distribution in the gel. Changes in viscosity data revealed the successful gelation of all formulations, as the measurements increased substantially from 215 cP to 598 cP before gelation and then to 3650 cP to 5587 cP after gelation. This sol-to-gel transition is vital for maintaining drug release and tissue retention within the nasal cavity.

Table 5
Physicochemical Characterisation of Ensifentrine In-situ Nasal Gel Formulations
F. Code	pH	Gelation Time (sec)	Viscosity before gelation (cP)	Viscosity after gelation (cP)	Drug Content (%)	Spreadability (g·cm/sec)
MF1	6.5 ± 0.2	45 ± 2.1	215 ± 8.2	3650 ± 52.4	97.8 ± 1.2	26.3 ± 1.4
MF2	6.4 ± 0.1	38 ± 1.9	342 ± 10.5	4128 ± 67.3	98.6 ± 0.9	21.7 ± 1.2
MF3	6.3 ± 0.2	32 ± 1.5	486 ± 12.8	4875 ± 75.2	99.1 ± 0.7	18.4 ± 0.9
MF4	6.6 ± 0.1	42 ± 1.8	287 ± 9.7	3982 ± 58.6	97.5 ± 1.3	23.5 ± 1.3
MF5	6.4 ± 0.2	35 ± 1.6	398 ± 11.3	4576 ± 70.1	98.9 ± 0.8	19.8 ± 1.1
MF6	6.2 ± 0.1	28 ± 1.3	537 ± 13.5	5214 ± 82.7	99.3 ± 0.6	16.2 ± 0.8
MF7	6.5 ± 0.2	39 ± 1.7	342 ± 10.1	4231 ± 63.5	97.2 ± 1.4	20.6 ± 1.2
MF8	6.3 ± 0.1	31 ± 1.4	465 ± 12.4	4895 ± 76.8	98.5 ± 0.8	17.3 ± 0.9
MF9	6.1 ± 0.1	25 ± 1.2	598 ± 14.7	5587 ± 89.3	99.5 ± 0.5	14.1 ± 0.7
Values are expressed as mean ± SD, (n = 3)

An investigation into the gelation temperature and mucoadhesive strength was conducted for nasal gel formulations (Table 6). Formulations containing lower PNIPAAm quantities (1–2%) achieved the suitable gelation temperature range of 32–35°C for nasal use. The temperature behaviour of PNIPAAm gels follows expectations because this polymer forms a gel above room temperature. The mucoadhesive strength increased from 0.98 N/cm² to 2.72 N/cm² when higher amounts of HPMC K4M were used in the formulations. Formulations containing PNIPAAm at 5% together with HPMC K4M at 0.6% demonstrated the strongest bond between the formulation and mucous membranes. The superior mucoadhesive properties enable the nasal gel to stay within the cavity longer, thus providing better drug absorption.

Table 6
Response Variables for Optimisation of Ensifentrine In-situ Nasal Gel Formulations
F. Code	Gelation Temperature (°C)	Mucoadhesive Strength (N/cm²)
MF1	34.2 ± 0.8	0.98 ± 0.06
MF2	30.5 ± 0.7	1.25 ± 0.08
MF3	28.1 ± 0.6	1.32 ± 0.07
MF4	33.7 ± 0.7	1.45 ± 0.09
MF5	29.8 ± 0.6	1.98 ± 0.12
MF6	27.5 ± 0.5	2.15 ± 0.13
MF7	34.1 ± 0.7	1.87 ± 0.11
MF8	29.2 ± 0.6	2.56 ± 0.15
MF9	27.1 ± 0.5	2.72 ± 0.16
Values are expressed as mean ± SD, (n = 3)

Optimization Results for PNIPAAm-based Ensifentrine In-situ Nasal Gel

Effect of Independent Variables on Gelation Temperature

Statistical analysis of the gelation temperature data revealed that the quadratic model was the most suitable for describing the relationship between formulation variables and gelation temperature. As shown in Table 7, the quadratic model demonstrated a sequential p-value of 0.0402, a non-significant lack of fit (p = 0.9898), and the highest adjusted R² value (0.9549) among all the evaluated models. The ANOVA results presented in Table 8 confirmed the significance of the overall model (F-value = 155.79, p = 0.0008), indicating that the selected quadratic model adequately described the variation in the data. PNIPAAm concentration (factor A) emerged as the most influential parameter affecting gelation temperature, with an exceptionally high F-value of 742.50 (p = 0.0001). HPMC K4M concentration (factor B) also exhibited a significant effect on gelation temperature (F-value = 11.48, p = 0.0428), albeit to a lesser extent compared to PNIPAAm. Notably, the quadratic term A² significantly influenced the response (F-value = 21.59, p = 0.0188), indicating a non-linear relationship between PNIPAAm concentration and gelation temperature.

The following polynomial equation expresses the mathematical relationship between the formulation variables and gelation temperature in terms of coded factors:

Gelation Temperature (°C) = + 29.70–3.22A − 0.4000B − 0.2250AB + 0.9500A² + 0.2000B²

(1)

The contour and response surface plots (Figs. 7A and 7B) illustrate the influence of polymer concentrations on the gelation temperature. A pronounced decrease in gelation temperature was observed with increasing PNIPAAm concentration, as evidenced by the high negative coefficient (-3.22) for factor A in the polynomial equation. The temperature decreased from approximately 34°C at 1% w/v PNIPAAm to around 27°C at 5% w/v PNIPAAm. The effect of HPMC K4M was relatively modest, with only a slight decrease in gelation temperature as HPMC K4M concentration increased from 0.2% to 0.6% w/v. The curvature observed in the response surface plot was primarily attributed to the significant quadratic term A², confirming that the effect of PNIPAAm on gelation temperature was not strictly linear, particularly at higher concentrations. The optimal gelation temperature range (32–35°C) for nasal application was achieved at lower PNIPAAm concentrations (1–2% w/v), with minimal influence from HPMC K4M concentration.

Effect of Independent Variables on Mucoadhesive Strength

The model fit summary for mucoadhesive strength indicated that the quadratic model was best, as it had a sequential p-value of 0.0529, a non-significant lack of fit (p = 0.9883), and an adjusted R² of 0.9466. The results of the ANOVA in Table 8 showed that the overall model was highly significant (F value = 136.35, p = 0.0010). Generally, compared to the gelation temperature, HPMC K4M concentration (factor B) had the highest F value of 508.12 (p = 0.0002) among all factors affecting mucoadhesive strength. However, mucoadhesive strength (F = 140.05, p = 0.0013) decreased significantly with PNIPAAm concentration (factor A), although to a lesser degree than with HPMC K4M. The interaction term AB also had a significant effect (F = 15.30, p = 0.0297), indicating a synergistic interaction between the two polymers. This was also significant with the quadratic term A² (F-value = 15.53, p = 0.0291) indicating that the mucoadhesive strength is apparently not linear with respect to PNIPAAm concentration.

The polynomial equation representing the relationship between formulation variables and mucoadhesive strength was:

Mucoadhesive Strength (N/cm²) = + 1.98 + 0.3150A + 0.6000B + 0.1275AB − 0.1817A² − 0.0767B²

(1)

The effects of polymer concentrations on mucoadhesive strength were visualised using polymer concentrations and contour plots, as well as response surface plots (Fig. 7C and 7D). The high positive coefficient (+ 0.6000) of factor B in the polynomial equation indicates a significant increase in mucoadhesive strength with an increase in the per cent concentration of HPMC K4M. The mucoadhesive strength were on the order of approximately 0.98 N/cm² at 0.2% w/v HPC K4 M and 2.72 N/cm² at 0.6% w/v HPC K4 M. Values of mucoadhesive strength increased from 0.98 N/cm² at 1% w/v PNIPAAm to approximately 1.32 N/cm² at 5% w/v PNIPAAm when HPMC K4M was fixed at 0.2% w/v positively correlated with PNIPAAm concentration. The contour plot revealed the evident presence of a positive coefficient for the interaction term AB (AB + 0.1275), as the highest mucoadhesive strength was obtained at the highest concentration of both polymers. For the significant quadratic terms, the slight curvature in the response surface plot was attributed to A² (-0.1817), which has a plateau effect, i.e., a plateau at higher PNIPAAm concentrations. Overall, the mucoadhesive strength was maximised at high concentrations of both polymers, but the dominant polymer was HPMC K4M.

Table 7
Model Fit Summary for Response Variables of Ensifentrine-loaded PNIPAAm In-situ Nasal Gel
Source	Sequential p-value	Lack of Fit p-value	Adjusted R²	Model Status
Gelation Temperature (°C)
Linear	< 0.0001	0.9523	0.9167
2FI	0.5218	0.9477	0.8419
Quadratic	0.0402	0.9898	0.9549	Suggested
Cubic	0.2995	0.9972	0.9373	Aliased
Mucoadhesive Strength (N/cm²)
Linear	0.0002	0.9288	0.8591
2FI	0.1166	0.9502	0.8439
Quadratic	0.0529	0.9883	0.9466	Suggested
Cubic	0.0148	1.0000	0.9998	Aliased

Table 8
ANOVA Results for Quadratic Models of Response Variables
Source	Sum of Squares	df	Mean Square	F-value	p-value	Significance
Gelation Temperature (°C)
Model	65.135	5	13.03	155.79	0.0008	significant
A-PNIPAAm	62.08	1	62.08	742.50	0.0001	significant
B-HPMC K4M	0.9600	1	0.9600	11.48	0.0428	significant
AB	0.2025	1	0.2025	2.42	0.2175	not significant
A²	1.81	1	1.81	21.59	0.0188	significant
B²	0.0800	1	0.0800	0.9568	0.4001	not significant
Mucoadhesive Strength (N/cm²)
Model	2.905	5	0.5796	136.35	0.0010	significant
A-PNIPAAm	0.5954	1	0.5954	140.05	0.0013	significant
B-HPMC K4M	2.16	1	2.16	508.12	0.0002	significant
AB	0.0650	1	0.0650	15.30	0.0297	significant
A²	0.0660	1	0.0660	15.53	0.0291	significant
B²	0.0118	1	0.0118	2.77	0.1949	not significant

Validation of the statistical model

The optimised formulation and its validation parameters are summarised in Table 9.

Table 9
Validation of Optimised Formulation of Ensifentrine-loaded PNIPAAm In-situ Nasal Gel
Factor/Response	Predicted Value	Experimental Value	Percentage Error (%)
Formulation Variables
PNIPAAm (% w/v)	1.0	1.0	-
HPMC K4M (% w/v)	0.6	0.6	-
Critical Quality Attributes
Gelation Temperature (°C)	33.89	34.1	0.62
Mucoadhesive Strength (N/cm²)	1.88	1.87	-0.53
Desirability	0.845	-	-
Ex vivo Permeation Study

Figure 8 and Table 10 present the ex vivo permeation results, illustrating an increase in the percentage of drug permeated over time. MF8 demonstrated the highest cumulative permeation in the studies performed (91.7 ± 3.1 after 8 hours), proving the effectiveness of the drug's diffusion through the nasal mucosa. It thus suggests that the ensifentrine-loaded nasal gels based on PNIPAAm are suitable and sustained carriers of ensifentrine better than other delivery systems, which might require low bioavailability or unadhesive.

Table 10
Ex vivo Permeation Study of Ensifentrine from In-situ Nasal Gel Formulations
Formulation	1 h	2 h	3 h	4 h	5 h	6 h	7 h	8 h
MF1	11.2 ± 0.8	19.5 ± 1.2	27.4 ± 1.5	35.8 ± 1.7	44.2 ± 1.9	52.7 ± 2.1	59.3 ± 2.3	65.3 ± 2.4
MF2	15.4 ± 0.9	26.8 ± 1.4	37.5 ± 1.8	48.2 ± 2.0	57.9 ± 2.3	65.7 ± 2.5	72.3 ± 2.6	76.8 ± 2.7
MF3	13.8 ± 0.8	24.3 ± 1.3	34.6 ± 1.7	45.1 ± 1.9	54.2 ± 2.1	62.5 ± 2.3	68.7 ± 2.4	72.4 ± 2.5
MF4	12.5 ± 0.9	21.7 ± 1.3	30.4 ± 1.6	39.2 ± 1.8	48.5 ± 2.0	56.8 ± 2.2	63.4 ± 2.4	69.1 ± 2.6
MF5	17.2 ± 1.0	29.5 ± 1.5	41.3 ± 1.9	52.7 ± 2.2	63.4 ± 2.5	72.8 ± 2.7	79.5 ± 2.8	83.5 ± 2.9
MF6	16.0 ± 0.9	27.8 ± 1.4	39.2 ± 1.8	50.5 ± 2.1	60.7 ± 2.3	69.6 ± 2.5	75.8 ± 2.7	79.2 ± 2.8
MF7	12.3 ± 0.8	21.5 ± 1.2	30.1 ± 1.6	38.7 ± 1.8	47.9 ± 2.0	56.3 ± 2.2	62.9 ± 2.4	68.2 ± 2.5
MF8	19.3 ± 1.1	33.6 ± 1.6	46.8 ± 2.0	59.7 ± 2.3	71.4 ± 2.6	81.3 ± 2.8	87.5 ± 3.0	91.7 ± 3.1
MF9	17.6 ± 1.0	30.4 ± 1.5	42.5 ± 1.9	54.3 ± 2.2	65.2 ± 2.5	74.5 ± 2.7	80.7 ± 2.8	84.3 ± 2.9
Values are expressed as mean ± SD, (n = 3)

Accelerated Stability Studies of Optimized Formulation

The stability studies of the optimised formulation (MF7) under accelerated conditions (Table 11) revealed no significant changes in physical appearance, gelation temperature, viscosity, or drug content over a 3-month storage period. The pH, viscosity before and after gelation, and mucoadhesive strength remained within acceptable ranges, indicating that the formulation is stable under both refrigerated and accelerated storage conditions. This stability profile ensures that the formulation maintains its efficacy and quality throughout its shelf life, supporting its potential for clinical use.

Table 11
Accelerated Stability Studies of Optimized Ensifentrine In-situ Nasal Gel Formulation (MF7)
Parameter	Initial (0 month)	1 month	3 months	6 months
Physical appearance	Clear, colourless solution	Clear, colourless solution	Clear, colourless solution	Clear, colourless solution
pH	6.5 ± 0.2	6.4 ± 0.1	6.3 ± 0.2	6.2 ± 0.1
Gelation temperature (°C)	34.1 ± 0.4	33.9 ± 0.5	33.7 ± 0.6	33.5 ± 0.7
Viscosity before gelation (cP)	342 ± 10.1	347 ± 11.3	352 ± 12.5	359 ± 13.2
Viscosity after gelation (cP)	4231 ± 63.5	4245 ± 67.2	4262 ± 70.8	4294 ± 75.3
Mucoadhesive strength (N/cm²)	1.87 ± 0.11	1.86 ± 0.13	1.84 ± 0.14	1.82 ± 0.15
Drug content (%)	97.2 ± 1.4	96.9 ± 1.5	96.2 ± 1.6	95.4 ± 1.8
% Drug diffused (after eight h)	68.2 ± 2.5	67.8 ± 2.7	66.9 ± 2.9	65.7 ± 3.1
Values are expressed as mean ± SD, (n = 3)

3.2. Discussion

Table 6 lists the gelation temperature of the PNIPAAm-based nasal gel formulations (~ 27.1°C to ~ 34.2°C), which are consistent with the known behaviour of thermoresponsive PNIPAAm to transition from a sol to gel at body temperature (~ 37°C). Results show that the temperature of gelation is suitable for nasal drug delivery (humidity, room temperature for ease of administration, and then becoming a gel when in contact with the patient's nasal cavity) and promoting prolonged retention. Variation in gelation temperature with changes in polymer concentration is indicative of the ability to fine-tune formulation properties and precisely control gelation characteristics. These results are consistent with prior studies showing thermoresponsive behaviour of PNIPAAm in nasal and other mucosal drug delivery systems [38].

The second important property for nasal drug delivery is mucoadhesive strength, which ensures the retention of the gel formulation at the site of action. The values of mucoadhesion strength obtained in this study were found to vary from 0.98 N/cm² to 2.72 N/cm² (Table 6). The values are higher with higher concentrations of HPMC K4M, which is consistent with the fact that this polymer increases mucoadhesion by forming H bonds with the mucosal surface [39]. The expectation is that formulations with a higher HPMC K4M concentration (0.6% w/v) will have stronger mucoadhesive properties, leading to a much longer retention time of the gel, that is, lower mucociliary clearance and continuous drug presence at the site of absorption. This is consistent with previous reports that HPMC-based formulations have better mucoadhesive properties and will lead to better retention of the drug in nasal and mucosal drug delivery systems [40].

An ex vivo permeation study (Table 10, Fig. 8) demonstrated that the developed formulations exhibited high drug permeation through the nasal mucosa, with a peak permeation of 91.7% achieved after 8 hours using the MF8 formulation. This result therefore substantiates that the nasal gels retained the drug in the nasal cavity for a prolonged period, and did so in a fashion that maximised the absorption of the drug [41]. Enhancing drug release and permeation through this combination of thermosensitivity and mucoadhesion was determined to be possible, with the thermosensitive polymers increasing permeation at higher concentrations of both PNIPAAm and HPMC K4M. This is in agreement with previous work where drug diffusion as well as absorption have been improved upon using thermosensitive and mucoadhesive polymers in the same formulation [42].

The accelerated stability of the optimised formulation (MF7) was determined under conditions where the formulation was deemed stable for at least 3 months, with no deviations from the gelation temperature, viscosity, or drug content. Such stability means that the gel platform is stable and possesses physicochemical properties that allow it to maintain those properties over time and provide consistent performance upon storage and use. As in other PNIPAAm-based gel formulations, these were similar findings where the polymer matrix not only provided physical stability but preserved the drug in its therapeutic properties over long-term storage [43].

In the study of solubility (Table 3), it was observed that phosphate buffer at pH 6.4 was a better solvent for ensifentrine than other solvents. This is particularly important for nasal formulations, where a phosphate buffer of pH 6.4 is commonly used to promote drug solubility and support optimal conditions for drug absorption via the nasal mucosa. Phosphate buffer enhances the solubility of ensifentrine, supporting the selection of this solvent for preparing nasal gel formulations that increase drug bioavailability and improve its systemic absorption potential. These observations are similar to other studies where it was found that the pH and the selection of solvent have an essential role in improving the solubility of the drug and its delivery [44].

Together, these results confirm that the nasal in-situ PNIPAAm gelled formulation exhibits favourable properties for drug delivery to the nose, including good gelation properties, improved mucoadhesion, sustained drug release, and stability. This flexibility also allows the concentration of PNIPAAm and HPMC K4M to be adjusted to optimise these properties, and make the system more suitable for a range of possible therapeutic applications.

The PNIPAAm-based nasal gel formulation created an environment to boost ensifentrine drug absorption and its therapeutic impact. The thermoresponsive formulation exhibited gelation temperatures between 27.1°C and 34.2°C, allowing the formulation to remain liquid until it reached body temperature, where it formed a gel structure to enhance its retention and absorption at the nasal mucosa. The mucoadhesive forces became stronger in the formulations due to an increase in HPMC K4M concentration from 0.98 N/cm² to 2.72 N/cm², leading to enhanced retention in the nasal cavities. The significant property facilitates better drug penetration and reduces the clearance of drug substances by mucus movement. The permeation study using nasal mucosa demonstrated enhanced drug delivery through the tissue when using formulations containing high proportions of PNIPAAm and HPMC K4M, which yielded maximum drug permeation of 91.7%. This shows that combined thermoresponsive and mucoadhesive polymers optimise drug absorption through the nasal tissue. The stability tests revealed that the optimised formulation maintained its stability features for three months without showing any changes in gelation temperature, viscosity level, or drug substance content. The solubility experimentation demonstrated phosphate buffer pH 6.4 as the best solution for ensifentrine thus making it suitable for nasal gel formulas. The PNIPAAm-based nasal gel provides excellent potential as an alternative to traditional drug delivery systems since it enables sustained drug release and enhanced mucoadhesion with stable characteristics, which are suitable for COPD and asthma treatment. Additional medical testing must verify the therapeutic value of this treatment.

PNIPAAm: Poly(N-isopropylacrylamide); PDE: Phosphodiesterase; COPD: Chronic Obstructive Pulmonary Disease; HPMC: Hydroxypropyl Methylcellulose; cP: Centipoise (unit of viscosity).

Acknowledgements

The authors sincerely appreciate the helpful contributions made by individuals and organisations that led to the study's accomplishment. This research was financially supported by Sciquaint Innovations (OPC) Pvt. Ltd. from Pune, which provided the required materials and research assistance. This manuscript has benefited significantly from the applicable evaluations delivered by the reviewers.

Author’s Contribution

All authors contributed equally to this work

Human Ethics and Consent to Participate Declarations:

Not applicable.

Conflict of interest

Authors declare no conflict of interest regarding this study

Funding

This research received no external funding

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Development and Optimization of PNIPAAm Based Ensifentrine Loaded In-situ Nasal Gel for Improved Bioavailability

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Methods

2.2.1. Calibration curve determination

2.2.2. Determination of solubility in different solvents

2.2.3. Differential scanning colourimetry (DSC)

2.2.4. Fourier Transform Infrared Spectroscopy

2.2.5. Experimental design

2.3. Formulation of PNIPAAm based ensifentrine loaded In-situ nasal

2.4. Characterization of hydrogel

2.4.1. Appearance and clarity

2.4.2. pH determination

2.4.3. Drug content uniformity

2.4.4. Gelation temperature

2.4.5. Gelation time

2.4.6. Viscosity

2.4.7. Spreadability

2.4.8. Mucoadhesive strength

2.4.9. Ex vivo permeation study using nasal mucosa

2.4.10. Stability studies under ICH conditions

3. Results

3.1. Calibration curve

3.2. Results of solubility studies

3.3. Differential scanning colourimetry

3.4. Fourier Transform Infrared Spectroscopy

3.5. Evaluation of In situ nasal gel

3.6. Results of physicochemical evaluation

3.2. Discussion

4. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

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