Microsphere-loaded dual-response in situ gel for enhanced ocular drug delivery

Abstract

Glaucoma is one of the most common blinding eye diseases worldwide. The limited ocular surface retention leading to a short duration of efficacy is the challenge in topical ocular drug delivery for glaucoma treatment. In this study, we developed a microsphere-in-situ gel composite drug delivery system (MPs-DSISG) containing Eudragit RS PO microspheres and a cation- and temperature-sensitive in situ gel, exhibiting dual-response properties. Precorneal fluorescence retention and tear elimination kinetics experiments confirmed that the ocular residence time of MPs-DSISG was longer than that of commercially available formulations and drug solutions. Besides prolonged retention time, the incorporated microspheres exhibited affinity for corneal epithelial cells and embedded themselves in the superficial layers of the conjunctiva, Tenon's capsule, episclera, and sclera. This enabled MPs-DSISG to achieve a 24 h intraocular pressure reduction, thus substantially reducing the dosing frequency. MPs-DSISG also demonstrated good biocompatibility‌ without ocular irritation and disturbing the tear film. In conclusion, this study demonstrates that incorporating microspheres into an insitu gel prolonged ocular retention and enhanced sustained drug efficacy, offering a promising improved treatment for anterior segment diseases.

Graphical abstract

Highlights

• •MPs-DSISGs extend drug residence by gelation—based on tear cations and ocular heat.
• •MPs released from MPs-DSISG create a drug reservoir, prolonging the retention time of the drug on the ocular surface.
• •MPs-DSISG boosts ocular drug bioavailability with better IOP lowering efficacy.

1. Introduction

Glaucoma is one of the leading causes of blindness worldwide and is characterized by progressive retinal ganglion cell loss, resulting in visual field defects [1]. Elevated intraocular pressure (IOP) is the primary factor in the development of glaucoma, driven by aqueous humour dynamics. Therefore, IOP reduction for glaucoma treatment typically involves either decreasing aqueous production or enhancing its outflow [2]. Common therapeutic approaches for glaucoma include pharmacological agents such as miotics [3] and IOP-lowering agents [4], laser treatments, including selective laser trabeculoplasty [5] and laser peripheral iridotomy [6], and anterior chamber paracentesis [7]. Topical administration of pharmacological agents is a cost-effective, non-invasive, and convenient method of ocular disease treatment, which is widely preferred by patients.

Common topical eye drops for glaucoma treatment include beta-adrenergic blockers, prostaglandin analogs, alpha-adrenergic agonists, carbonic anhydrase inhibitors, and cholinergic agents [8,9]. Among these, beta-adrenergic blockers are characterized by fewer adverse effects, lower cost, and relatively good efficacy. Representative drugs include the selective beta-adrenergic blocker betaxolol hydrochloride (BH) and the non-selective beta-adrenergic blocker timolol maleate (TM). Although they have similar chemical structures and physiological effects and have been used in clinical studies for the treatment of glaucoma for a long time [10,11], there are still differences between them. The LogP and pKa values of betaxolol are greater than those of timolol [12]. Consequently, these differences in physicochemical properties may lead to variations in their behaviour—both individually and combined formulation—following administration. For example, BH exhibited approximately twice the bioavailability of TM, whereas TM had a half-life around 1.4 times longer than that of BH [13]. Furthermore, the combination of tafluprost and BH achieved greater IOP reduction than that obtained by tafluprost and TM [14]. Therefore, BH and TM may also show differences in the same drug delivery system due to their own physicochemical properties.

Traditional aqueous eye drops, which are generally drug solutions, upon administration to the ocular surface, are prone to rapid clearance due to tear film dynamics, blinking reflexes, and nasolacrimal drainage [15], resulting in an actual bioavailability of <5% [16].

In situ gel (ISG) can prolong drug retention at the site of administration by adapting to the physiological environment [17]. Gellan gum (GG), a natural polymer often used in ISG, undergoes phase transition from a solution to a semi-solid in the presence of cations such as Ca²⁺, Mg²⁺, Na⁺, and K⁺ [18] and serves as a component of the commercially available Timoptic XE [19]. GG can inhibit the expression of pro-fibrotic genes induced by transforming growth factor beta 1 (TGF-β1) in corneal fibroblasts, which is helpful for reducing scarring and improving wound healing [20]. Gelatin (Gel) is another biocompatible material, which can reduce inflammation and has been widely employed in tissue engineering, wound healing, and drug delivery [21]. Poloxamer 407 (P407) is also a polymer often used in ISG which can create a thermosensitive ISG that exists as a solution at low temperatures but rapidly transforms into a three-dimensional gel upon temperature elevation to prolong drug retention at the site of administration [22]. In addition, P407 can protect corneal endothelial cells and eliminate corneal swelling [23]. However, it has been reported that in commercially available in situ gel eye drops for glaucoma treatment, free drug molecules released after administration may cause some adverse reactions. For instance, Timolol GFS [24] and Timoptic-XE have shown that approximately one in eight of patients’ eyes experienced burning and stinging upon instillation, and Rysmon TG [25] has also reported different degrees of ocular irritation. Our previous studies indicated that encapsulating drugs within Eudragit RS PO microspheres or nanoparticles can mitigate such irritation [26,27]. Moreover, microspheres, being larger in particle size than nanoparticles, were less susceptible to being cleared by the tear film or the blinking reflex. This resulted in a longer ocular retention time compared to nanoparticles [16].

Building on these concepts, Eudragit RS PO was employed to fabricate microspheres (MPs) loaded with BH and TM, which were subsequently incorporated into a composite GG, Gel, and P407 ISG to produce a microsphere-loaded dual-response in situ gel (MPs-DSISG) drug delivery system in this study. The safety of MPs-DSISG was systematically assessed through comprehensive in vitro and in vivo investigations, and the duration of drug on the ocular surface was evaluated by anterior corneal retention experiments, as well as the drug effect was determined by IOP-lowering pharmacodynamics experiment. This study showed that the characteristics of MPs-DSISG and the incorporated drugs BH and TM, as revealed by in vitro and in vivo experiments, provide some valuable references designing ocular preparations.

2. Materials and methods

2.1. Materials

BH was purchased from Zhengzhou JACS Chemical Products Co., Ltd. (Zhengzhou, China) and TM was from Tianjin Minxiang Biomedical Co., Ltd. (Tianjin, China). The BH commercially available formulation, Betoptic, was purchased from Alcon Laboratories, Inc. (Hünenberg, Switzerland). The TM commercially available formulation, Timolol Maleate Eye Drops (TM eye drops), referred to as TM solution, was purchased from Jiangsu Farever Pharma. Co., Ltd. (Xuzhou, China), and Gel was from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). GG was supplied by CP Kelco Inc. (Atlanta, GA, USA), and P407 by BASF AG (Florham Park, NJ, USA), and Eudragit RS PO was by Shanghai Chineway Pharma Tech Co., Ltd. (Shanghai, China). Triethyl citrate, sodium fluorescein, polyvinylalcohol (PVA) and hyaluronic acid (HA) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Trypan blue was obtained from MYM Biological Technology Company Limited (Beijing, China). Dichloromethane and formamide were purchased from Tianjin Damao Chemicals Reagent Factory (Tianjin, China). Methanol and ethanol were purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). Fertilized eggs were sourced from Xinxing Dahua Agricultural Poultry (Yunfu, China). Disposable intravenous infusion needles were purchased from Jiangxi Hongda Medical Equipment Group Ltd. (Nanchang, China), and disposable human venous blood sample collection containers were from Liuyang SANLI Medical Technology Development Co., Ltd. (Liuyang, China).

New Zealand white rabbits, male or female, weighing 2–2.5 kg, were acquired from Southern Medical University (Guangzhou, China). All rabbits were healthy and housed at the temperature of 25 ± 2 °C with a 12-h light/dark cycle. Animal studies were approved by the Guangdong Pharmaceutical University Experimental Animal Ethics Committee (Approval No.: gdpulaccvf2022010).

2.2. Preparation of MPs-DSISG

BH-MPs or TM-MPs were prepared using an emulsification-solvent evaporation method (Fig. 1A). The drug was mixed with 0.3% (w/v) HA solution at a ratio of 1:10 (w/v), then added to dichloromethane. The mixture contained 3.5% (w/v) drug, 1% (w/v) Span 80, 4.5% (w/v) Eudragit RS PO and 0.24% (w/v) triethyl citrate solution, which was subsequently poured into 1% (w/v) PVA-Gel solution at the volume ratio of 1:8 shearing (IKA T18 digital ULTRA TURRAX, IKA Instrument & Equipment Co., Ltd., Baden-Württemberg, Germany) at 3000 r/min for 5–10 min. Then the suspension was stirred at 100 r/min at 20–30 °C for 4–12 h, followed by vacuum drying (DZF-6032, Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) to obtain drug-loaded MPs. Blank MPs were prepared using the same method without any drug. The prepared MPs were then formulated into an MP suspension with a drug concentration equivalent to that of the commercially available formulations (BH: 2.8 mg/mL [11]; TM: 3.4 mg/mL [19]).

Fig. 1.

Characterizations of the formulations. (A) Schematic diagram of the preparation of microspheres-loaded dual-response in situ gel (MPs-DSISG). (B) Zeta potential diagram of the preparations (n = 6). (C) Polydispersity index (PDI) of the preparations (n = 3). ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, ns: no significance. (D, E) The cumulative release rate curves of betaxolol hydrochloride (BH) (D) and timolol maleate (TM) (E) preparations (n = 3). (F) Optical microscopy images and particle size distribution diagrams of the preparations (n = 3). MPs: microspheres; DSISG: dual-response in situ gel; Gel: gelatin; GG: gellan gum; P407: poloxamer 407; W1 phase: drug-hyaluronic acid solution; W2 phase: polyvinylalcohol-gelatin solution; W/O/W: drug-hyaluronic acid in Eudragit RS PO in polyvinylalcohol-gelatin solution.

GG and Gel were weighed and dissolved in deionized water, followed by heating and stirring at 65 °C. Subsequently, the mixtures were cooled to 20–25 °C. P407 was dissolved in deionized water under ice bath conditions. After dissolving, the prepared GG, Gel, and P407 solutions were mixed uniformly to form the composite DSISG containing 0.7% (w/v) GG, 0.4% (w/v) Gel, and 16.2% (w/v) P407. Drugs (BH or TM or inside MPs) were weighed and added into the gel to prepare BH-DSISG, BH-MPs-DSISG, TM-DSISG, or TM-MPs-DSISG.

2.3. Detections of MPs-DSISG properties

All formulations were mixed with mannitol to adjust the osmotic pressure within the range of 280–320 mOsmol/kg, as measured by a freezing point osmometer (OSMOMAT 3000 basic, Gonotec, Niedersachsen, Germany). Subsequently, the pH of the formulations was measured using a pH meter (PHS-3C, Shanghai Lei Ci Instrument, Shanghai, China), and tris buffer was added to adjust the pH to the range of 6–8. The morphology of formulations was observed under an optical microscope (BK6000, Chongqing Aote Optical Instrument, Chongqing, China), and their particle size was recorded. The zeta potential analyser (JS94, Shanghai Zhongchen Digital Technology Equipment, Shanghai, China) was used to measure their zeta potential.

The prepared MPs were centrifuged (H1850, Hunan Xiangyi Centrifugal Instruments, Changsha, China), and the supernatant was filtered through a 0.22 μm filter membrane. Subsequently, the absorbance of BH and TM in the supernatant was measured at 273 and 294 nm, respectively, using a UV spectrophotometer (UV-1800, Shanghai Meipuda Instrument, Shanghai, China) to determine the free drug concentration. The encapsulation efficiency ($\text{EE}\%$) and drug loading ($\text{DL}\%$) of the MPs were calculated as follows:

$$\begin{array}{c}EE\%=\frac{M_1-M_0}{M_1}\times 100\end{array}$$ (1)

$$\begin{array}{c}DL\%=\frac{d}{D}\times 100\end{array}$$ (2)

Where $M_1$ represents the total amount of drug input, $M_0$ denotes the amount of free drug in the supernatant, $d$ is the mass of the drug in the MPs, and $D$ stands for the total mass of the MPs.

2.4. Gelation temperature and time of the formulations

The blank DSISG, BH-DSISG, TM-DSISG, BH-MPs-DSISG, and TM-MPs-DSISG were individually placed into several vials (3 vials per formulation). Vials were then submerged in a water bath, with the temperature gradually increased from room temperature. For each 0.1 °C increment, vials were maintained at that temperature for 10 min. The temperature at which the phase transition occurred was recorded as the gelation temperature. Subsequently, the same formulations were placed into vials, and simulated tear fluid (STF; containing 6.78 g NaCl, 1.38 g KCl, 2.18 g NaHCO₃, and 0.084 g CaCl₂·2H₂O, 1000 mL H₂O [28]) was added to each vial (formulation:STF = 40:7 (v/v) [29]). The mixtures were then evaluated at both the room temperature (25 °C) and ocular surface temperature (34 °C [29]). The time required for the mixtures to transition from a flowable solution state to a non-flowable semi-solid state was recorded as the gelation time.

2.5. Rheology and gelling ability test

The viscosity of each formulation was measured at 25 and 34 °C under various shear rates using a viscometer (DV2TLVCJ0, Brookfield Corporation, MA, USA). Additionally, the viscosities of BH-DSISG, TM-DSISG, BH-MPs-DSISG, and TM-MPs-DSISG were determined after mixing with STF (formulation:STF = 40:7 (v/v)) at both 25 and 34 °C under different shear rates—ranging from 3 to 101 1/s. The viscosity-shear rate curves were then fitted into the Ostwald-de Waele power-law equation [26] to determine the rheological behaviour of the formulations. If n < 1, it indicates a pseudoplastic fluid suitable for ocular drug delivery. A value of n = 1 corresponds to a Newtonian fluid, typically an aqueous solution prone to drainage through the nasolacrimal duct, while an n > 1 represents a dilatant fluid, which exhibits shear-thickening behaviour and may cause discomfort during blinking.

$$\begin{array}{c}\tau =K{\gamma }^n\end{array}$$ (3)

Where $\tau$ represents the shear stress, $K$ denotes the viscosity coefficient of the fluid, $\gamma$ is the shear rate, and $n$ is the non-Newtonian index of the fluid.

The gelation properties of BH-DSISG, TM-DSISG, BH-MPs-DSISG, and TM-MPs-DSISG were also evaluated using a texture analyser (TA-XT plus, Stable Micro Systems, Guildford, UK) at 25 and 34 °C, with and without the STF.

2.6. In vitro drug release

A volume of 100 mL of STF was added to 250 mL beakers (n = 3 per formulation), which were then sealed and placed in a water bath shaker (THZ-82, Shanghai Xiaohan Industrial Development BH formulation, Shanghai, China) set to 120 r/min at 34 °C. Dialysis bags (MWCO 8000–14,000 Da), measuring 8 cm in length, were prepared by securing one end with string. 4 mL of the formulation was added through the open end of the dialysis bag which was then tightly sealed to ensure no leakage occurred. The dialysis bags were subsequently submerged in the preheated dialysis medium, and 5 mL of the release medium was withdrawn and replaced with an equivalent volume of the fresh release medium at predetermined time points. The collected dialysis medium was filtered through a 0.22 μm pore size membrane and analysed using a UV spectrophotometer (BH at 273 nm, TM at 294 nm). The cumulative drug release percentage ($\mathrm{W}\%$) was calculated and the results were plotted as percentage of cumulative drug release over time.

$$\begin{array}{c}W\%=\frac{C_n\times V+\sum _{i=1}^{n-1}{C}_i\times V_0}{W}\times 100\end{array}$$ (4)

Where $V$ represents the total volume of the release medium; $C_n$ denotes the concentration of the drug in the release medium during the $n$ th sampling; $V_0$ is the volume of the medium removed during each sampling point; and $W$ represents the total drug amount contained in the formulation inside the dialysis bag.

2.7. Haemolysis experiment of red blood cells

Blood was collected from the marginal ear vein of rabbits using disposable intravenous infusion needles and transferred into disposable human venous blood sample collection containers. Each tube was gently shaken to ensure thorough mixing of the blood with sodium heparin, preventing coagulation. The red blood cells (RBCs) were then diluted and divided into the following groups: negative (normal saline), positive (deionized water) control, BH solution, Betoptic, BH-MPs, BH-DSISG, BH-MPs-DSISG, TM eye drops, TM-MPs, TM-DSISG, and TM-MPs-DSISG groups. Each group included three centrifuge tubes containing the RBCs suspension and corresponding formulations. All tubes were incubated in a shaking incubator at 34 °C and 120 r/min for 2 h. After incubation, the tubes were centrifuged, and the supernatants were separated and measured for absorbance to calculate haemolysis rates [30]. The morphologies of the precipitated RBCs were examined under an optical microscope.

2.8. Hen's egg test on the chorioallantoic membrane (HET-CAM)

Fertilized eggs were incubated, and the shell corresponding to the marked air cell location was carefully peeled off, exposing the white egg membrane (three eggs per formulation). A few millilitres of normal saline were gently applied to moisten the membrane, facilitating its careful removal to expose the CAM. 0.3 mL of each formulation, as well as the positive (0.1 M NaOH solution) and negative (normal saline) control, was respectively, applied dropwise onto the CAM. The vascular responses were then observed for 5 min and the irritation score (IS) was calculated [31].

After the observation, 0.5 mL of 0.1% (w/v) trypan blue solution was immediately applied onto the CAM surface for 1 min. Subsequently, the CAM within the marked area was excised and rinsed thoroughly with normal saline. Filter paper was used to dry any excess moisture before weighing the sample and incubating it overnight in 5 mL of formamide for extraction. The mixture was centrifuged, and the absorbance of the supernatant was measured to determine the amount of trypan blue absorbed.

2.9. Rabbit blink experiment

New Zealand white rabbits were randomly divided into ten groups (three eyes per group): BH solution, Betoptic, BH-MPs, BH-DSISG, BH-MPs-DSISG, TM eye drops, TM-MPs, TM-DSISG, TM-MPs-DSISG, and normal saline control groups. A volume of 50 μL of the corresponding formulation was respectively administered into the conjunctival sac of each rabbit eye, then the blinking frequency was recorded for 1 min, and this procedure was repeated thrice.

2.10. Draize test and stability of tear film

Draize test includes single and multiple-irritation tests, with procedures similar to those of the rabbit blink test. In the single-irritation test, prior to drug administration, photographs of each rabbit eye were taken under a slit lamp (YZ5S, Suzhou 66 Vision Technology Co., Ltd, Suzhou, China). Subsequently, the drug was administered into the conjunctival sac, and the eyes were closed for 10 s. Photographs were then acquired at 1, 2, 4, 24, 48, and 72 h post-administration, and an ocular IS was assigned [32]. For the multiple-irritation test, each drug was administered twice daily (8.30 a.m. and 8.30 p.m.) for seven days, with scoring performed before each dosing as well as 1, 2, 4, 24, 48, and 72 h after the final dose.

Subsequently, a volume of 20 μL 0.2% sodium fluorescein solution was administered into the lower conjunctival sac and eyelids were manually blinked to distribute fluorescein spread over the ocular surface. Subsequently, the tear film was examined using a slit-lamp to determine the time at which the first dry spot appeared on the cornea, which was recorded as the tear breakup time (TBUT).

2.11. Haematoxylin and eosin (H&E) staining ocular tissues

After the tear film stability test, rabbits were euthanized, and their eyeballs were removed, washed, and immersed in 4% paraformaldehyde. Following fixation, tissues underwent ethanol dehydration, paraffin embedding, sectioning, and drying. Subsequently, the sections underwent dewaxing, staining, rehydration, and finally, mounting for optical microscopic examination.

2.12. Ocular tissue penetration

New Zealand white rabbits were randomly divided into nine groups (three eyes per group): BH solution, Betoptic, BH-MPs, BH-DSISG, BH-MPs-DSISG, TM eye drops, TM-MPs, TM-DSISG, and TM-MPs-DSISG groups. Corneas with a 2-mm scleral rim were carefully dissected and preserved, while the remaining sclera was retained for further use. The freshly isolated tissues were immediately placed between the donor and receptor chambers of Franz diffusion cells (Shanghai Kai Li Medical Technology Co., Ltd, Shanghai, China). Subsequently, 0.5 mL of the test formulation was added to the donor chamber, while the receptor chamber was filled with 34 °C pre-warmed glutathione bicarbonate Ringer's (GBR; containing 0.62 g NaCl, 0.0358 g KCl, 0.0103 g NaH₂PO₄·H₂O, 0.2454 g NaHCO₃, 0.0115 g CaCl₂·H₂O, 0.0159 g MgCl₂·6H₂O, 0.09 g glucose, 0.0092 g oxidized glutathione, and 100 mL H₂O [28]) solution and stirred at 120 r/min. At predetermined timepoints, 0.5 mL of drug-containing sample was withdrawn from the receptor chamber and replaced with an equal volume of fresh GBR. The collected samples were centrifuged and filtered through a 0.22 μm pore size membrane and analyzed for drug content using high-performance liquid chromatography (HPLC; LC-20AT, Shimadzu, Kyoto, Japan). Following the completion of the permeation experiments, the eye tissues were immediately rinsed several times with deionized water, minced, homogenized, and mixed with 100 μL of methanol. The mixture was then vortexed and centrifuged, and the supernatant was analyzed for drug content using HPLC to calculate the cumulative drug penetration and apparent permeability coefficient (Papp) [33].

2.13. Precorneal fluorescence retention test

New Zealand white rabbits were randomly divided into nine groups the same as ocular tissue penetration experiment. A volume of 50 μL of the respective 0.2% sodium fluorescein-containing formulation was administered into the conjunctival sac. Subsequently, photographs of the rabbits' eyes were taken under the cobalt blue light of a slit-lamp at predetermined timepoints until the fluorescence disappeared.

2.14. Tear pharmacokinetics

New Zealand white rabbits were randomly divided into nine groups the same as precorneal fluorescence retention test. 100 μL of the formulation was applied into the rabbit eyes, and the pre-weighed filter paper strips (3 × 8 mm) were placed in the conjunctival sac at predetermined timepoints and then removed and re-weighed. The saturated paper strips were dried using nitrogen gas, followed by the addition of 70 μL methanol. Subsequently, the samples were vortexed, sonicated, and centrifuged, and the supernatant was analyzed by HPLC to determine the drug content.

The experimental data was used to calculate the area under the curve (AUC), which reflects the amount of drug retained on the corneal surface for each formulation. Moreover, the pseudo-relative bioavailability ($F^{\prime }$) was determined as the relative amount of drug retention on the ocular surface. In this context, the aqueous solution of the drug served as the reference formulation, while other formulations were considered as the test formulations. The formula for $F^{\prime }$ was as follows:

$$\begin{array}{c}{F}^{\prime }=\frac{{AUC}_t\times V_t}{{AUC}_r\times V_r}\end{array}$$ (5)

Where $t$ denotes the test formulation, $r$ is the reference formulation, $V_t$ represents the dose administered of the test formulation,and $V_r$ represents the dose administered of the reference formulation.

2.15. Pharmacodynamic study

A chronic elevated IOP model was established using compounded carbomer [34]. Briefly, 0.1 mL of aqueous humour was extracted from the rabbit eye, and then 0.1 mL of the compounded carbomer containing 0.025% dexamethasone and 0.3% carbomer was injected. IOP was measured every day, and if it exceeded 22 mmHg for seven consecutive days, it was considered that the elevated IOP model had been successfully established. The rabbits with high IOP were randomly divided into ten groups (six eyes in each group): BH solution, Betoptic, BH-MPs, BH-DSISG, BH-MPs-DSISG, TM eye drops, TM-MPs, TM-DSISG, TM-MPs-DSISG groups and normal saline control. A volume of 50 μL of the corresponding formulation was administered, and the IOP was measured at different timepoints using a handheld tonometer (Tonovet plus, iCare, Helsinki, Finland). The percentage IOP reduction was calculated as follows:

$$\begin{array}{c}\Delta IOP=\frac{{IOP}_0-{IOP}_t}{{IOP}_0}\times 100\end{array}$$ (6)

Where ${IOP}_0$ represents the IOP at baseline and ${IOP}_t$ denotes the IOP at predetermined timepoint.

2.16. Statistical analysis

All data were indicated as mean ± standard deviation (SD). Origin2021 software was used for statistical analysis. The difference among groups were assessed using one-way analysis of variance with Tukey's post hoc test. The differences were considered significant for ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, and ^⁎⁎⁎⁎P < 0.0001.

3. Results and discussion

3.1. Properties of the preparations

The MPs-DSISG delivery system was composed of MPs and DSISG. MPs were fabricated using an emulsification-solvent evaporation method. GG, Gel and P407 were dissolved in deionized water to formulate DSISG (Fig. 1A). The zeta potential of the blank MPs was measured at −61.47 ± 6.26 mV. A reduction in this negative charge was observed when the MPs were either loaded with drugs or incorporated into the DSISG (Fig. 1B). These results can be attributed to two primary factors. First, the pKa values of BH and TM are 9.4 [35] and 9.2 [36], respectively. Consequently, both drugs carry positive charges in HA, PVA and Gel solution, which results in a reduction of the negative charge on the blank MPs. Furthermore, Gel in DSISG formulation contributes to this effect, which forms monomer α-chains, dimers and trimers containing positively charged amino acids such as lysine that can neutralize the negative charges on the surface of BH-MPs and TM-MPs. The polydispersity index (PDI) values of the preparations were showed in Fig. 1C. Betoptic group exhibited a significantly higher PDI than the other groups, indicating that its nonuniform particle diameters. The in vitro drug release experiment (Figs. 1D and E) demonstrated that BH-MPs-DSISG and TM-MPs-DSISG showed sustained-release characteristics, with a cumulative drug release of was less than 40% within 0.5 h. Moreover, the total release time of them was significantly longer compared to the drug solution, drug-MPs and commercially available formulations. In the drug release model fitting [37], an R² value closer to 1 indicates a better fit between the model and the data. For both self-prepared and commercially available formulations, the R² values were greater than 0.9 (Table S1). Overall, BH solution, Betoptic, BH-MPs, TM eye drops, TM-MPs, and TM-DSISG exhibited better compliance with the first-order kinetic model, whereas BH-DSISG, BH-MPs-DSISG, and TM-MPs-DSISG showed better alignment with the Higuchi model.

The Ritger–Peppas equation [38] can be used to determine whether carrier erosion contributes to the drug release mechanism, using the diffusion exponent (n) as an indicator. When n ≤ 0.45, the release mechanism follows Fick diffusion; when 0.45 < n ≤ 0.89, the release mechanism indicates non-Fick diffusion, where drug diffusion and matrix erosion occur concurrently; when n > 0.89, the release mechanism is dominated by matrix erosion. In the Ritger–Peppas model fitting (Table S1), the n value for MPs was <0.45, which meant that the drug release mechanism was Fick diffusion, while the n value for BH-DSISG was 0.48 and that for BH-MPs-DSISG was 0.46, suggesting that the drug release mechanism for these two formulations was non-Fick diffusion, where drug diffusion occurred simultaneously with the erosion of the DSISG matrix. The morphology and particle size of the prepared blank MPs, BH-MPs, TM-MPs, BH-MPs-DSISG, and TM-MPs-DSISG are exhibited in Fig. 1F. The average particle sizes of these formulations were 5.23 ± 3.40, 4.63 ± 2.25, 5.00 ± 3.37, 2.66 ± 1.63 and 4.82 ± 1.71 μm, respectively. Ophthalmic formulations typically require microparticles smaller than 10 μm [39] to avoid foreign body sensation in the eye; all the above formulations met this requirement. The MPs of each formulation exhibited spherical surfaces, while the ones of Betoptic were irregularly shaped. The average particle size of Betoptic was 4.93 ± 5.55 μm, but its PDI value was 1.22 ± 0.14, and some Betoptic microparticles exceeded 30 μm which may cause discomfort during ocular administration. The $\text{EE}\%$ of the prepared BH-MPs and TM-MPs was 70.08% ± 0.02% and 68.47% ± 0.03%, respectively, while the $\text{DL}\%$ was 5.67% ± 1.33% and 5.43% ± 1.56%, respectively.

3.2. Phase transition of MPs-DSISG

The BH-DSISG, TM-DSISG, BH-MPs-DSISG, and TM-MPs-DSISG prepared in this study exhibited dual responsiveness to cations and temperature. Both the blank DSISG and the formulation groups remained in a flowable solution state at 25 °C but exhibited varying degrees of gelation following the addition of STF (Fig. 2A) owing to the presence of cations such as Ca²⁺, K⁺, and Na⁺ in STF which can form chemical bonds and hydrogen bond with GG and Gel, suggesting that the formulations were sensitive of ions. It was notable that all DSISG formulations underwent gelation after changing the temperature to 34 °C, which may be due to the micellar aggregation of P407, indicating their ability to sensitively undergo phase transitions in response to temperature changes. These properties demonstrated MPs-DSISG dual-responsive characteristics to STF and temperature. Notably, both TM-MPs-DSISG and BH-MPs-DSISG exhibited a higher gelation degree at 34 °C alone than at the same temperature in the presence of STF. This is because the gelation of these preparations was primarily governed by the micellar aggregation of P407 via hydrophobic bonds at 34 °C without STF, whilst GG and Gel hardly contribute to gel under this condition. Although the addition of STF introduced ions that formed bonds with GG and Gel to aid gelation, the large proportion of water in STF diluted the system. This reduction in gelling polymer concentration ultimately weakened the integrity of the composite three-dimensional networks.

Fig. 2.

Gelling properties of the preparations. (A) The gelation state of the preparations under different conditions. (B) The gelation temperature of the preparations. (C–E) Gelation time of the preparations at 34 °C (C), 25 °C mixed with simulated tear fluid (STF) (D), and 34 °C mixed with STF (E) (n = 3). ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, ns: no significance. (F, G) Viscosity at 25 °C of betaxolol hydrochloride (BH) (F) and timolol maleate (TM) (G) formulations. (H, I) Viscosity at 25 °C mixed with STF of BH (H) and TM (I) formulations. (J–N) Gelation strength under the different conditions for the blank dual-response in situ gel (DSISG) (J), betaxolol hydrochloride-dual-response in situ gel (BH-DSISG) (K), betaxolol hydrochloride-microspheres-loaded dual-response in situ gel (BH-MPs-DSISG) (L), timolol maleate-dual-response in situ gel (TM-DSISG) (M), and timolol maleate-microspheres-loaded dual-response in situ gel (TM-MPs-DSISG) (N).

It has been shown that there is tear evaporation on the ocular surface and that the corneal temperature is lower than that of other parts of the human body, around at 34 °C [40], while the temperature of the conjunctival sac is about 35 °C [41]. In Fig. 2B, the gelation temperature of the blank DSISG was relatively low at 32.5 ± 0.5 °C. The gelation temperatures of the BH-MPs-DSISG and TM-MPs-DSISG groups were at 34.4 ± 0.8 °C and 35.2 ± 1.2 °C, respectively. This meant that the formulation was able to gel in the conjunctival sac, where it was stored, facilitating MPs-DSISG spread over the entire ocular surface with blinking and reducing nasolacrimal duct loss. Additionally, the results showed that the gelation temperature of BH-MPs-DSISG was lower than that of TM-MPs-DSISG, indicating that it was more sensitive to temperature. However, the gelation temperature of TM-MPs-DSISG is closed to the temperature of the conjunctival sac, which may still be in the gel transition stage following administration and risk incomplete gelation. The gelation time of BH-MPs-DSISG and TM-MPs-DSISG at 34 °C was significantly longer than those of the blank DSISG group (Fig. 2C). In contrast, no significant differences were observed between the BH-DSISG, TM-DSISG, and blank DSISG groups. These findings indicated that the drugs had minimal impact on the phase transition of the gel, while the MPs were larger in size and carried a negative charge, which may affect the strength of molecular alignment in the gel matrix. When subjected to 34 °C mixed with STF, all formulations exhibited a shortened gelation time compared to the condition of 25 °C with STF, this result was attributed not only to the electrostatic interactions driven by ion responsiveness but also to the formation of micelles by P407, further enhancing the three-dimensional network structure (Figs. 2D and E). Moreover, the hydrophobic segments on the surfaces of BH-MPs and TM-MPs promote the rapid phase transition of P407.

According to Ostwald-de Waele power-law equation, the n values for the BH solution and the TM eye drops were 1.03 and 1.06, respectively, which were close to 1, classifying these solutions as Newtonian fluids. For the remaining formulations, the n values were less than 1, categorizing them as pseudoplastic fluids (Table 1 and Figs. 2F–I).

Table 1.

Rheological behaviour of various formulations before and after mixing with simulated tear fluid (STF) at 25 °C.

Formulation		Equation	n	Fluid type
Without STF	BH solution	τ = 0.01γ1.03	1.03	Newtonian fluid
	Betoptic	τ = 31.66γ0.26	0.26	Pseudoplastic fluid
	BH MPs	τ = 1.05γ0.47	0.47	Pseudoplastic fluid
	BH DSISG	τ = 34.14γ0.40	0.40	Pseudoplastic fluid
	BH MPs-DSISG	τ = 16.62γ0.53	0.53	Pseudoplastic fluid
	TM eye drops	τ = 0.01γ1.06	1.06	Newtonian fluid
	TM MPs	τ = 0.56γ0.55	0.55	Pseudoplastic fluid
	TM DSISG	τ = 4.78γ0.75	0.75	Pseudoplastic fluid
	TM MPs-DSISG	τ = 66.79γ0.21	0.21	Pseudoplastic fluid
With STF	BH DSISG	τ = 14.01γ0.50	0.50	Pseudoplastic fluid
	BH MPs-DSISG	τ = 33.02γ0.34	0.34	Pseudoplastic fluid
	TM DSISG	τ = 15.82γ0.43	0.43	Pseudoplastic fluid
	TM MPs-DSISG	τ = 27.32γ0.30	0.30	Pseudoplastic fluid

BH: betaxolol hydrochloride; MPs: microspheres; DSISG: dual-response in situ gel; TM: timolol maleate.

The gelation of eye drops should not affect eye blinking. Generally, the force required for blinking is about 0.2 N, and the forceful blink is 0.8 N [42]. At 34 °C mixed with STF, the gel strength of all formulations was below 0.1 N. Even at 34 °C without STF, the maximum gel strength of both BH-MPs-DSISG and TM-DSISG was only 0.12 N. Therefore, the low magnitude of gel strength suggested that the formulated preparations would not impede normal blinking (Figs. 2J–N). At 25 °C, the gelation strength of all formulations was relatively low. However, after the addition of STF, the gelation strength of each formulation increased due to the spontaneous aggregation of GG molecules with the cations present in STF, which form double-helix structures [43]. The stability of these structures primarily depended on intermolecular hydrogen bonding. As further aggregation occurred, a three-dimensional network structure was formed [44], effectively entrapping water molecules and inducing gel formation. Both monovalent ions, such as Na⁺ and K⁺, and divalent ions, such as Ca²⁺, present in STF can effectively promote gel formation. Divalent cations can directly interact with two carboxyl groups to form more stable chemical bonds, thereby further enhancing the stability of the gel. At 34 °C, the gelation strength of all formulations was stronger than at 25 °C, as P407 and hydrophobic interactions gradually strengthened, leading to micelle formation [45]. When the temperature reached the gelation point, the micelles further interconnected to form a three-dimensional network structure, causing the system to transition from a liquid state to a gel state.

At 34 °C, BH-DSISG showed significantly weaker gel strength than TM-DSISG (Figs. 2K and M). Conversely, upon the incorporation of MPs, the gel strength of BH-MPs-DSISG became significantly stronger than that of TM-MPs-DSISG (Figs. 2L and N). These results may be attributed to the fact that the LogP value of betaxolol is greater than that of timolol. As both GG and Gel in the DSISG were hydrophilic, the more lipophilic betaxolol interacted less effectively with them, resulting in a weaker gel structure. However, upon incorporating microspheres, betaxolol in BH-MPs-DSISG appeared to facilitate the formation of a stronger three-dimensional network. This enhanced structure, formed with the hydrophobic segments of the MPs and P407 micelles, resulted in a greater gel strength than that of TM-MPs-DSISG. Notably, the gel strength of TM-DSISG was higher than that of TM-MPs-DSISG (Figs. 2M and N), possibly because the embedded microspheres disrupted the uniformity of the gel network, thereby reducing its overall mechanical strength. In conclusion, these observed differences in in vitro gelation were anticipated to influence the subsequent in vivo drug ocular surface retention.

3.3. In vitro irritation tests

To evaluate the safety of the formulations, a haemolysis test was performed. As observed in Fig. 3A, while the red blood cells (RBCs) in the Betoptic group maintained a circular shape, most had lost their characteristic biconcave morphology, suggesting partial lysis of the cells, which resulted in the blood-red appearance of the supernatant. Quantitative analysis revealed that the haemolysis rate for the Betoptic group reached 66.27% ± 1.64% (Fig. 3B). The flocculent material observed in the centrifuge tube likely resulted from the adhesion of irregularly shaped Betoptic microparticles to RBCs, thus disrupting the RBC membrane structures and increasing the haemolysis rate. A distinct phenomenon was observed in the BH-MPs-DSISG group (Fig. 3A), where the liquid in the centrifuge tube separated into three layers: a clear supernatant layer without obvious blood red colour, an intermediate layer with slight blood coloration, and a dark red sediment layer of RBCs. The RBCs in the BH-MPs-DSISG group maintained their intact morphology, exhibiting a biconcave disc shape with a low haemolysis rate of 1.32% ± 1.09%, showing no significant difference compared to the negative control. The observed phenomenon may be attributed to the formation of a network structure by BH-MPs-DSISG in the normal saline solution of RBCs. This network structure potentially entraps free RBCs, preventing them from being fully sedimented during centrifugation, thereby resulting in the appearance of the blood-red intermediate layer. The RBCs in the BH-MPs, TM-MPs, BH-DSISG, and TM-DSISG groups were similarly intact, displaying a biconcave disc shape, with clear supernatants post-centrifugation. The haemolysis rates among these groups did not significant compared with the negative control. For all experimental groups except Betoptic (Figs. 3B and C), the haemolysis rates were less than 5%, indicating no irritancy [46].

Fig. 3.

In vitro irritation of the preparations. (A) Haemolytic status of the preparation (n = 3). (B, C) Haemolysis rates of betaxolol hydrochloride (BH) (B) and timolol maleate (TM) (C) formulations (n = 3). (D) Vascular responses to the formulations. (E, F) Scoring and irritation grade assessment for BH (E) and TM (F) formulations (n = 3). (G, H) Trypan blue attachment on the chorioallantoic membrane (CAM) following treatment with BH (G) and TM (H) formulations (n = 3). ^⁎⁎⁎⁎P < 0.0001, ns: no significance. MPs: microspheres; DSISG: dual-response in situ gel; Non: no irritation.

In Hen's egg test on the chorioallantoic membrane (HET-CAM), 0.1 M NaOH (positive control) caused vessel rupture within 5 min, with cauliflower-like diffusion, indicating a strong irritation (Fig. 3D). In contrast, 5 min after administration, the CAM vasculature in the negative control (normal saline) and other treatment groups remained intact and clearly visible. The scoring results in Figs. 3E and F also showed consistent findings, indicating no irritation from the reagents in the experimental groups. Subsequently, trypan blue staining was applied to the CAM, measuring the amount of trypan blue absorbed to further evaluate irritation. Trypan blue is a vital stain that only stains dead cells. As shown in Figs. 3G and H, no significant difference was observed between the experimental groups and the negative control, while the positive control group exhibited the highest trypan blue uptake.

3.4. In vivo safety evaluation

Although in vitro irritation tests have demonstrated that MPs-DSISG did not cause any severe irritation, these tests were not reflective of the physiological environment of the eye. Therefore, more clinically relevant measurements, such as in vivo rabbit blink and tolerability test, should be conducted to allow for a more effective assessment. Rabbit eyes are more sensitive to foreign substances than human eyes and exhibit stronger reactions, making them preferred for evaluating formulation irritancy at the site of administration [47]. In the rabbit blink test, no significant difference was observed between the experimental groups and saline control, whereas significant difference was noted compared to Betoptic (Figs. 4A and B).

Fig. 4.

In vivo biocompatibility of the preparations. (A, B) Blink frequency of rabbit eyes within 1 min for betaxolol hydrochloride (BH) (A) and timolol maleate (TM) (B) formulations (n = 3). (C, D) Heatmap of scoring results from the formulations in single-irritation test (C) and multiple-irritation test (D) (n = 3). (E, F) Tear breakup time (TBUT) of the rabbit eyes after completion of multiple-irritation test for BH (E) and TM (F) formulations (n = 3). (G, H) Central corneal thickness of BH (G) and TM (H) formulations (n = 3). ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, ns: no significance. (I) Haematoxylin and eosin (H&E) staining images of the cornea, iris, sclera, and retina. MPs: microspheres; DSISG: dual-response in situ gel.

In Draize test, the score did not exceed 3 in the single- or multi-stimulation tests for tolerability (Figs. 4C and D), suggesting that all formulations were non-irritating. Finally, after fluorescein application, MPs, DSISG, and MPs-DSISG showed better tear film stability than that observed with the commercially available formulations, such as Betoptic and TM eye drops (Figs. 4E and F), likely due to less irritation.

Following the experimental procedures, all rabbits were euthanized, and ocular tissue sections were H&E-stained. Both Betoptic and TM eye drops demonstrated significantly increased central corneal thickness compared with the saline group (Figs. 4G and H), indicating they had irritation, whilst BH-MPs-DSISG and TM-MPs-DSISG had no significant difference with saline control, suggesting that their biocompatibility was better. The central corneal thickness of the BH-DSISG and the TM-DSISG groups was also thicker, which could be due to the higher gel matrix content in the ISG leading to prolonged retention and thus higher corneal hydration. The central corneal thickness was observed to be lower in the microsphere groups, and the central corneal thickness of MPs-DSISG was lower than that of DSISG, indicating that the addition of microspheres can mitigate the corneal hydration effect induced by the DSISG, thus lowering the potential risk of corneal oedema from the in situ gel. Compared with control group (normal saline), no morphological abnormalities were noted in all experimental groups, indicating they do not have severe irritation (Fig. 4I). In summary, across various safety evaluation tests, BH-MPs-DSISG and TM-MPs-DSISG met safety requirements and were therefore suitable for further in vivo evaluation.

3.5. Ex vivo eye tissue penetration test

Both BH and TM achieve IOP-lowering effect by acting on the β-receptors of ocular tissues to reduce ciliary body atrial aqueous production. The tissue permeability of drugs is one of the factors influencing their efficacy. In ex vivo eye tissue penetration test (Fig. 5A), the ocular tissue penetration of BH was higher than that of TM (Figs. 5B–E). The observed difference can be attributed to the higher LogP value of betaxolol compared to timolol, which indicates greater lipophilicity. Given that ocular tissue cell membranes are phospholipid bilayers, the more lipophilic betaxolol exhibits a higher binding affinity for these tissues, thereby enhancing its penetration ability form the formulation. BH-DSISG, TM-DSISG, BH-MPs-DSISG, and TM-MPs-DSISG showed lower cumulative corneal and scleral drug penetration, primarily because of the static condition of the preparations in the ocular tissue permeation device at the donor chamber (Figs. 5B–E). At 34 °C, gelation of DSISG and MPs-DSISG occurred, slowing drug diffusion and reducing penetration. The sclera permeability coefficient (Papp) of each group was higher than the corneal Papp (Figs. 5F–I). Compared with Betoptic, BH-MPs demonstrated lower corneal drug permeation and corneal Papp was not significantly different (Fig. 5F). However, BH-MPs exhibited superior sclera permeation to Betoptic (Fig. 5H), which may account for the subsequent BH-MPs, showing a greater intraocular pressure-lowing effect than Betoptic.

Fig. 5.

Franz diffusion of ex vivo eye tissues. (A) Schematic illustration of the ocular tissue permeation experiment. (B, C) Cumulative corneal drug penetration of betaxolol hydrochloride (BH) (B) and timolol maleate (TM) (C) formulations (n = 3). (D, E) Cumulative scleral drug penetration of BH (D) and TM (E) formulations (n = 3). (F, G) The apparent permeability coefficient (Papp) of BH (F) and TM (G) formulations at the cornea (n = 3). (H, I) The Papp of BH (H) and TM (I) formulations at the sclera (n = 3). (J, K) BH (J) and TM (K) formulations retained in the cornea (n = 3). (L, M) BH (L) and TM (M) formulations retained in the sclera (n = 3). ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎⁎P < 0.0001, ns: no significance. MPs: microspheres; DSISG: dual-response in situ gel.

Notably, both BH and TM were detected in MPs groups (Figs. 5J and K), which can be attributed to the presence of hydrophobic polyacrylates in the MPs, having affinity to the hydrophobic corneal epithelial cells. The sclera also revealed having drug for both BH-MPs and TM-MPs (Figs. 5L and M) groups. This may be the collagen bundles of the conjunctiva, Tenon's capsule, and superficial sclera on the surface of the sclera are thin and loose, with sinusoidal patterns [48]. Therefore, the BH-MPs and TM-MPs can be embedded in the interstices of these loose collagen bundles, enabling drug delivery to form a drug reservoir.

3.6. Ocular surface retention capability

The retention capability of a formulation on the ocular surface determined how long a drug can be maintained at the site of administration. Both BH solution and TM eye drops exhibited the shortest pre-corneal fluorescence retention time, and the MPs also demonstrated relatively short retention time (Figs. 6A–C). However, the retention time of BH-DSISG was significantly prolonged—nearly four times that of the respective aqueous solutions and 2.46 times that of the commercially available formulation, Betoptic. The pre-corneal fluorescence retention of BH MPs-DSISG was further extended, reaching 3.47 times that of Betoptic and BH-MPs and 6.56 times that of the BH solution, but no significant difference with BH-DSISG. Similarly, the pre-corneal fluorescence retention of TM MPs-DSISG was 1.97 times that of TM-MPs. These results indicated that DSISG can prolong the retention time of MPs on the ocular surface. Notably, the fluorescein retention of TM-MPs-DSISG was shorter than that of BH-MPs-DSISG due to the weaker gelation capability, as illustrated in Figs. 2L and N. The gelation temperature of TM-MPs-DSISG was 35.2 ± 1.2 °C, closely matching the conjunctival sac temperature of approximately 35 °C. As a result, the formulation risked incomplete gelation, and with blink and tear dilution, its retention time was further shortened.

Fig. 6.

Ocular surface drug retention ability. (A) Fluorescein retention from the formulations. (B, C) Ocular surface retention time of betaxolol hydrochloride (BH) (B) and timolol maleate (TM) (C) formulations (n = 3). (D, E) Tear pharmacokinetics of BH (D) and TM (E) formulations (n = 3). (F, G) The area under the drug concentration versus time curve (AUC) of BH (F) and TM (G) formulations (n = 3). (H, I) The time required for a 90% reduction in drug concentration in tears (T_0.1) for BH (H) and TM (I) formulations (n = 3). (J, K) The pseudo-relative bioavailability (F′) of BH (J) and TM (K) formulations (n = 3). ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, ns: no significance. MPs: microspheres; DSISG: dual-response in situ gel.

Tear pharmacokinetics experiment was conducted to assess the drug retention on the ocular surface. The drug from the BH-MPs-DSISG group remained on the ocular surface for the longest duration (Fig. 6D), with detectable levels maintained up to 6 h, consistent with the results of the precorneal fluorescence retention. After 5 h, the concentration was still recorded at 54.59 ± 36.59 μg/mL. In contrast, the drug concentration after the application of the BH solution declined rapidly, reaching its lowest level within the shortest time (45 min). For other formulations, the drug concentration had already declined below 25 μg/mL after 2 h. Compared with the BH formulations, the drug concentration of TM formulations was declined fast (Fig. 6E). The TM-MPs reached its minimum level at 45 min with a concentration of 35.55 ± 8.29 μg/mL, while both TM-MPs-DSISG and TM-DSISG reached their minimum levels at 2 h. The AUC_{0–6 h} of BH-MPs-DSISG was 56,108 μg·min/mL, which was 10.76 times higher than that of the BH solution and three times higher than that of Betoptic (Fig. 6F). The AUC_{0–6 h} of TM-MPs-DSISG was 32,767 μg·min/mL, which was 2.38 times that of TM eye drops (Fig. 6G). In addition, the time required for a 90% reduction in drug concentration in tears for BH-MPs-DSISG was significantly longer than that for BH solution, Betoptic, BH-MPs and BH-DSISG (Fig. 6H). However, the groups of TM formulations were no statistically significant differences (Fig. 6I). When the BH solution was used as the reference formulation, BH-MPs-DSISG exhibited the highest relative drug amount retained on the ocular surface, showing significant differences compared with Betoptic, BH-MPs and BH-DSISG (Fig. 6J). Conversely, Fig. 6K showed that when the TM eye drops served as the reference formulation, no significant differences were observed among the various formulations.

This experiment has shown that the ocular drug retention time of BH-MPs-DSISG was longer than that of TM-MPs-DSISG, which can be attributed to the following three principal factors. Firstly, the gelation temperature of TM-MPs-DSISG exceeded the physiological ocular temperature, existing incomplete gelation probability as shown in Fig. 2B. Secondly, compared with timolol, betaxolol's higher LogP value [12] denotes greater lipophilicity, promoting a stronger affinity for both ocular tissues and micelles formed by the hydrophobic P407 molecules. Finally, the pKa value of TM is 9.2 [36] and that of BH is 9.4 [35]. According to the Henderson-Hasselbalch equation, in the physiological environment of tears, there are more ionic forms of TM than BH, making it more likely to escape into tears and be lost, and its disappearance rate is faster than that of BH.

The MPs-DSISG system consistently demonstrated excellent ocular surface retention in both the precorneal fluorescence retention test and tear pharmacokinetics study. These results indicate its potential for prolonging the therapeutic efficacy of the ocular drugs.

3.7. Pharmacodynamics for lowering IOP

An elevated IOP model was established using compounded carbomer (Fig. 7A). Rabbits with successfully modelled were selected for pharmacodynamics experiment (Fig. 7B). BH solution exhibited the shortest duration of action, lasting approximately 4 h (Fig. 7C), attributing to the BH solution having a short residence time on the ocular surface. However, Fig. 7D shows that TM eye drops demonstrated an IOP-lowering effect for 8.5 h. This sustained effect may be because TM is a non-selective beta-adrenergic blocker that acts on multiple receptors in ocular tissues. The IOP-lowering effect of BH-DSISG and BH-MPs lasted for 23 h, significantly surpassing Betoptic (10 h) and BH solution. Furthermore, BH-MPs-DSISG exhibited a therapeutic effect exceeding 24 h (Figs. 7C and E), and TM-MPs-DSISG extended this effect to 24 h (Figs. 7D and F). The average IOP-lowering time of TM MPs and TM DSISG was 20 h and 21 h, respectively, both shorter than that of TM-MPs-DSISG. These results attributed to the reasons that DSISG has dual response of tear cations and ocular heat as well as BH-MPs which adhere to the corneal epithelial cells and embed within the interstices of the loose scleral collagen bundles, thereby forming a drug reservoir that achieved sustained drug release. Additionally, BH-MPs-DSISG maintained a greater amplitude of IOP-lowering over an extended duration compared to both Betoptic and BH solution (Fig. 7G). The AUC of TM-MPs-DSISG was significantly greater than those of the other three preparations in Fig. 7H.

Fig. 7.

The effect of lowering intraocular pressure (IOP). (A) Schematic illustration of the modelling process for chronic high intraocular pressure. (B) Trend chart of IOP during the modelling. (C, D) Efficacy of IOP reduction of betaxolol hydrochloride (BH) (C) and timolol maleate (TM) (D) formulations (n = 6). (E, F) The efficacy time of BH (E) and TM (F) formulations (n = 6). (G, H) The area under the average IOP-lowering versus time curve of BH (G) and TM (H) formulations (n = 6). ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, ns: no significance. AUC: area under the curve; MPs: microspheres; DSISG: dual-response in situ gel.

In conclusion, combining with the data in Fig. 6, these results demonstrated that MPs-DSISG can reduce the loss of the drug and enhance drug ocular surface retention capability, thereby significantly prolonging the IOP-lowering time comparing to the drug solution and commercially available preparations.

4. Conclusion

In this study, MPs-DSISG drug delivery systems showed differences depending on the distinct physicochemical properties of the small molecule drugs. In the in vitro release and tear pharmacokinetics experiments, BH-MPs-DSISG exhibited superior performance compared to TM-MPs-DSISG. This difference is attributed to their distinct pKa values. Timolol maleate, having a lower pKa, is more likely to be ionized and released into the tears, making it more prone to precorneal loss. Furthermore, the LogP value of betaxolol is greater than that of timolol which resulted in significantly greater drug penetration of BH than that of TM. Moreover, BH-MPs-DSISG outperformed TM-MPs-DSISG in the ocular surface retention experiments.

Overall, MPs-DSISG exhibited favourable properties. On the one hand, the incorporation of microspheres improved the biocompatibility of MPs-DSISG system compared to DSISG alone. On the other hand, DSISG served to prolong the ocular retention time of microspheres and drug. These advantages allow BH or TM to improve their efficacy when used to treat glaucoma. This enhancement was likely due to the following reasons: MPs-DSISG underwent a phase transition triggered by the cations present in tears and ocular surface temperature, transitioning from a solution state to a three-dimensional gel network (Fig. 8A); during this process, MPs within the delivery system bound to the corneal epithelial cells and were embedded in the loose collagen bundles of the conjunctiva, Tenon's capsule, and superficial sclera, enabling the cornea and conjunctiva to act as a drug reservoir, providing sustained drug release (Figs. 8B and C). In summary, MPs-DSISG demonstrated prolonged ocular surface retention and a sustained IOP-lowering efficacy, suggesting a promising drug delivery system for anti-glaucoma drugs.

Fig. 8.

Schematic diagram of the drug delivery system on the ocular surface. (A) Microspheres-loaded dual-response in situ gel (MPs-DSISG) undergoes phase transition due to the cations present in the tears and the temperature on the ocular surface, transitioning from a solution state to a three-dimensional network gel state; (B) The microspheres (MPs) within the delivery system bind to the corneal epithelial cells, enabling the cornea to act as a drug reservoir; (C) MPs in the MPs-DSISG system are embedded in the loose collagen bundles of the conjunctiva, Tenon's capsule, and superficial sclera, providing sustained drug release. BH: betaxolol hydrochloride; TM: timolol maleate; DSISG: dual-response in situ gel; STF: simulated tear fluid; Gel: gelatin; GG: gellan gum; P407: poloxamer 407.

CRediT authorship contribution statement

Jinlan Mao: Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Kangyiran Pan: Writing – review & editing, Validation, Investigation, Formal analysis. Suling Bei: Validation, Project administration, Investigation, Data curation. Haidong Guo: Validation, Project administration, Investigation, Data curation. Shuai Wang: Writing – review & editing, Visualization, Investigation. Bowen Pan: Visualization, Project administration, Data curation. Yuqian Liang: Validation, Project administration, Data curation. Yiqing Dai: Validation, Project administration, Data curation. Ilva D. Rupenthal: Writing – review & editing, Validation, Supervision, Resources. Li Liu: Validation, Resources, Methodology, Investigation. Xifeng Teng: Writing – review & editing, Supervision, Resources, Methodology. Dongzhi Hou: Writing – review & editing, Validation, Supervision, Resources, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: