HSPiP and QbD oriented optimized nanocubosomes for ameliorated absorption of tolterodine tartrate: In vitro and in vivo evaluations

Abstract

The study explored HSPiP and QbD-(quality by design) enabled optimized cubosomes for sustained drug release, improved permeation, and enhanced oral bioavailability. OCUB1 (the optimized product) was characterized for size, zeta potential (ZP), thermal analysis, and surface roughness. In vitro drug release and hemolysis studies were carried out using a dialysis membrane and rat erythrocytes (4 % suspension), respectively. An ex vivo non-everted intestinal permeation study (180 min) compared permeation potential between DS (suspension) and OCUB1. In vivo pharmacokinetic (PK) study investigated PK parameters in rats whereas hematological and biochemical assays ensured the safety of OCUB1. HSPiP predicted glyceryl monooleate (GMO), poloxamer−188, and polyvinyl alcohol (PVA) as optimal excipients based on minimum RED (relative energy difference) values while QbD identified OCUB1 as the most optimized formulation with desirable attributes such as low size (169 nm), high ZP (−29.2 mV), low polydispersity index (0.23), and maximum entrapment efficiency (85.3 %). Thermal analysis confirmed solubilization of TOTA in OCUB1, and atomic force microscopy (AFM) technique confirmed its cubical shape. OCUB1 showed extended drug release (98.1 % over 48 h) and sustained ex vivo permeation (J_ss = 6.69 μg/cm²/min, steady state flux) across rat intestine as compared to DS (J_ss = 9.172 μg/cm²/min). In vivo PK parameters exhibited significant improvement, with 3.2-fold increase in C_max as compared to the DS. In vitro hemolysis, along with biochemical and hematological assays, ensured the safety of OCUB1 for oral delivery. Conclusively, OCUB1 presents a promised alternative to conventional capsule, offering reduced side effects and enhanced patient compliance.

Graphical abstract

1. Introduction

A sudden and uncontrollable urge to urinate is commonly associated with overactive bladder (OAB) syndrome and enuresis in children. OAB and enuresis significantly impact the quality of life in patients (Leron et al., 2018). Its prevalence varies across countries. Middle income countries show higher prevalence than high-income countries. In eight developed countries (France, Italy, Japan, UK, USA, Spain, Germany, and China), the total cases were estimated as 363 million in 2020 which was expected to be 401.6 million in 2030 (Cheng et al., 2024). The frequency and the associated consequences are significant in elder patients. The disease assessment and treatment strategies are still challenging due to embarrassment, ignorance, and unknown risk factors (Leron et al., 2018).

Chemically, tolterodine tartrate (TOTA) is a benzhydryl compound (2R,3R)-2,3-dihydroxybutanedioic acid;2-[(1R)-3-[di(propan-2-yl)amino]-1-phenylpropyl]-4-methylphenol) with molecular formula of C₂₆H₃₇NO₇. Pharmaceutically, low molecular weight (475 g/mol), high lipophilicity (log p ∼ 1.8 of salt free compound), weak basic compound (pKa of ∼9.9), and good solubility of the salt form (12 mg/mL), are making it suitable for sustained delivery using cubosomes (hydrophilic inner chamber) (Detrol label US FDA, 2025). Weak basic nature (pKa of 9.6) of the drug is sensitive to acidic and oxidation based degradation. Cubosomes are stable in acidic labile condition for protecting TOTA in gastric lumen. TOTA is hydrophilic and lipophilic in nature, ideal for high entrapment in hydrophilic inner chamber (for salt form) and lipophilic domain (for tolterodine). Thus, the investigated cubosomal nanovesicle was ideal for oral delivery of low dose TOTA (2–4 mg). The aforementioned physicochemical properties of TOTA (moderate lipophilicity, high aqueous solubility, and acid labile nature) make it highly suitable for cubosome-based delivery.

Oral administration causes high hepatic metabolism (an active metabolite by CYP2D6), resulting in several side effects and high plasma drug fluctuation. Controlled and sustained delivery of the drug is considered safe and well-tolerated over immediate release capsule due to flatter serum concentration-time profile (Drutz et al., 1999). TOTA and its active metabolites (5-hydroxymethyltolterodine) are nonselective competitive muscarinic receptor antagonist (M₂ and M₃) and antispasmodic in therapeutic activities. Pure TOTA is sparingly soluble in water than its tartrate form (Detrol label US FDA). Two doses (2 mg twice a day and 4 mg once a day) are recommended as first-line treatment using immediate release (2 mg) or extended release (4 mg) oral capsule. Garely and Burrows provided a comprehensive review report on the benefits-risk profile of the drug intended for oral capsule (Garely and Burrows, 2004). Despite therapeutic benefits, the drug results in constipation, headache, and mouth dryness after oral administration. Moreover, it is difficult to administer children (unable to swallow) or advanced age patients. Considering these constraints, few researchers recommended transdermal applications in terms of solution, patch, topical proniosomal gel, and microemulsion (Elshafeey et al., 2009; Rajabalaya et al., 2016; Rajabalaya et al., 2017; Sun et al., 2013; Zhao et al., 2009). However, these transdermal products have been challenged to achieve commercial success due to the cost of the development, stability, and scale up at large scale production. It was reported that the side effects, efficacy, and tolerability of the drug were dependent upon the release pattern of the drug in vivo. Therefore, extended release TOTA was well tolerated than immediate release in human (Gacci et al., 2014). In this context, the side effects of the drug can be mitigated using biocompatible sustained release liquid crystals (cubosomes) as nanocarrier.

Several lipid-based nanocarriers (liposomes, niosomes, transfersomes, lipid NPs, and microemulsions) have been well explored for hydrophilic and lipophilic drug delivery. However, these nanocarriers face multiple challenges (poor storage stability and degradation by gastric lipases). Nanoemulsions, liposomes, and niosomes are predominantly prone to physical instability (high drug leakage), chemical oxidation, and limited drug loading efficiency. In contrast, cubosomes (possessing bicontinuous cubic phase) with high internal surface area, offer superior drug loading and high stability across a wide range of pH, ionic strength, and temperature (Sivadasan et al., 2023; Lakshmi et al., 2014). Microemulsions, nanoemulsions, lipid nanoparticles, and SNEDDS are well explored for rapid drug release whereas cubosomes provide controlled and sustained drug release, making cubosomes ideal for oral delivery in acidic gastric conditions (Abourehab et al., 2022; Lakshmi et al., 2014).

Cubosomes can be prepared using biodegradable, bio-adhesive, and biocompatible monoolein by emulsification of the cubic lipid phase in water (Salah et al., 2017). Among various nanocarriers, cubosomes have proven promising benefits for oral delivery of drugs with inspiring characteristics such as stability, bio-adhesion mediated improved absorption and bioavailability, and substantial mechanical rigidity as compared to liposomal products. The nanocarrier is reported to facilitate oral absorption through bio-adhesion properties, interacting with intestinal cell membrane, and secretion of physiological surfactant in the gastrointestinal tract. Notably, cubosomes maintain the drug in soluble form by entrapping into the mixed micelle (formed from digestion of cubosomes) and subsequently results in enhanced oral bioavailability (Mohsen et al., 2021).

HSPiP and QbD are software to be used in preliminary studies. Hansen solubility parameters predicted theoretical solubility and physicochemical interaction with carriers and solvent. QbD is an experimental design tool for achieving quality product by identifying material and processing attributes under given set of constraints. In this study, an attempt has been made to address HSPiP and QbD based TOT loaded cubosomes for oral delivery in the treatment of OAB. The optimized formulation was evaluated for formulation characteristics (size, zeta potential, polydispersity index, morphology, topography using atomic force microscopy, and thermal analysis), in vitro drug release, ex vivo permeation, and in vivo assessment as compared to the drug suspension in rat model.

2. Materials

Tolterodine tartrate (TOTA, ≥ 98 % pure) was a gift sample received from J.B. Chemicals & Pharmaceuticals Ltd., Thane, India. Polyethylene glycol 400 (PEG400), propylene glycol (PG), and oleic acid were procured from CDH Fine Chemical, Mumbai, India. Distilled water was generated using Millipore system (Merck Millipore, California, USA). Dialysis membrane of 12,000 − 14,000 Da molecular weight cut-off was procured from HiMedia Laboratories Private Limited, Mumbai, India. Polyvinyl alcohol (PVA) was purchased from Merck, USA. Poloxamer 188 (hydrophilic polyoxyethylene-polyoxypropylene block copolymer) served as stabilizer and it was procured from Thermo Scientific Chemical., Mumbai, India. Glyceryl monooleate (GMO) and glyceryl mono stearate (GMS) were procured from Sigma-Aldrich (Saint Louis, MO, USA). All required solvents were of AR grade in the study. Buffers solutions were freshly prepared wherever required.

3. Methods

3.1. HSPiP based theoretical solubility and interaction between the drug excipients

HSPiP and QbD are well established software to predict theoretical solubility and optimization (material and process), respectively. HSPiP is basically working on innate cohesive energy of materials distributed over dispersion (hydrophobic portion of molecule such as hydrocarbon expressed as δ_d, hydrogen bonding as δ_h, and polarity (dielectric constant value as δ_p) energies. These are termed as Hansen solubility parameters and mathematically, it is defined as the sum of square of each energy as provided in Eq. (1)

$${\left[\mathrm{\delta }\right]}^2={\left[{\mathrm{\delta }}_{\mathrm{d}}\right]}^2+{\left[{\mathrm{\delta }}_{\mathrm{p}}\right]}^2+{\left[{\mathrm{\delta }}_{\mathrm{h}}\right]}^2$$ (1)

where “δ” is the total cohesive energy in the equation. The three parameters of eq. (1) are considered as co-ordinates for a point in a 3-dimensional space which is called as the Hansen space (R_a). The predictive program categorized solvents, surfactants, co-surfactants, and polymers as “bad” or “good” depending upon estimated RED (relative energy difference) value. It is usually >1 for insoluble solute in a particular solvent(s) or polymer(s) at fixed temperature and vice versa (Hansen, 2007). RED is the ratio of R_a to R_o where R_o and R_a are Hansen sphere radius (by default value) and Hansen space parameter, respectively. These can be calculated for formulation.

$${\left[{\mathrm{R}}_{\mathrm{a}}\right]}^2=4{\left[{\mathrm{\delta }}_{\text{d2}}–{\mathrm{\delta }}_{\text{d1}}\right]}^2+{\left[{\mathrm{\delta }}_{\text{p2}}–{\mathrm{\delta }}_{\text{p1}}\right]}^2+{\left[{\mathrm{\delta }}_{\text{h2}}–{\mathrm{\delta }}_{\text{h1}}\right]}^2$$ (2)

Targeted HSP can be attained using the program input parameters and by-default setting.

$$\mathrm{RED}={\mathrm{R}}_{\mathrm{a}}/{\mathrm{R}}_{\mathrm{o}}$$ (3)

3.2. Experimental solubility of tolterodine tartrate (TOT)

The experimental solubility data were gathered by estimating real solubility of the drug in the predicted excipients before formulation development (lipids, surfactants, mixtures, water, and organic solvents). The solubility measurement of the drug was measured in targeted excipients (distilled water, glyceryl mono oleate, glyceryl mono stearate, polyvinyl alcohol (PVA), polyethylene glycol 400 (PEG), oleic acid, propylene glycol, and equimolar blend of poloxamer 188 and PVA). In brief, a fixed volume (10 mL) of pure solvents (PEG400, PG, oleic acid, and water) was used to dissolve the drug. GMO, GMS, and PVA are solid excipients. Therefore, these were used in the formulation. Excess amount of TOTA was added in a separate flask and the tested solvent was added into it. The mixture was stirred on water bath shaker (50 rpm) at constant temperature (40 ± 1 °C) for 24 h to attain an equilibrium point. Then, the mixed content was centrifuged (6000 rpm for 8 min) to settle down the insoluble content at the bottom. The separated supernatant solution was removed carefully and filtered it with a membrane fitter before dilution. Each sample was individually quantified using a UV–Vis spectrophotometer (Shimadzu UV-1280, Japan) at λ_max of 223 nm (Rajabalaya et al., 2017). The solubility results are expressed as mean ± SD (standard deviation).

3.3. Central composite design: an optimization tool

Experimental design tool is based on the systematic and statistically oriented approach to optimize materials and methods under set conditions. Several compositions of the selected factors were predicted after feeding input range of factors (at varied levels). The CUB-TOTA were systematically optimized by two significant factors at varied levels using central composite design (CCD) model of Design Expert 13 software (Trial version) (Stat-Ease Inc., Minneapolis, USA). The model is reliable and precise as compared to D-optimal design for investigating interactions and quadratic effects. Various experimental runs were carried out in CCD as it is more efficient and executed few outcomes (13 runs) to determine the optimal values of the fed factors variables (Ramzan et al., 2021). The fed factors were assigned as X₁ for the lipid (GMO) and X₂ as stabilizers concentration (poloxamer 188: PVA in 1:1 ratio) against the three dependent variables (responses) such as particle size (Y₁: nm), % entrapment efficiency (Y₂: %EE), and polydispersity index (PDI as Y₃). The goals were set for independent variables (Y₁, Y₂, and Y₃) (minimum, maximum, targeted, and in range) for getting the most desirable value of overall desirability value (Y₁, Y₂, and Y₃). A general polynomial equation for CCD model is given below:

$$Y_{\mathit{1,2,3}}=B_{\mathit{0}}+B_{\mathit{1}}{X}_{\mathit{1}}+B_{\mathit{2}}{X}_{\mathit{2}}+B_{\mathit{3}}{X}_{\mathit{1}}{X}_{\mathit{2}}+B_{\mathit{4}}{X_{\mathit{1}}}^2+B_{\mathit{5}}{X_{\mathit{2}}}^2$$ (4)

where Y_1,2,3 is a common representation of a dependent variable (X) with two coefficients (B₁ and B₂) for the factors (X₁ and X₂). B₀ is an intercept. B₃ is a coefficient of interaction between factors, whereas B₄ and B₅ are the coefficients of quadratic terms “X₁” and “X₂”, respectively.

The data obtained from the experimental design was statistically analyzed using analysis of variance (ANOVA) and the best suited model was fitted into regression (Hussain et al., 2023). Furthermore, the optimized CUB-TOTA formulation was identified through the numerical point of prediction method and inspecting numerical function parameter (overall desirability function). The estimated overall desirability may be positive, negative, and zero depending upon set constraints. The positive and negative terms in the polynomial equation represent the synergistic or antagonistic impact of the factor on the responses. Zero desirability confirms the assessed factor or response is out of the fit while optimization. It does not contribute in the optimization of quality prediction (Attimarad et al., 2022).

3.4. Preparation of optimized OCUB-1

The experimental design predicted OCUB1 with maximum desirability value. Its composition was predicted as 10 % w/v of X₁ and 3 % of X₂ (P407: PVA) for the promised product with desired and expected formulation quality attributes. This was prepared following a procedure reported in literature (Lai et al., 2009). Initially, X₁ and X₂ were simultaneous heated to the melting temperature of 65 °C to get a homogeneous melt. The drug was added into the molten mixture under a continuous stirring condition. The obtained isotropic blend was gradually emulsified in an aqueous phase maintained at the same temperature (65 °C) under vigorous stirring rate (600 rpm). This results in a primary coarse dispersion of cubosomes. For further size reduction, the coarse dispersion was forced to high speed homogenization (12,000 rpm) for 8 mins. This led to formation of nanoscale cubosomes dispersion. Final strength of the formulation was 0.4 %w/w. The prepared formulation was kept aside overnight to cool it gradually and afterwards OCUB-1 was stored in a glass vial for further characterization. Furthermore, a suspension of TOTA was prepared with 1 % carboxymethyl cellulose in water and stirred for 1 h using magnetic stirrer. The drug suspension (SUS-TOTA) was use as a control formulation along with OCUB1. OCUB1 was transferred into a gelatin capsule (capsule #000) (4 mg/g).

3.5. Optimized OCUB-1 characterizations

OCUB-1 was subjected to determine particle size (PS), polydispersity index (PDI), and zeta potential (ZP) using a dynamic light scattering-based technique (Malvern ZS, Nano, UK). PDI was measured to understand the size distribution pattern and peak intensity. The nano-cubosomal dispersions were diluted with Milli-Q water just before analysis at room temperature (25 ± 1 °C). The diluted sample avoided interference in results (Hussain et al., 2016a, Hussain et al., 2016b). The ZP was determined employing a special capillary sample holder of zeta-sizer (Malvern ZS, Nano, UK) at 25 °C. The sample was transferred to fill the capillary (U-shaped capillary sample holder) using a syringe. Both ends were completely closed and electrodes were kept dried. The results were interpreted accordingly. All measurements were conducted in triplicate. The experiment was replicated for mean and standard values (mean ± SD).

3.6. Thermal behavior using differential scanning calorimeter (DSC)

Thermal behavior of the excipients and the drug were studied using a DSC technique (DSC-50, Shimadzu, Japan). The sample was individually processed over constant temperature range. Each sample was accurately weighed (∼ 2–3 mg) and transferred to an aluminum pan. Notably, moisture was avoided during weighing and the sample transfer. Moreover, the pan with the test sample was completely crimped and sealed. The sealed sample was placed inside the thermal furnace against a blank pan (as reference). The sample was slowly heated with constant heating rate (10 °C/min) to achieve a final maximum temperature of 320 °C. Then, the heating was reduced by cooling process with the same rate. Cooling process was run using a Nitrogen chiller (a gas flow rate of 20 mL/min). Similar method was adopted in our published report (Malik et al., 2024).

3.7. Atomic force microscopy (AFM): topographical assessment

Topological profile (surface roughness profile) of OCUB1 was studied for the surface roughness behavior as compared to the blank OCUB1 under AFM. For this, a glass coverslip was used to hold the sample. The surface of the coverslip was coated with poly-L-lysin (a fixative) and dried under air. The coated surface served as fixative to hold the sample against the force applied during analysis by the AFM probe (cantilever). The sample (a drop) was placed and smeared on the coated surface of the glass coverslip to make a fine film. The cubosomes were immobilized with poly-L-lysin to protect the sample and its movement while processing (Altamimi et al., 2022). The samples were completely dried to avoid high noise in scanning. Therefore, air drying was recommended for overnight. A wet sample is unable to be scanned at right position against the cantilever (V-shaped micrometer-sized lever) due to constant movement from the targeted location (Quintanilla, 2013). The tip remains in semi-contact mode while image acquisition. Each sample was separately scanned at room temperature of 25 °C, scanning rate of 0.8 Hz, and under semi-contact angle mode employing AFM Solver Pro 47 (Saint Petersberg, Russia). The in-built software was used to process the data. Various topological parameters were estimated such as average roughness (nm), skewness, coefficient of kurtosis, root mean square of roughness (nm), and enthalpy. The roughness curve (hill and valley) was estimated to confirm the surface behavior.

3.8. In vitro drug release and release kinetics studies

In vitro drug release pattern of OCUB1 and SUS-TOTA (suspension) formulations, were investigated employing a dialysis bag as reported in the literatures (Ramzan et al., 2022; Shahid et al., 2022). A fixed content (0.5 g) of the formulation containing 2.0 mg of TOTA was transferred into the dialysis bag and suspended in a beaker (PBS, pH 7.4, and 50 mL). The in vitro drug release study was performed at constant temperature (37 ± 1.0 °C) and stirring speed (100 rpm) using three plate magnetic stirrer (Multi-Hotplate Stirrers-3, DAIHAN Scientific, South Korea). The samples (2 mL) were collected and replaced with fresh PBS pH 7.4 at regular time intervals (0.5, 1, 2, 3, 4, 5, 6, 8, 24, and 48 h) from each container with an equal volume followed by analysis using HPLC method. The % cumulative TOTA released was calculated and reported as a comparative assessment. The study was carried out in triplicate (n = 3, ± SD). Furthermore, the drug release from optimized OCUB1 was subjected to different release kinetic models (zero order, first, Higuchi, and Korsmeyer-Peppas) to understand release behavior of the drug from the nanocarrier and the suspension. The diffusion coefficient value “n” dictated release mechanism (Fickian or non-Fickian release kinetics).

3.9. In vitro hemolysis study

In general, new formulation or drug may exhibit hemolysis after oral or parenteral delivery. Particularly, ionized products are responsible to interact with negatively charged erythrocytes, leading to hemolysis and reduced hemoglobin content in patients. In vitro hemolysis test is considered as a preliminary toxicity assessment for any product to negate its incompatibility. Orally delivered nanoscale cubosomes enter systemic circulation through modulated intestinal membrane and lymphatic pathway (Sivadasan et al., 2023; Lakshmi et al., 2014). To confirm hemocompatibility, red blood cells (RBCs) hemolysis study was carried out at different concentrations. Furthermore, regulatory agency of European Commission (Scientific Committee on Consumer Safety: SCCS) often recommend hemolysis testing for any drug development, especially for novel formulations or those with a high potential for systemic absorption (Bernauer et al., 2020).

In vitro preliminary RBCs toxicity of OCUB1 was conducted to compare against SUS-TOTA. PBS and triton X100 served as negative and positive controls, respectively. The method was followed as per our reported procedure (Shahid et al., 2022). Briefly, varied concentrations (0.625, 1.25, and 2.5 μg/mL) of the test samples were allowed to interact with 2 mL of RBCs suspension (5 % suspension of RBCs separated from plasma). A final volume (5 mL) was made with PBS. All the test samples were added with the RBCs suspension followed by incubation for 2 h at 37 ± 1 °C. Then, the tubes were taken out from the incubator and subjected to centrifugation at 9000 rpm for 6 min. The collected supernatant was employed to estimate the released hemoglobin (RBC hemolysis indicator) during incubation. The absorbance (λ_max = 540 nm) was taken using an UV − Vis spectrophotometer (UV-1601, Shimadzu, Japan).

3.10. Ex vivo permeation study using rat intestine

Ex vivo permeability study was carried out to compare permeability potential of the drug ferrying nanocubosomes (OCUB1) against the drug suspension. This was conducted using a non-everted sac of rat intestine obtained from sacrificed rats. The animals were approved from the Institute for conducting the ex vivo study as per the protocol and the procedure reported in the literature with slight modification (approval number: IOP/IAEC/2023/Octo/15) (Dixit et al., 2012; Parsa et al., 2013). The rats were randomly grouped as group-I (DS) and group-II (OCUB1), and housed in a temperature controlled condition (25 °C) with 12 − 12 h dark-light cycle. Each group contained 6 rats and they were fasted for 12 h before ethical sacrifice. The rats were ethically sacrificed (CO₂-anesthesia) and jejunum was excised from each rat group. The inner intestinal content was rapidly flushed with buffer solution (PBS, pH 7.4) to remove intestinal lumen under constant supply of oxygen and maintained temperature (37 ± 1 °C). The jejunum was gently transferred into the medium and divided into small segment (4.5 cm) to fill the test sample (0.5 mL equivalent to 2 mg). The ends were closed with a clip. A constant effective surface area for the drug diffusion was maintained for each group. Each sac was pre-incubated at the same temperature and the permeation medium (200 mL) for 5 min, previously maintained at constant temperature (37 ± 1 °C), stirring (100 rpm), and aeration (slow rate). Each mL of DS (4 mg/mL) and OCUB1 (4 mg/mL) was separately transferred into the sac (Dixit et al., 2012; Parsa et al., 2013). At predetermined time points (30, 60, 90, 120, and 180 min), the sample (0.5 mL) was taken out from the medium and fresh buffer was used to replace the sampled volume. The withdrawn sample was used to assay the drug diffused from lumen to the medium (200 mL). The drug permeated was estimated using our validated HPLC method (Patil et al., 2024).

3.11. In vivo study: pharmacokinetics profile

The in vivo oral bioavailability study of the optimized OCUB1 relative to SUS-TOTA was carried out in male Wister rats (220 ± 10 g) to provide a preclinical in vivo data of intrinsic impact of the nano-cubosomes after oral administration. The protocol of the study was reviewed and approved (approval number: IOP/IAEC/2023/Octo/15) by the Institute Animal Ethics Committee (IAEC) of SVKM's Institute of Pharmacy, Dhule, and Maharashtra, India) and conducted in accordance with the guidelines of the IAEC. The rats were randomly grouped, labeled, and housed according to the guidelines (ARRIVE guideline). All rats were kept in an animal care facility under standardized conditions of temperature (24 ± 1.5 °C) and humidity (45–50 ± 5 %). They were fed on standard pellet diet ad libitum.

Twelve adult male Wistar rats were randomly assigned to two groups with equal number (n = 6). Prior to commence the bioavailability experiment, the rats were on fast condition overnight (12h). Group-I and II received SUS-TOTA and OCUB1, respectively. Both groups were dosed at 4 mg/kg of body weight (Pahlman et al., 2001) without making any dosing error. The blood (cumulative volume of 1 mL) sample was collected at different time intervals (0.5, 1, 2, 4, 6, 8, 12, 16, 20, and 24 h) from the anesthetized rats. The blood samples were immediately stored into 2 mL of Eppendorf tubes containing EDTA (ethylenediaminetetra acetic acid) sodium salt, followed by centrifugation under cold condition (4 °C) for 8 min at 8000 rpm. The plasma samples were separated and kept in the labeled tubes and stored at −20 °C till further analysis.

The proteins present in the plasma sample (150 μL) were precipitated by adding methanol (a precise volume as per need). Then, the processed plasma samples were treated with a mixture of organic solvent (methanol and chloroform, 2:1) to extract TOTA by lysis of cubosomal vesicles. The mixture was vortexed and centrifuged to separate the organic layers. Finally, a vacuum was applied to evaporate the excess solvents and the residues were left in the tube. The dry residues were reconstituted with the suitable volume of the mobile phase. The reconstituted samples were filtered and subjected to HPLC for quantitative analysis of TOTA. The drug-plasma concentration time curves were plotted and the drug content were analyzed with PK solver software (an add-in program for PK parameters and pharmacodynamics data analysis in Microsoft excel). Several PK parameters were estimated. These were the peak plasma concentration (C_max, ng/mL), the time required to reach C_max (T_max, h), the mean residence time (MRT, h), and the area under the drug-plasma concentration time curve from zero to infinity (AUC), and area under first moment curve (AUMC). The data were statistically analyzed using ANOVA (analysis of variance) model with multiple comparisons to assume the statistical significance at p < 0.05 (significant level).

3.12. Biochemical and hematological analysis

The study was performed for acute toxicity assessment. The rats were grouped and received formulations for 14 days at the same dose (aforementioned). It was imperative to observe any variations in biochemical and hematological biomarkers. A blood sample (0.2 mL at a time from one rat) was taken from retro orbital plexus of anesthetized rats for hematological and biochemical analysis. The vials were kept at room temperature for 100 min and then centrifuged a 6000 rpm for 10 min to separate serum and plasma (Hussain and Singh, 2016). Both the components (plasma and serum) were used for further evaluation of various biochemical parameters such as glucose, cholesterol, urea, ALT (alanine transaminase, SGPT), AST (aspartate transaminase, SGOT), total proteins, ALP (alkaline phosphatase from liver and bone as main source), creatinine, and bilirubin. The blood samples collected in the sodium citrate tubes were used for analyzing red blood cell count (RBC), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (Plt), total leukocytes count (TLC), polymorphs, lymphocytes, monocytes, and eosinophils count using an auto clinical analyzer.

4. Results and discussions

4.1. HSPiP based theoretical solubility and interaction between the drug excipients

Hansen solubility parameters are used to understand possible interactions existing between the solute and solvent. These interactions are innate energies of materials due to polarity, dispersion, and hydrogen bonding (functional group based interaction) (Hansen, 2007). Theoretical solubility of TOTA was predicted in the lipids, surfactants, and co-surfactant. Results are compiled in Table 1. Various estimated HSP and RED values are presented to screen suitable excipients. The predicted theoretical solubility values of TOTA in the excipients are exhibited in the same table. Maximum predicted solubility of the drug was found to be in water which may be attributed to salt nature of TOTA. This predicted solubility is quite reliable and rationalized based on the literature value (Detrol, US FDA label). Moreover, the predicted solubility of TOTA in GMS and GMO is underestimated by the program due to solid nature of excipients. Therefore, HSP estimated values were used to re-calculate HSP values in 10 % aqueous solution of GMS and GMO individually (Table 1). Notably, HSP values of TOTA were found to be closely related to the HSP values of GMO as shown in Table 1 which can be selected to formulate the cubosomes. This can be correlated to preferential lodging of the drug in the lipid matrix (bicontinuous phase) of cubosomes as well as aqueous inner chamber. This results in maximum drug entrapment, protection from acidic degradation, and stability in cubosomes for slow and sustained drug release. Poloxamer 188 and PVA served as stabilizer in the formulations (Yakaew et al., 2021).

Table 1.

Summary report of HSPiP software estimated HSPs values (enthalpy = 42.13 kJ/mol and fusion temperature = 215 °C).

Name			HSP values estimated
Excipients	Ra estimated		δd (MPa1/2)	δp (MPa1/2)	δH (MPa1/2)	δt (MPa1/2)	M.Vol (cc3/mol)
TOTA	Predicted solubility (%w/w)	RED	17.9	3.1	6.0	19.1	297.2
PG⁎⁎	1.53	1.4	16.8	10.4	21.3	29.8	74.3
PEG400⁎⁎	0.49	0.72	16.5	8.5	13.0	22.6	210.8
OA⁎⁎	0.3	0.42	16.0	2.8	6.2	17.6	318.7
GMO⁎	0.21	0.49	16.4	4.4	9.3	19.3	371.3
GMS⁎	0.19	0.57	16.2	4.2	10.3	19.7	377.6
Water	1.108	4.12	15.5	16.0	42.3	47.6	18.0
PVA	–	2.0	25.8	6.9	17.0	31.7	36.6
βPoloxamer 188	–	0.82	17.49	10.23	8.74	22.10	–
Validation parameter
	Predicted HSP and RED⁎ parameters for the suggested combination of solvents
Combinations			Sol. w/w	RED	δd	δp	δH
GMO (10 %)					1.64 + 13.95	0.44 + 14.4	0.93 + 38.3
GMS (10 %)					1.62 + 13.95	0.42 + 14.42	1.03 + 38.3
PVA (1.5 %)
Poloxamer 188 (1.5 %)
OCUB1
	SMILE
TOTA	“Cc1ccc(c(c1)[C@H](CCN(C©C)C©C)c2ccccc2)O”
PG	“CC(CO)O”
PEG400	“C(COCCOCCOCCOCCO)O”
OA	“CCCCCCCC/C=C\CCCCCCCC(=O)O”
GMO	“CCCCCCCC/C=C\CCCCCCCC(=O)OCC(CO)O”
GMS	“CCCCCCCCCCCCCCCCCC(=O)OCC(CO)O”
PVA	“XCC(O)X”
Water	“HO”

^⁎RED = The relative energy difference (Ra/Ro) where Ro = 18.0. (0) and (1) were flagged as bad and good solvent, respectively as per HSPiP. These were flagged as “good, (1)” due to experimentally obtain as “soluble” in the mixed solvents.

^⁎⁎(Ezati et al., 2020; Hansen, 2007; Abbott, 2012; ^βKitak et al., 2015).

4.2. Experimental solubility analysis

The actual solubility of the drug was investigated in the predicted excipients as summarized in Table 1 to simulate the predicted theoretical data. The result is illustrated in Fig. 1. The predicted solubility (0.2 %w/w = 2 mg/mL) of the drug in GMS (HLB of 11) and GMO are unrelated due to solid nature of the excipients to the experimental solubility values (Fig. 2) at the explored temperature (45 °C). In buffer (phosphate buffer, pH 7.4), TOTA solubility is approximately 12.0 mg/mL whereas it is 11.08 mg/mL in neat water. This difference can be correlated to salt mediated improved solubility of Detrol (containing TOTA) in the buffer (Detrol US FDA label). Moreover, the predicted solubility values of TOTA in water (11 mg/mL) and OA (3.0 mg/mL) are in good agreement with the published report (12 mg/mL in water and 5.0 mg/mL in OA) (Elshafeey et al., 2009). OA (oleic acid) is a highly lipophilic (HLB = 1.0) oil and considered to have limited solubility of the drug in the salt form (hydrophilic). Therefore, the experimental and the predicted solubility values are in good agreement with the program fitness (Table 1 and Fig. 1). Notably, combination of two excipients (low and high HLB) may result in enhanced drug solubility and stability (Elshafeey et al., 2009). Therefore, the combination of 3.0 % aqueous solution of PVA and poloxamer 188 (1:1) rendered remarkable increase in TOTA solubility. The experimental solubility of TOTA in 10 % PVA was 11.5 mg/mL whereas it was reached to 18.9 mg/mL in combination with poloxamer 188 (HLB = 29) (Fig. 1). It can be rationalized based on PVA (hydrophilic surfactant with high HLB, 18) mediated improved solubility (Ghitman and Stan, 2019). The combinations of the excipients (including water as an aqueous) showed HSP values close to water. This indicates the prime interactive forces associated with water (hydrogen bonding, polarity, and London force) are working in the aqueous solution of PVA, poloxamer 188 or combination of them in tandem. Various primary physicochemical properties including particle size, particle morphology, and drug-excipient interaction influence the drug solubility (Manaia et al., 2017). The selection of a suitable excipient for high solubility of TOTA is desirable step in formulation development with quality product for improved oral bioavailability. It is clearly evident from the result of the experimental solubility analysis that GMO, distilled water, and mixture of poloxamer 407 and PVA (1:1) exhibited high solubility of TOTA at given conditions.

Fig. 1.

Solubility (mg/mL) of tolterodine tartrate (TOTA) in various excipients/ingredients for oral drug delivery.

Fig. 2.

2D and 3D contour plots generated in response surface methodology. Fig. 3A, B, and C illustrated 2D plots of Y₁, Y₂, and Y_3, respectively whereas 3C, 3D, and 3E elicited 3-D contour plots for Y₁, Y₂, and Y₃, respectively. All of these responses are quadratic models.

4.3. Preparation of cubosomes and optimization using CCD model

Investigating solubility in different excipients is essential for ensuring the efficacy, safety, and enabled controlled release at the target site (Ezike et al., 2023). Moreover, these ingredients exhibit better biocompatibility and maintain the drug stability in the cubosomes throughout in vivo journey (Nazem et al., 2023). The selection of CCD model was based on the several reasons. The adopted model with 2 factors (at three levels) renders a systematic and statistically powerful method to get an optimized formulation. The model identifies a linear effect, an interaction between the studied factors (critical to understand the joint impact of the factors on the responses), and generally quadratic effects (Singh and Lillard, 2009). The model is efficient with a comprehensive study considering set constraints, assisting to locate an optimal region in the design space. Notably, the high value in CCD design table underscores the robustness of the optimization and enables user to differentiate prominent signals from noise. In other design, it may be acceptable with compromised precision. Thus, the model is related with rotatability and stability, improving the reliability of the predicted data (Aly et al., 2025).

The predicted formulations were formulated (CUB-TOTA1 – CUB-TOTA13) using varied ratio of GMO and the blend of poloxamer 188 and PVA (Table 2). The levels of the design model were selected based on the experimental solubility and the benchtop stability of the drug at room temperature. Optimization was run under given sets of constraints (Table 2) to achieve maximum desirability. X₁ and X₂ had profound impact on Y₁, Y₂, and Y₃ as predicted in the program. Particle size (Y₁) of cubosomal formulation is an important characteristic for efficient drug dissolution, absorption, high surface area, stability, and shelf-life of nanoformulations (Ramzan et al., 2022). The model equation for Y₁ is given as follows:

$$Y_{\mathit{1}}=\mathit{267.80}–\mathit{8.49}{X}_{\mathit{1}}+\mathit{63.93}{X}_{\mathit{2}}–\mathit{15.33}{X}_{\mathit{1}}{X}_{\mathit{2}}+\mathit{46.35}{X_{\mathit{1}}}^2–\mathit{5.12}{X_{\mathit{2}}}^2$$ (1)

Table 2.

Optimization of different factors involved in the preparation of CUB-TOTA using CCD.

Level of factors
Code and factors	−α (%)	−1 (%)	0 (%)	+1 (%)	+α (%)
X1: GMO	8.9	10.0	12.5	15.0	16.1
X2: Poloxamer: PVA (1:1)	0.9	1.4	2.5	3.0	5.1
Constraints
Code and responses	Low		High		Goal
Y1: Particle size (nm)	163.4		431.8		Minimum
Y2: Entrapment efficiency (%)	43.2		81.8		Maximum
Y3: Polydispersity index	0.231		0.373		In-Range
Formulation code	X1	X2	Y1	Y2	Y3
CUB–TOTA1	0	0	313.6	67.2	0.312
CUB–TOTA2	0	0	289.9	74.7	0.248
CUB–TOTA3	0	0	265.5	72.4	0.272
CUB–TOTA4	−1	+α	431.8	43.2	0.373
CUB–TOTA5	–1	+1	276.5	73.4	0.251
CUB–TOTA6	0	+1	271.2	72.1	0.363
CUB–TOTA7	0	–α	351.2	44.7	0.340
CUB–TOTA8	–α	0	211.8	77.6	0.267
CUB–TOTA9	0	–1	256.2	73.0	0.301
CUB–TOTA10	–1	+1	163.4	81.7	0.232
CUB–TOTA11	+α	0	321.2	55.6	0.298
CUB–TOTA12	0	0	413.7	41.4	0.321
CUB–TOTA13	+1	+1	245.2	76.7	0.367

The negative sign of coefficient X₁ indicates the factor (GMO concentration) needs to be decreased to get desired (optimized) response. A positive sign of X₂ must be increased to an optimal level. Thus, adjusting both factors to an optimal level may result in the lowest particle size, optimal PDI, and high %EE. The 3D and 2D plots for Y₁-Y₃ are illustrated in Fig. 2A-F wherein the combined impact of the factors on the studied responses demonstrated quadratic effects without interaction in the design space with high precision and reliability (Aly et al., 2025).

It is clearly evident from Fig. 2A, B, and C that both factors exhibited quadratic relationship with the size, %EE, and PDI, respectively. The particle size was found to be decreased with increasing content of the lipid till 12.5 % (X₁ = GMO) as shown in Fig. 3D. Beyond 12 %, the size was progressively increased. Therefore, it would be better to recommend X₁ concentration about up to 12.5 % w/w. X₂ was linearly related with the size. Overall, the trend was quadratic with X₁ as shown in Fig. 2A and D. The impact of X₂ was quite undesirable beyond 3 % as per set goal. Y₁ was found to be increasing with increased content of X₂ and it was better to opt minimum concentration. Adverse effect of X₂ at higher concentration can be correlated to the probable chance of precipitation of poloxamer or PVA in the aqueous phase at the explored temperature and pH (Kitayama et al., 2023). Therefore, X₂ was optimized at low concentration (3 % w/w). In contrast to the development of oral formulation, OCUB1 having low particle size to get absorbed substantially from intestinal lumen and extended circulation half-life, the drug loaded lipidic cubosomes should have stabilized for controlled drug release at physiological pH (Nazem et al., 2023).

Fig. 3.

(A-C) Interaction plots and (D—F) predicted versus actual plots for the investigated responses.

In general, Y₂ depends upon on various innate properties of the drug, formulation characteristics, and temperature. These are physicochemical properties of TOTA (hydrophilic nature, molecular structure, interaction forces, and pKa), particle size, drug interaction with lipid matrix of cubosomes, solubility stability inside inner chamber of cubosomes, and integrity of lipid bilayer to control the drug leakage (Sivadasan et al., 2023). Eq. (2) shows positive effect of GMO concentration (X₁) and negative effect of stabilizer concentration (X₂) (Fig. 2B & 2E). Overall result showed that there was initial increase in %EE followed by steeper reduction on increasing the concentration of X₁ whereas %EE was proportionally reduced with increased content of X₂. The higher concentration of PVA and poloxamer may result in adverse effects such as high viscosity by PVA and facilitated self-emulsification of poloxamer to form micelle. Moreover, the high concentrations of poloxamers and PVA as X₂ can alter the partitioning behavior of hydrophilic TOTA for promoted diffusion into the aqueous phase rather than entrapment within bicontinuous cubic phase of cubosomes (Lai et al., 2009). Nevertheless, optimizing the concentration of X₂ is crucial for maximizing %EE of hydrophilic TOTA. Therefore, balancing X₁ and X₂ can avoid the adverse effects (high viscosity and unintended formation of micelle) of poloxamer and PVA associated with high concentration (Sivadasan et al., 2023). The unique quadratic behavior of X₁ and X₂ on %EE can be correlated to relative formation of micelle beyond X₁ = 12.5 % and X₂ = 5.0 % rather cubosomes (Sivadasan et al., 2023). The model was fit as evidence with the low p-value (0.0008) and high F-value (61.96). The model equation for Y₂ is given below:

$$Y_{\mathit{2}}=+\mathit{62.00}+\mathit{1.07}{X}_{\mathit{1}}–\mathit{10.55}{X}_{\mathit{2}}+\mathit{4.20}{X}_{\mathit{1}}{X}_{\mathit{2}}–\mathit{11.17}{X}_{\mathit{1}}–\mathit{2.33}{X_{\mathit{2}}}^{\mathit{2}}$$ (2)

Polydispersity index (Y₃) denotes the size distribution of the developed cubosomal particles in a defined area. It is clearly evident from Fig. 2C and F that X₁ showed positive effect whereas X₂ had negative impact on the PDI of the developed cubosomal formulation. In brief, Y₃ followed the same pattern as observed in Y₁ due to particle size and its related population distribution. Statistical analysis also confirmed the best fit of the model (quadratic) as the low p (0.0008) and high F-values (45.56). Notably, the summary of statistical analysis was compiled in Table 3. The model equation for Y₃ is given below:

$$Y_{\mathit{3}}=+\mathit{0.31}–\mathit{3.33}{\mathit{4X}}_{\mathit{1}}–\mathit{0.02}{\mathit{4X}}_{\mathit{2}}+\mathit{2.01}{X}_{\mathit{1}}{X}_{\mathit{2}}–\mathit{0.01}{{\mathit{8X}}_{\mathit{1}}}^2–\mathit{0.02}{{\mathit{2X}}_{\mathit{2}}}^{\mathit{2}}$$ (3)

Table 3.

Characteristics of the models fitted to the responses.

Responses	Model fitted	R2	Adjusted R2	Predicted R2	Precision
Particle size (Y1)	Quadratic	0.7471	0.5665	0.6970	6.872
EE (Y2)	Quadratic	0.7536	0.5776	0.7235	6.277
PDI (Y3)	Linear	0.7772	0.5133	0.7060	5.832

Fig. 3 illustrated interaction curves for all the investigated responses.

4.4. Overall desirability as numerical validation parameter

The software resulted in overall desirability as shown in Fig. 4. The most optimized formulation obtained was OCUB1 with the highest desirability function parameter as compared to other suggested formulations. The numerical function parameter was estimated as 0.93, suggesting the studied responses (Y₁, Y₂, and Y₃) were statistically significant under set constraints. The model was the best fit in the optimization. This can be evidenced with the high correlation established between the predicted and the actual values of the characterized parameters for OCUB1. The optimization showed the lack of interaction between X₁ and X₂. Moreover, the impact of the investigated factors on the responses were identified.

Fig. 4.

Desirability bar graph as numerical function parameter.

4.5. Characterization parameters

Optimized (OCUB1) was evaluated for the particle size, ZP, and PDI. The size is the most critical parameter to affect in vitro drug release, %EE, stability, and in vivo absorption through lymphatic system. OCUB1 was associated with optimal size of 169.4 ± 1.7 nm and low PDI value <0.3 (0.232 ± 0.03). The distribution of cubosomes was broadened as the PDI value increases due to increased content of the lipid (Abousamra and Mohsen, 2016). PDI and TEM report corroborated uniform particle size and aggregation free product. The optimization process revealed that the size mainly depended upon X₁ and X₂. There may be several factors not described here. ZP is a significant parameter as a measure of stability (Worle et al., 2007). The value of ZP was – 29.2 ± 1.9 mV, suggesting OCUB1 possessed kinetically and thermodynamically stable product. The negative ZP values are attributed to the interaction established between the hydroxyl component of P407, the lipid, PVA, and aqueous medium (Salah et al., 2017).

4.6. Thermal analysis

Thermal analysis of the excipients and the drug was studied to determine characteristic fusion temperature as shown in Fig. 5A-E. The technique provides the purity, solid state characteristics, and thermal behavior (thermally induced degradation or probable chance of recrystallization after fusion temperature) heated over a wide range of temperature. The pure TOTA was a crystalline in nature with sharp endothermic peak at 219.3 °C without any recrystallization over the studied temperature range (Fig. 5A). The result is in good agreement to the published report (219.3 °C) (Malik et al., 2024). GMO showed fusion temperature at 56.2 °C whereas poloxamer melted at 62.4 °C (closely related to published value) (Fig. 5B and C). Both excipients confirmed pure form of the excipients without any contamination or degradation (Yousaf et al., 2023). Polymeric PVA portrays a broad endothermic peak with mean fusion temperature of 238 °C as shown in Fig. 5D. In is noteworthy that PVA (as a crystalline surfactant/stabilizer) is thermally more stable than poloxamer and GMO. The value is slightly different from the published report (229 °C) which may be attributed to instrumental error or variation (Martínez-Hernández et al., 2010). It is apparent that no visible and remarkable peaks were observed in OCUB1 which suggested the soluble form of the drug maintained in the semisolid bicontinuous phase of cubosomes and non-crystalline OCUB1 (Fig. 5E). Thus, the drug was completely solubilized in the lipid matrix and the surfactant blend of cubosomes.

Fig. 5.

Thermal behavior of the excipients and the drug used in OCUB1: (A) DSC thermograms exhibiting endothermic peaks with characteristic fusion temperature of TOTA (at 219.3 °C), (B) GMO (56.2 °C), (C) Poloxamer (62.4 °C), (D) PVA (238.8 °C), and (E) the optimized OCUB1 (no prominent observed endothermic peaks).

4.7. Atomic force microscopy (AFM): topographical assessment

Cubosomes are soft liquid crystal nanocarrier which could be better imaged using AFM for the surface roughness profile as compared to cryo-transmission electron microscopy (freezing affects the sample) (Neto et al., 1999). This is an advanced and sophisticated technology for surface roughness profile, shape, and size. Blank OCUB1 and the drug loaded OCUB1 were scanned under AFM (contact mode) and the result is portrayed in Fig. 6A-H and Table 4. Fig. 6A and B are 3-D and 2-D images of blank OCUB1, respectively. It is apparent that cubosomes were cubical in morphology with marginal surfaces (sharp edges) (Fig. 6C). This sharpness in shape can be further supported with skewness and kurtosis values as the most reliable and real topological parameters as compared to average roughness (Ra) and rms (root mean square as rms) values. Rms values indicated the morphologically homogeneous nature of cubosomes prepared (Akhtar et al., 2015). The surface roughness curve (Fig. 6C) exhibited smooth surface of cubosomes ending with sharp edge as shown in 3D and 2D images. There were no negative values of skewness and kurtosis (Table 4) in the blank OCUB1. In general, negative skewness is characterized with long tail in roughness histogram (Fig. 6D). Skewness values are the real and reliable topological surface roughness parameter as compared to average roughness value. This can be explained based on the fact that two different surface may have same Ra value. Similar surface profile is characterized using rms and the coefficient of kurtosis. Kurtosis measures the sharpness of a surface profile whereas skewness measures the symmetry of surface profile. Negative values of kurtosis and skewness suggest surface roughness as compared to smooth surface profile (Duboust et al., 2016). Kurtosis value ˂ 3 recommends zig zag surface pattern. Skewness is the indicator of surface damage, crack, fissures, and roughness. Negative skewness can be correlated to long tail in the roughness histogram towards negative scale on x-axis. However, the negative value was absent in blank OCUB1 and the drug loaded OCUB1. This might be correlated to smooth surface properties of cubosomes and successful entrapment of the drug in the aqueous cavity. These events can be explained in sequence. Fig. 6E is the 3D contour image of TOT loaded OCUB1 wherein multiple cubosomes were imbedded with characteristic shaped crystals. A single cubosome crystalline vesicle was scanned under AFM and it has been illustrated in Fig. 6E. The single cubosomal vesicle was about 70 nm in length and 55 nm in height (Fig. 6E-H). The surface roughness curve and histogram confirmed that there is significant change in surface roughness after loading TOTA in the cavity. Moreover, it shows the absence of surface drug adsorption. Positive skewness and kurtosis values further supported the absence of crack or surface abnormality (blunt ended peak of histogram) (Duboust et al., 2016). Table 4 summarized the estimated surface roughness parameters for the blank and the drug loaded OCUB1. The drug loaded OCUB1 showed relatively higher values of roughness parameters as compared to the blank OCUB1. This may be attributed to the fraction of unentrapped drug adsorbed onto the surface.

Fig. 6.

Topographical analysis of OCUB1 using AFM: (A) 3-D image of blank OCUB1 with multiple bunches of cubosomes, (B) 2-D image of blank OCUB-1, (C) surface roughness analysis report of blank cubosomes OCUB-1, (D) histogram report of roughness analysis of 3D image (panel A), (E) TOTA loaded 3-D image of OCUB1, (F) 2D image of TOTA loaded single cubosome in zoomed image, (G) roughness analysis report, and (H) histogram of panel F.

Table 4.

Summary report of AFM based topographical data.

AFM parameters	TOTA-OCUB1	Blank-OCUB1
Amount of sampling	195	195
Peak to peak, py	79.91	39
Average roughness (nm)	37.77	8.7
rms (root mean square) (nm)	18.16	9.9
Surface skewness, ssk	0.124	0.08
Coefficient of kurtosis, ska	0.7	1.1
Entropy	7.3	5.7

4.8. In vitro drug release

The release of TOTA from the OCUB1 and SUS-TOTA were analyzed up to 48 h using cellulose membrane (dialysis bag). The release pattern of TOTA from OCUB1 and SUS-TOTA are portrayed in Fig. 7A. The drug was released approximately 98.2 % from SUS-TOTA within initial 8 h which may be attributed to its aqueous solubility at explored pH. Furthermore, the drug release was quite slow and sustained from OCUB1 comprised of GMO and X₂ stabilized liquid crystalline matrix of cubosomes (Salah et al., 2017). Generally, hydrophilic TOTA was entrapped in the hydrophilic channel (inner compartment of cubosomes). Thus, lipid layer of cubosomes serve as rate limiting barrier for diffusion (concentration gradient as driving force for the drug diffusion) from inner to outer side. Moreover, several factors can be taken into consideration to explain the drug release mechanism and pattern. These are diffusion coefficient, the drug solubility, log P, shape (cubic or hexagonal geometry), size distribution, pore size, interface curvature, pH, temperature, and ion strength of buffer medium (Sivadasan et al., 2023).

Fig. 7.

(A) In vitro release of TOTA from optimized OCUB1 and SUS-TOTA (control) formulations and (B) In vitro hemolysis toxicity assessment as preliminary toxicity test.

OCUB1 exhibited biphasic release pattern of TOTA in the release medium at the explored temperature and ionic strength. Initially, the drug release was moderate up to 8 h (43.5 %) followed by slow drug release for a period of 48 h (98.1 %). This biphasic release patter from OCUB1 was in agreement with the cubosomes to deliver hydrophilic 5-fluorouracil (5-FU) (Nasr et al., 2015) where burst release was observed due to low affinity of hydrophilic drug to the lipophilic domain of cubosomes and poor drug adsorption. The initial moderate release can be attributed to the presence of weakly bound drug within lipid layer whereas slow drug release phase may be related to the weak diffusion of hydrophilic TOTA across lipophilic layer of cubosomes (El-Laithy et al., 2018). Conclusively, OCUB1 successfully extended the drug release up to 48 h for high patient compliance and mitigated adverse effects.

The drug release from cubosomes is correlated with diffusion theory and the drug molecules were migrated from the internal phase of cubosomes to the outer environment (release medium) (Lara et al., 2005; Nasr et al., 2015). Various release kinetics model were applied to analyze the drug release mechanistic aspects from OCUB1. The maximum regression value (R² = 0.9261) was obtained when Korsemeyer-Peppas model was applied whereas Higuchi (R² = 0.8724), first order (R² = 0.6754) and zero order (R² = 0.4528) models were poorly fit in the data. Therefore, it is evident from statistical analysis that Korsemeyer-Peppas model was the best fit model for non-Fickian diffusion (including erosion) based release kinetics as evident with “n” value of 0.65. The results obtained from in vitro release study is accordance with previously reported release mechanisms of nano-cubosomes (Venkatesh et al., 2014).

4.9. In vitro hemolysis

The hemocompatibility assessment was recognized to be as an essential to ensure acute preliminary toxicity potential of the nanocubosomal system on RBCs for oral administration. Therefore, it was conducted at three different concentrations (0.625, 1.25, and 2.5 μg/mL). The results obtained from RBC hemolysis study were portrayed in Fig. 7B. The percent hemolysis produced by OCUB1 at 2.5 μg/mL, 1.25 μg/mL, and 0.625 μg/mL were 16.74 ± 2.43 %, 11.49 ± 2.4 %, and 7.65 ± 2.3 %, respectively. Significant (p < 0.001) differences were found from the positive control (98.25 ± 1.03 %). The positive control caused 100 % lysis which may be attributed to high binding capacity of triton X100 (detergency) to the lipid bilayer of biological membrane and gradually distorted the geometry of acyl chains in lipid bilayer of the cell membrane (Bjørnestad and Lund, 2023). Triton X100 exhibits as a detergent property due to conical molecular shape in polar compartment and its short hydrophobic tail resulted in more penetration in the cellular bilayer caused local curvature of the biological membrane and distort its geometry (Rodi et al., 2014; Bjørnestad and Lund, 2023). In contrast, a significantly (p < 0.001) low hemolysis (16.64 ± 2.4 %) toxicity was claimed even at the highest concentration of OCUB1 (2.5 μg/mL) as compared to SUS-TOTA (2.5 μg/mL: 82.45 ± 3.1 %) due to protective nature of GMO based cubosomal system (Fig. 7B). The high hemolysis activity of SUS-TOTA was attributed to the distilled water of the suspension (serving as positive hemolysis), suspending agent in the suspension, and self-induced in vitro hemolysis of the RBC cells during incubation.

4.10. Ex vivo permeation study

The drug permeation across the intestinal membrane was conducted for the DS and OCUB1. As expected, OCUB1 exhibited slow and sustained delivery of the drug as compared to DS which may be attributed to cubosomes lipid bilayer acting as controlled barrier for the drug diffusion from inner bicontinuous matrix. The cumulative amount permeated from OCUB1 and DS were estimated as 992.74 μg/cm² and 1598.88 μg/cm², respectively, at 180 min (Fig. 8). The DS showed rapid drug absorption over OCUB1 for the studied time period. The estimated J_ss (steady state permeation flux) was 9.172 μg/cm²/min (r² = 0.97) for DS whereas it was found to be 6.69 μg/cm²/min for OCUB1 (r² = 0.98). The estimated permeation coefficient values for DS and OCUB1 were calculated as 4.5 × 10⁻³ cm/s and 3.3 × 10⁻³ cm/s, respectively. Thus, OCUB1 successfully controlled the drug permeation across intestinal membrane under ex vivo conditions.

Fig. 8.

Ex vivo permeation of the drug through jejunum of rat over period of 180 min. The permeation profile was relatively high from the drug solution (DS) due to solution nature whereas the optimized OCUB1 controlled and sustained the drug permeation across rat intestine under similar experimental conditions (n = 3, data expressed as mean ± standard deviation).

The drug salt was soluble in DS and it was ready for immediate permeation at the intestinal mucosal surface. Therefore, the solution form of the drug available maximally at the apical mucosal layer for permeation towards the distal side (from apical to distal region). The mucosal layer is hydrophilic in nature and it assists to dissolve hydrophilic salt form of TOTA. However, tolterodine (without salt) after solubilization is highly lipophilic and it creates a hurdle to be dissolved in the hydrophilic mucosal layer. Considering the salt based solubility, the drug was maintained in the soluble form inside lumen. The cubosomes encapsulated the drug within the lipid matrix and the drug needs to be diffused out of the lipid bilayer serving as rate limiting step (Sivadasan et al., 2023). Moreover, the blend of poloxamer and PVA increased the viscosity of the product to render slow and sustained drug diffusion across interfacial layer (impeded the drug desorption across the bicontinuous layer of cubosomes) (Kojarunchitt et al., 2011). The OCUB1 might have interacted with the mucosal layer, slowing down its movement and the concentration gradient across the intestinal layer. Another possible reason for slow and sustained drug permeation from OCUB1 may be attributed to less drug partitioning from the lipid matrix to the aqueous medium (PBS 7.4).

Multiple permeation mechanisms worked to facilitate its permeation for rapid drug absorption. Ex vivo condition avoids the role of active enzymatic degradation before absorption in gastric or intestinal juice, enterocytes mediated degradation, hepatic first pass metabolism, and systemic degradation (Zheng et al., 2024). Thus, permeation study revealed relatively high drug permeation from DS whereas OCUB1 exhibited controlled drug release for extended drug availability to the systemic circulation to avoid plasma drug variation as observed in the conventional dosage form, hepatic metabolism, pre-and post-absorption metabolism (Zheng et al., 2024).

Considering the short term viability of cellular integrity in ex vivo study, long term permeation (12–24 h) study is challenging in the absence of aeration, physiological conditions, and the lack of such model (Azman et al., 2022). Therefore, DS elicited relatively high cumulative permeation and J_ss as compared to OCUB1. However, OCUB1 offered sustained drug permeation, protected the drug degradation, and enhanced oral bioavailability over time (in vivo condition). Notably, short term ex vivo study (∼ 3–4 h), rapid and immediate drug permeation often outperform slow and sustained drug permeation in cumulative permeation metrics (Sallam and Marín Boscá, 2015).

4.11. Pharmacokinetics study

Pharmacokinetic (bioavailability) studies were carried out in male Wister rats (250–300 g) using the two different formulations (OCUB1 and SUS-TOTA) administered orally. The time course of the plasma drug concentrations is summarized in Fig. 9. The pharmacokinetic parameters calculated from the plasma drug concentration-time profiles were listed in Table 5. Following oral administration, the PK of SUS-TOTA (treatment I) and OCUB1 (treatment II) were evaluated in rats. The maximum drug concentration (C_max) of 783.8 ± 17.7 ng/mL was achieved at a T_max of 12.54 ± 2.2 h for OCUB1 whereas C_max was 243.3 ± 11.2 ng/mL at 8.21 ± 3.4 h for SUS-TOTA. Several factors attributed for significant (p < 0.05) C_max value after OCUB1 treatment wherein the amorphous nature of the loaded TOTA, the large surface area to volume ratio offered by cubosomes and the drug stability in cubosomes are of prime importance (Tayel et al., 2016).

Fig. 9.

Plasma drug concentration-time profile of OCUB1 and SUS-TOTA after oral administration at dose of 4 mg/Kg (rats).

Table 5.

Pharmacokinetic parameters of OCUB1 and SUS-TOTA after given oral treatment at dose 4 mg/Kg BW.

Pharmacokinetic Parameters	SUS-TOTA	OCUB1
Cmax (ng/mL)	243.3 ± 11.2	783.8 ± 17.7
Tmax (h)	8.21 ± 3.4	12.54 ± 2.2
AUC0–∞ (ng.h/mL)	2136.5 ± 167.3	7787.4 ± 213.1
AUMC0–∞ (ng.h2/mL)	29,367.6 ± 192.5	98,243.7 ± 928.7
MRT (h)	9.57 ± 1.9	16.32 ± 2.2
Ke (h−1)	0.006 ± 0.002	0.002 ± 0.001

There was significant (p ˂ 0.001) increment in MRT value of OCUB1 (16.32 ± 2.2 h) as compared to SUS-TOTA (9.57 ± 1.9 h) which indicated the controlled-release characteristics of the optimized nanocubosomal system. Moreover, OCUB1 might have protected the drug from in vivo hepatic degradation resulting in about 364.7 % and 334.5 % increase in AUC_0–∞ and AUMC respectively, for OCUB1 as compared to SUS-TOTA (AUC_0–∞: 2136.5 ± 167.3 ng.h/mL; AUMC_0–∞: 29367.6 ± 192.5 ng.h²/mL). As a result of this, improved bioavailability might be attributed to OCUB1 escaped from reticuloendothelial system (RES) mediated clearance (Lai et al., 2010). Furthermore, nanonization and stable GMO based OCUB1 result in prolonged circulation time and delayed opsonization by the immune system for different in vivo fate and enhanced therapeutic efficacy to achieve high patient compliance (Hosny, 2020).

The possible mechanisms of facilitated oral absorption of TOT-loaded nanocubosomes may be explained based on formulation characteristics and physicochemical nature of the drug. These are summarized as (a) increased apparent solubility in bicontinuous cubic phase structure, (b) protection from gastric acid induced degradation due to lipid matrix, (c) high internal surface area for increased solubility and permeation across intestinal membrane, (d) nanoscale size and lipid nature of cubosomes facilitate interaction with mucosal membrane and lymphatic access for reduced hepatic metabolism and enhanced systemic availability, and (e) unique nature of the structure sustained TOT release in the gastrointestinal tract (GIT) (Sivadasan et al., 2023; Lakshmi et al., 2014). In brief, the studied carrier improved oral BA of TOT by protecting the drug, enhanced solubility, modulated intestinal permeability, and sustaining its release across GIT.

4.12. Hematological and biochemical analysis

The optimized formulation (OCUB1) was intended for oral delivery. Therefore, it was required to monitor the normal level of biological markers in the rats in acute toxicity. The biochemical and hematological markers were assayed at the end of 14 days of treatment. The result of hematological parameters are summarized in Table 6. Notably, OCUB1 treated group didn't show any significant (p < 0.05) changes in the hematological parameters as compared to the value of the control group at explored dosing strength. These findings are quite relevant, reliable, and in good agreement with the published report for rat model (Ochiai et al., 2018). Normal values of white blood cells and RBCs suggested safety aspects of the product for oral administration at the studied dose and dosage form.

Table 6.

Effect of OCUB1 on hematological parameters of rat.

Treatment (n = 3)
Parameters	Control	OCUB1
Hemoglobin (gm/dL)	14.5 ± 0.41	12.3 ± 0.5
RBC (106/mm3)	6.5 ± 1.01	5.8 ± 1.3
PCV (%)	52.8 ± 0.85	54.7 ± 0.8
MCV(fL)	55.1 ± 1.93	53.9 ± 1.9
MCH (pg)	18.7 ± 0.78	20.8 ± 0.92
MCHC (g/dL)	31.2 ± 2.21	33.6 ± 3.13
RDW (%)	13.6 ± 1.02	12.9 ± 2.14
NEUT (%)	21.2 ± 1.11	20.9 ± 2.08
LYMP (%)	77.5 ± 3.03	79.2 ± 2.11
EOSI (%)	2.0 ± 0.11	2.9 ± 0.62
BASO (%)	0.3 ± 0.02	0.21 ± 0.01
PTC (103/μL)	732 ± 86.21	747 ± 110.23
TLC (cells/mm3)	5700 ± 321.23	5800 ± 213.76

Additionally, there was no significant difference (p < 0.05) in the biochemical parameters of treated group as compared to the control group (untreated) (Table 7). The safety of the OCUB1 was attributed to the maximum TOTA entrapped in the optimized vesicles and there was no direct contact of TOTA to biological components. These estimated values were found to be in normal range (Ochiai et al., 2018). ALT and AST are the most common liver biomarkers to ensure physiological functionality at normal level or pathological conditions. Generally, these biomarkers are found to be raised in liver related abnormality such as inflammation, infection, and liver cirrhosis (McGill, 2016). AST is also related to cardiac and skeleton muscle. However, both groups exhibited normal range of these biomarkers in the blood of the rats. ALP (homodimeric protein enzyme) is the most critical biomarker used in clinical and industrial setup. It is associated with multiple physiological processes (gene expression, molecular transportation, and metabolism) of various organs such as liver, intestine, kidney, prostate, endocrine, and placenta. Its main function is dephosphorylation of nucleic acids and protein (Shaban et al., 2022). Normal values of APL in the treated and the control groups corroborated biocompatible nature of OCUB1. The total protein and serum creatinine were assessed to confirm normal functionality of kidney whereas triglycerides were estimated to detect probable chance of the cardiovascular risk. Elemental analysis of calcium and phosphorus is related to identification of any possible abnormality in the heart and kidney. These inorganic elements were rapidly increased in the blood due to chronic cardiovascular and renal diseases (Cubbon et al., 2015; Lai et al., 2010).

Table 7.

The effect of OCUB1 on biochemical parameters of rats.

Treatment (n = 3)
Parameters	Naïve control	OCUB1
Calcium (mg/dL)	10.8 ± 0.31	11.2 ± 0.56
BUN (mg/dL)	19.41 ± 1.21	23.9 ± 1.87
Serum creatinine (mg/dL)	0.47 ± 0.02	0.41 ± 0.01
Alkaline phosphatase (U/L)	745 ± 8.91	768 ± 6.56
Phosphorus (mg/dL)	7.17 ± 0.21	6.93 ± 0.67
Bilirubin (mg/dL)	0.05 ± 0.003	0.06 ± 0.001
Blood glucose (mg/dL)	159 ± 2.14	148 ± 5.61
Triglycerides (mg/dL)	123 ± 1.78	116 ± 2.11
Total cholesterol (mg/dL)	75 ± 9.11	79 ± 8.21
S.G.O.T(AST)(U/L)	55 ± 5.22	59 ± 4.32
S.G.P.T(ALT)(U/L)	33 ± 3.18	39 ± 2.88
Total proteins (mg/dL)	7.3 ± 0.33	6.9 ± 0.51

5. Conclusion

The study was focused to address an alternative strategy of oral drug delivery for slow and sustained delivery of TOTA using stable cubosomes based oral administration. Conventional tablet/capsule is associated with multiple negative consequences as result of rapid drug dissolution and short of elimination half-life of the drug. Moreover, hepatic degradation and intestinal metabolism caused adverse effects limited clinical use despite directly acting muscarinic potential antagonist in all age groups. Cubosomes are well established lipidic biocompatible nanocarriers for several benefits as compared to conventional dosage form. An attempt was made to overcome the consequences associated with tablet (at the same dose) by slow and extended drug release using GMO based cubosomal formulation. HSPiP software predicted interactive forces responsible to solubilize the drug in the investigated excipients at preliminary stage of excipients screening and simulated the actual data. Trial formulations set the levels of factors against the set goals of targeted responses. High desirability decided a right composition of the optimized formulation under desired constraints. The optimized OCUB1 was associated with desired formulation characteristics (cubical and smooth as revealed in AFM) for improved oral bioavailability and the drug release at predetermined rate. This may be prudent to correlate non-Fickian diffusion mechanism. The composition and the dose of formulation were safe and non-hemolytic. Biochemical and hematological biomarkers were found to be within range. These finding suggested the safety aspect of OCUB1 as compared to the suspension as the control. Hence, the optimized OCUB1 can be the state-of-the-art oral product for oral delivery with high patient compliance and low adverse effects.

CRediT authorship contribution statement

Afzal Hussain: Investigation, Formal analysis, Data curation, Conceptualization. Tasneem Khan: Visualization, Software. Mohd Usman Mohd Siddique: Formal analysis, Data curation, Conceptualization. Danishuddin: Writing – original draft, Visualization, Validation. Mohammad A. Altamimi: Writing – review & editing, Writing – original draft, Funding acquisition, Formal analysis. Mohhammad Ramzan: Validation, Methodology, and Data curation.

Funding

The authors acknowledge and extend their appreciation to the Ongoing Research Funding Program (ORF-2025-524), King Saud University, Riyadh, Saudi Arabia.

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.

Data availability

Data will be made available on request.