Introduction

Hypertension (HT) is a persistent malady defined by a dramatic increase in arterial blood pressure, causing various cardiovascular disorders such as atherosclerosis and stroke [1]. Hypertension damages more than 1 billion individuals globally, as it is the cause of 58% of hemorrhagic stroke cases, 50% of ischemic stroke cases, 55% of ischemic heart disease cases, and 58% of other cardiovascular disorders [2]. Moreover, throughout the following ten years, more individuals are anticipated to be afflicted by HT globally. Therefore, hypertension management has been categorized as a global health priority [3].

Angiotensin II receptor blockers (ARBs) are primarily employed for the management of high blood pressure, diabetic nephropathy, and coronary artery diseases [4]. Recently, several researchers have focused on their pleiotropic properties, such as glioblastoma [5], cancer [6], and COVID-19 infection [7].

Losartan potassium (LP) reduces blood pressure by being an ARBs drug that blocks the binding of angiotensin II to the angiotensin II receptor type 1 (AT1) [8]. ARBs also preserved cerebral blood flow, reduced neurological and cognitive impairment, and disrupted the blood–brain barrier in rodent models of Alzheimer's disease, traumatic brain injury, and cerebral ischemia [9, 10]. Furthermore, LP has a neuroprotective effect on resistance to premature damage risks in astrocytes, neuronal microglia, and cerebrovascular endothelial cell culture [11]. Despite its numerous attributes, LP has poor bioavailability, approximately 33% [12]. Such low bioavailability might be due to it undergoing the initial metabolic cycle by the CYP-450 enzyme, with 14% of LP being transformed into an active metabolite [13]. Therefore, the intranasal route is a plausible alternative to the traditional oral route. The intranasal route administration allows active molecules' safe and efficient transit through the BBB [14]. Furthermore, the nasal route can inhibit the liver's initial metabolic cycle and transmit medications via an olfactory route immediately to the brain. This could be the cause of its ability to enhance absorption and limit adverse effects [15]. Despite its numerous advantages, the nostril route of administration removes drugs from the absorption site due to poor permeability of the epithelial cells, limited space on the surface, enzymatic breakdown, minimal capacity utilized for drug delivery, and elimination of mucociliary [16]. Thus, nanocarriers such as spanlastics have been developed to overcome their limitations [17,18,19].

The amazing stretchiness and deformability of nonionic surfactant-structured spanlastic nanovesicles (SNVs) is due to the two-layer membrane of surfactant macromolecules that surrounds a drug solution in water [20]. SNVs are composed mainly of a Span® and an edge activator (EA) that enhance their flexibility and deformability, allowing drugs to pass through biological membranes more easily. Moreover, SNVs can efficiently deliver the hydrophilic and hydrophobic drugs enclosed in an interior hydrophilic layer and an outer lipid layer [18]. Furthermore, SNVs are osmotically active, have biochemical rigidity, an extended shelf life, and excellent customer satisfaction [21]. Interestingly, SNVs are nontoxic nanovesicles relative to other vesicles composed of cationic surfactants [22], Moreover, they were reported to enhance drug permeation via various routes like transdermal [20, 22, 23], ocular [24, 25]and intranasal routes [17, 19, 26, 27].

A Full factorial design is demonstrated. This method minimizes the number of experiments and eliminates the necessity for a significant number of experiments. Full factorial design permits the examination of several variables at once while preserving the caliber of the data gathering process. A Full factorial design can be used at the outset of a subsequent response [28]. 3122 full factorial design has three factors: two factors have two levels, and one factor has three levels. The independent variables were under investigation. The pattern and power of significant factors in every experiment are assessed using the data for variance analysis (ANOVA; using Design-Expert® software (Version 10, Stat-Ease Inc., Minneapolis, MN).

An attempt has yet to be made to investigate the potential role of LP-loaded SNVs in managing hypertension via the nasal route. Therefore, the current investigation aimed to formulate LP-SNVs and incorporate them into an in-situ gel to enhance its pharmacokinetic criteria. The in-situ gel formulation containing optimal LP-SNVs was exposed to rheological, in-vitro drug release, ex-vivo permeation, histopathological, and pharmacokinetic studies.

Materials and Procedures

Materials

National Organization for Drug Control and Research (NODCAR) in Cairo, Egypt, graciously donated losartan potassium (LP); Sigma Aldrich (St. Louis) auctioned acetonitrile HPLC grade, Span 60® (sorbitan monostearate), Span 20® (sorbitan monolaurate), and Poloxamer 407 (P-407). The suppliers of chloroform and methyl alcohol were EL-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). Dialysis bags from SERVA Electrophoresis GmbH (Heidelberg, Germany) were acquired with a molecular weight cutoff of 12000 Da. EL-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt) supplied the potassium dihydrogen orthophosphate and the disodium hydrogen orthophosphate.

Experimental Design and Optimization of LP-loaded SNVs

Using Design-Expert® software (Version 10, Stat-Ease Inc., Minneapolis, MN), a full 3122 factorial experimental design was conducted to examine the influence of the independent variables on the properties of SNVs dispersions. Span® type (A) with two levels, EA type (B) with two levels, and amount of span® with three levels (C) were the independent variables. The dependent variables under investigation were entrapment efficiency (EE %) (Y1), Vesicle size (VS) (Y2), the percentage of LP liberated from different formulations after 8 h (Q8h) (Y3), and the total amount of LP permeated per cm2 after 24 h (Q24 h) (Y4). Table I.

Table I Independent Variable and their Levels Along with Dependent Variables and their Constraints in 3122 Design of LP–loaded SNVs
Full size table

Formulation of LP-loaded SNVs

Employing the thin film hydration approach, SNVS was produced, as indicated by a previous report [20], with minor modifications. In a nutshell, Span 20® or 60® and EA were eradicated in 2:1 v/v (chloroform: methanol) organic solvents. After that, a Heildolph Laborota 4000 Series rotary evaporator from Heizbad, Germany, was used to make a thin, dry layer on the flask wall by evaporating the organic solvents at a speed of 90 revolutions per minute while working at 60°C and lower pressure. Then, the dry film was hydrated with 10 ml of phosphate-buffered saline (pH 7.4) containing 10 mg of LP for 30 min—Table II.

Table II The 3122 Full Factorial Design of LP-loaded SNVs
Full size table

Evaluation of Fabricated SNVs

Assessment of EE%

A cooling centrifugation method (Sigma Laborzentrifugen, Osterode am Harz, Germany) was used to separate EE%-free LP from LP-SNVs that were stuck. In brief, LP-SNV dispersions were centrifuged at a 14000 rpm rotation speed at 4°C for 2 h [29]. The amount of free LP in the separated supernatant was analyzed using a Shimadzu UV-1800 (Tokyo, Japan) spectrophotometer with a λmax of 205 nm.

$$\mathrm{EE\% }\;=\;(\mathrm{total\;amount\;of\;drug}\;-\;\mathrm{free\;drug\;amount})\;/\mathrm{\;total\;amount\;of\;drug\;x\;}100$$
(1)

VS and Polydispersity Index (PDI) Measurements

A Malvern Zeta sizer (Malvern Instruments, Malvern, UK) was used to estimate the VS and PDI of LP-SNVs at an angle constant of 90° and a temperature of 25°C [30]. In advance of analysis, 10 ml of deionized water was added to 0.1 ml of each SNV's dispersion to guarantee that the magnitude of light diffraction was within the tolerance range of the instrument. Three separate measurements were made.

In-vitro Drug Release Investigations

The in-vitro release characteristics of LP –SNVs formulations were performed by diffusion technique using Franz cells with an effective area of 5 cm2 [30] to compare LP release from LP-SNVs and LP solution [31]. A definite volume of each LP-SNVs formulation (equivalent to 3 mg LP) and free LP solution (equivalent to 3 mg LP) were positioned within the donor hall. In the next step, fifty milliliters of simulated nasal electrolyte solution (SNES) containing 7.45 mg/mL of NaCl, 1.29 mg/mL of KCl, and 0.32 mg/mL of CaCl2.2H2O were added to the receptor part to act as a recipient medium. The pH was adjusted to 5.5 using 0.1 N of hydrogen chloride [15]. A semi-permeable barrier was introduced among the donor and recipient chambers. The media was agitated at 50 rpm and kept at 37°C ± 0.5. Next, at predefined times (1, 2, 3, 4, 5, 6, and 8 h), 1 ml of samples were collected, and the receiving chamber was immediately refilled with an equivalent volume buffer to retain the sink condition. Eventually, sample analysis was done using spectrophotometry (Shimadzu UV-1800, Tokyo, Japan) at λmax of 205 nm. Three analyses of the samples were conducted, and the percentage of LP liberated was reported as mean values (± SD). The total amount of LP liberated from LP-SNVs was plotted against time to determine the LP release pattern [32].

Ex-vivo Permeability

The Franz diffusion cell was implemented to assess the nasal permeation of LP-SNV formulations and LP solutions. A slaughterhouse provided the camel's nasal mucosa, which was meticulously dissected [33]. It was positioned with the donor compartment and 3mg of LP towards the diffusion cell mucosal surface amid the chambers of the donor and recipient. The receiver chamber was filled with 50 ml of pH 6.4 buffer at 37 ± 0.5°C and rotated at 50 rpm. At various intervals (1, 2, 3, 4, 6, 8, 12, and 24 h), one milliliter of samples was removed, and the medium was promptly supplied, adding a comparable quantity of PBS to sustain the sink condition. Finally, sample analysis was done using spectrophotometry (Shimadzu UV-1800, Tokyo, Japan) at λmax of 205 nm. The analysis of samples was performed in triplicate [34]. The whole quantity of LP permeated per unit area (µg/cm2) was plotted graphically against time (h). In addition, steady-state flux in µg/cm2/h (Jss) and permeability coefficient in cm/h (Kp) were established for all LP-SNVs formulations and free drugs. Where the lag time was determined from the x-axis intercept (the linear portion of the graph), the permeability coefficient (Kp) in cm/h can be estimated by dividing the slope of the straight line portion of the curve (flux) by the concentration of drug originally added, and the enhancement index (EI) was calculated applying the following equation:

$${\text{EI}}=\mathrm{KP\;of\;SNVs\;formulation}/\mathrm{KP\;of\;control\;LP\;solution}$$
(2)

Optimization of LP-SNVs

The ideal combination of independent variables was selected by Design Expert® software using the desirability function as a basis for maximizing EE%, % of LP liberated from SNVs formulation after 8 h (Q8h), and the total quantity of LP permeated/cm2 after 24 h (Q24h), and minimizing VS. The optimum formulation was constructed, tested, and compared with the values that the used software implied [35].

Transmission Electron Microscopy (TEM)

TEM (JEOL, JEM-1230, Tokyo, Japan) with an electrical discharge power of 80 kV was used to investigate optimal LP-SNVs formulation morphology. A tweaked drop was stained with phosphotungstic acid (2% w/v) using carbon grids, and it was subsequently examined using a TEM [36].

Assessment of the Optimal Formulation's Physical Stability

For 90 days at 4°C, the optimal LP-SNVs formula was enclosed in a glass vial. To assess formulation stability, EE%, drug release, VS, and zeta potential were measured in withdrawn samples at 0, 30, 60, and 90 days [37].

Fabrication of an In-situ Mucoadhesive Gelling System with the Optimal SNVs

A cold method was used to make an in-situ mucoadhesive gelling system with the best SNVs plain and LP-SNVs in-situ gels. The thermosensitive polymers P-407 (20% w/w) and P-188 (10% w/w) were mixed with the mucoadhesive polymer Carbopol 971P (0.5% w/w) [38]. Firstly, precisely weighted amounts of P-407 (20% w/w), P-188 (10% w/w), and Carbopol 971P (0.5% w/w) were distributed in the cold (4–6°C) purified water under constant stirring. One drop of triethanolamine was added to the gel solution to make sure that all of the carbopol 971P would dissolve and to get a clear polymer mixture. Finally, the mixture was stored in a refrigerator for twenty-four hours to ensure all the polymers were dissolved.

Characterization of In-situ Gel

Sol–gel Transition Temperature (Tsol–gel) and Gelling Time Measurements

A visual inspection approach was used to ascertain the gelation temperature of the manufactured in-situ gels [32]. In summary, vials (5 ml) that included 2 ml of the investigated formulas were fixed with parafilm and submerged in a water bath with a thermostat. The temperature rose 0.5°C/min from 20 to 40°C. The samples were permitted to acclimate at each new destination. The specimens were inspected for gelation when the meniscus stopped moving upon orientation at a 90° angle. Gelation time was defined as the moment of the first discovery of gelling.

Determination of Mucoadhesive Strength

By using a modified balancing method to measure the force needed to separate each formulation from nasal mucosal tissue, the mucoadhesive strength of each formulation was ascertained. Cyanoacrylate glue was used to attach a 1 cm2 piece of camel nasal mucosa to each of the two glass slides. The first slide, which was positioned beneath the pan with an adjustable height, held 50 mg of gel. A second slide, which contained the mucosal portion, was affixed to the underside of the same pan in an inverted configuration. For two minutes, the two slides containing the gel formulation were held in close proximity to one another to guarantee close contact. The second pan's weight was then increased continuously until the slides separated from one another. Based on the lowest weight required to separate the mucosal tissue from each formulation's surface, the mucoadhesive force—which is expressed as the detachment stress in Dynes/cm2—was calculated [39].

$$\mathrm M\mathrm u\mathrm c\mathrm o\mathrm a\mathrm d\mathrm h\mathrm e\mathrm s\mathrm i\mathrm v\mathrm e\;\mathrm s\mathrm t\mathrm r\mathrm e\mathrm n\mathrm g\mathrm t\mathrm h\;(\text{Dynes/cm}^2)\;=\;\;\mathrm m\mathrm g/\mathrm A$$
(3)

where, m = weight required for detachment in grams, g = acceleration due to gravity (980 cm/sec2). A = Area of mucosa exposed.

Rheological Characterizations

A cone and plate viscometer (DV-III Ultra viscometer, RV model, Brookfield, USA) was used to examine the in-situ gel's rheological characteristics. The aliquot (0.5 g) of the investigated formulation was moved to the bottom panel of the viscometer. The viscometer also has a rotating bath that measures the viscosity of the mixture at two different temperatures: 25°C and 37°C ± 0.5. Spindle 52 and shear rates ranging from 20 to 200 s are used [40]. The equation was used to determine the viscosity coefficient.

$$\mathrm{Log\;G\;}=\mathrm{\;N\;Log\;F}\;-\;\mathrm{\;Log\;\eta }$$
(4)

G is the shear rate (sec−1), F is the shear stress (dyne/cm2), and N is Farrow's constant. To determine the value of N, which denotes the departure from Newton's law, log G was plotted against log F.

In- vitro Drug Release (Q8h) and Ex-vivo Permeability Investigations (Q24h)

Utilized the same methodology previously mentioned (i.e., "In-vitro Drug Release Investigations" section and Ex-vivo permeability section), 0.5 g of LP-SNVs loaded in-situ gel and control gel (equal to 3 mg LP) were used in the in-vitro release and ex-vivo permeation assays.

Pharmacodynamics Study

The pharmacodynamics studies were performed in conformity with the institutional requirements of the Animal Rights and Ethical Behavior Committee of Nahda University for Experimental Animals, Beni-Suef, Egypt. The study was approved under the approval number NUB-023–011. The researchers adhered to the principles outlined in the Declaration of ARRIVE and its accompanying guidelines for the ethical treatment and utilization of animals. The investigation involved using thirty male albino Wistar rats, with an average weighing between 200 and 250 g. Five groups of these rats were created; each group encompassed six rats. The rats were housed in a climate-controlled space with a typical room temperature and between 40 and 45% humidity. Additionally, a twelve-hour light-and-dark cycle was maintained. The food was easily accessible to the animals [41]. Dexamethasone (0.1 mg/kg) was given subcutaneously for 2 weeks to induce hypertension in all groups except group 1 [42]. Group 1 served as the control group, Group 2 served as the diseased control, Group 3 received LP solution orally, Group 4 received LP solution intranasal in-situ gel, and Group 5 received LP-SNVs intranasal in-situ gel. Before and after the recommended medication, the systolic blood pressure was monitored using a blood pressure arrangement based on the tail-cuff method (NIBP 200 A; Biopac System, Inc., Goleta, CA, USA) [43] at various days 0, 1, 3, 5, 7, 9, and 14. In a nutshell, the tail was positioned inside an enlarged, piezoelectric pulse sensor-equipped cuff that was elongated. Once that sensor detected the first wave, the blood pressure was measured while gradually lowering the pressure on the cuffs [41].

Histopathological Studies for Safety and Tolerance

A histopathological investigation was conducted to estimate the LP-SNVs in-situ gel safety after intranasal administration [44]. Six male albino rats weighing between 200 and 250 g each were split into two squads. The starting squad received 20 µ l of LP-SNVs in-situ gel via nasal route one time per day for a total of fourteen days, while the other squad acted as a control group [45]. Next, ketamine (20 mg/kg) was administered to numb the rats. Then, the sacrificed rats' nasal mucosa was removed, immersed in 10% formaldehyde for 24 h, imposed within paraffin blocks, and separated into pieces that were 5 mm thick. Eventually, specimens were examined after being stained with hematoxylin and eosin (H&E) under an optical microscope equipped with a camera [46, 47].

Bioavailability and Pharmacokinetic Studies

Three groups of rats, with six rats each, were employed in the investigation. Diethyl ether was used to sedate the rats and limit sneezing while delivering in-situ gel into both nostrils. Group (A) received LP-SNVs intranasal in-situ gel (10 mg/kg) [48]; Group (B) received LP solution intranasal in-situ gel, and Group (C) received LP solution orally (10 mg/kg). In the beginning, throughout a variety of periods (0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 h), each rat's retro-orbital venous plexus yielded one milliliter of blood into a heparinized tube. After a 20-min centrifugation at 3000 rpm, the plasma was extracted from the blood samples. Then, a 1 ml aliquot of plasma was mixed with 5 ml of acetonitrile and agitated for 3 min to ensure the mixture was well mixed. The resultant blend was centrifuged for 25 min at 11,000 rpm to separate the plasma protein. The supernatant plasma was ultimately maintained for analysis at -4°C [49].

Chromatographic Conditions

An established HPLC strategy was adopted to determine the quantity of LP [48, 50, 51]. Agilent Eclipse C18 column (250 × 4.6 mm), a mobile phase composed of acetonitrile and water (pH modified to 3.0 with phosphoric acid) (55:45 V/V), was adjusted at a flow rate of 1 ml/min. UV detection was carried out at 30°C and 230 nm [52, 53], and the retention time was 3.5 min for LP [54], while the retention time for the valsartan internal standard was 5 min [55]. In addition, the study demonstrated a linear calibration curve across the concentration range (0.1–10 µg/ml) with R2 (0.997).

Pharmacokinetic Analysis

The LP pharmacokinetics were estimated using a non-compartmental model [44]. The PK solver was utilized for computing the pharmacokinetic parameters: peak LP concentration (Cmax), time needed to focus its peak concentration (Tmax), elimination half-life (t1/2), elimination rate constant (Ke), and area under the curve (AUC).

Results and Discussion

Assessment of Factorial Design

The model's fitness and validity were tested by analyzing the data obtained using a factorial design, measuring the design criterion, and evaluating the impact of every distinct factor and its interactions on the underlying responses using Design-Expert® software. The generated design was statistically validated using analysis of variance (ANOVA), F measures, and correlation coefficients at a 95% confidence level (p 0.05). The desired ratio was > 4 for all responses (EE%, VS, Q8h, and Q24h). Furthermore, the corrected and anticipated R2 values must be comparable for improved accuracy.

Determination of EE% of LP-SNVs

In the produced LP-SNVs formulations, the EE% of LP was assessed and displayed in Table II. The EE% equivocated between 34.8 ± 3.41 and 83.8 ± 2.92%. The adjusted R-square (0.9747) and the predicted R-square (0.9865) were close. ANOVA and residual estimation validated the model's applicability. The adequacy/precision ratio of 45.500 indicated the signal's capacity to navigate the design space. All independent variables had a significant influence on the EE% values (p < 0.05).

$$EE\%\;=\;+\;59.60\;+\;10.00A\;+\;5.32B\;+\;10.32C1\;-\;1.82C2$$
(5)

EE% at the middle level of the third independent variable (Conc. of Span®) is affected by span® type (A) and EA (B). This is shown in Fig. 1. Notably, the Span® type exhibited a favorable effect on the EE%. The EE% of SNV formulations that contained Span 60® were higher than those that contained Span 20®, and this could be explained by the extended saturated alkyl chain (C16) of Span 60® [56]. In addition, Span 60® has a higher phase transition temperature (53°C) in comparison with Span 20® (16°C), which gives the SNVs contained in Span 60® a more stable bilayer [57]. Furthermore, Span 60® is a hydrophobic surfactant with an HLB value of 4.7; therefore, it can accommodate a higher amount of LP and exhibit a higher EE% [58]. This finding agrees with previous literature [59]. In order to elucidate these results, the hydrophilic-lipophilic balance of the edge activator is used. For Cremophor RH-40 and P-407, it is 14 and 22. Values of HLB are correlated with the extended saturated alkyl chain in the edge activator; the greater the alkyl chain length, the lower the HLB value, indicating that the edge activator is lipophilic and, as a result, forms fewer hydrophilic pores and has a lower bilayer amphiphilic characteristic, which reduces the fluidity of the vesicles’ shell, ending up raising the EE% [60, 61]. Therefore, SNVs elaborated with Cremophor Rh 40 had a lower HLB value [14] and a higher EE% than SNVs elaborated with P-407, which had a higher HLB value [22, 62]. Additionally, the P-407-based SNVs formulation exhibited low EE%, possibly due to P-407's high molecular weight, which can destroy the credibility of the vesicles' membrane, allowing the drug to escape into the surrounding aqueous medium [36]. These results are in harmony with previous findings [62].

Fig. 1
figure 1

3D-response surface plot for the effect of span® type, and Edge activator at the middle levels of the 3rd factor (span concentration) on a, EE%, b vesicle size, c Q8h, and d Q24

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Vesicle Size Assessment

Vesicle size is an essential criterion for evaluating nano-vesicles, as it immediately impacts the physical stability of the drug, bio-distribution, cellular uptake, and drug release. In addition, small particles provide better bodily accessibility because they can freely reach their target via systemic circulation [63]. The VS of fabricated SNVs formulations varied between 205.5 ± 6.5 and 445.1 ± 13.52 nm (Table II). It is well established that vesicles containing EAs usually acquire small sizes due to their lack of inclination for agglomeration and spherical shape [20]. The LP-SNVs formulations' PDI values fluctuated between 0.12 and 0.41. Table II could reflect the vesicles' congruent distribution [62].The recommended model after transformation for VS was the main effect, with adjusted R2 equaling 0.9583 and being relatively close to the predicted one of 0.9219.

$$Vs\;=\;+\;312.88\;-\;63.17A\;-\;19.00B\;+\;31.47C1\;+\;7.04C2$$
(6)

Our findings revealed that the investigated variables significantly impacted SNVs—mean size (p < 0.05). Notably, SNVs formulations based on Span 60® were smaller than those based on Span20®. Therefore, it seems reasonable to assume that the lower HLB produced a small vesicle size, as the HLB of span 60® is (4.7), while the HLB of span 20® is (8.6) [64]. You can see the connection between VS and the surfactant HLB in the outermost power drop. This is because lowering the HLB values and making the vesicles smaller makes them more hydrophobic [20, 65]. Such results agree with previous literature [58]. Comparable to the Span® type effect, the EA type had a significant impact on VS, with noticeably smaller VS in the case of SNVs containing Cremophor-RH-40 rather than those containing P-407; this could be partly referred to the physical condition of the edge activator as Cremophor-RH-40 is usually a paste, and P-407 is a solid at ambient temperature that might have caused the size difference [66]. Additionally, Cremophor-Rh40 [14] has a lower HLB value than P-407 [22], which may have made it less hydrophilic and smaller, which may have led to a smaller surface area [66]. Our findings are parallel to a previous study [67]. Our findings are parallel to a previous study [66, 68].

Q8h of LP from SNVs

Figure 2 shows the release plots of LP from the SNV formulations that were made and the control solution in PBS (pH 5.5). The LP-SNVs formulation exhibited sustained release of LP over 8 h of the experiment. The percentage of LP released from SNVs after 8 h fluctuated between 30.7 ± 3.10 and 69.5 ± 1.45% (Table II), while the LP solution released 86.9 ± 7.9%. Residual analysis and ANOVA were performed to confirm the model's adequacy, with an adequate precision of 15.39 and an adjusted R2 of 0.8851. This showed close confirmation with a predicted R2 of 0.7852, as shown in Table III.

Fig. 2
figure 2

The release plots of LP from generated SNVs formulations and the control solution

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Table III Blood Pressure in Millimeter-Hg for each Rat Group Both Before and After Therapy
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$$\%\;of\mathit\;LP\;released\mathit\;=\;+48.28\;-\;7.77A+5.02A-\;7.40\text{C}1\;+\;0.24\text{C}2$$
(7)

The independent variables significantly impacted LP release (P < 0.5) among dispersions. The results showed that SNVs made of Span 20® had much higher Q8h values than SNVs made of Span 60®. This may be because the unsaturated alkyl chain in Span 20® makes it easier for LP to leak out of vesicles [69]. Another explanation for the observed boosted release of SNVs based on Span 20® is its lower phase transition temperature (16°C) compared to Span 60® (53°C) [55, 63]. As the liberation of LP from the LP-SNVs formulation was conducted at 32 ± 0.5°C, SNVs composed of Span 60® had lower release values due to their higher transition temperature, which probably renders them in a well-ordered gel state. These results agreed with previous literature [23, 65, 70]. Similarly Cremophor-Rh40-decorated SNVs had higher release values than P-407-decorated SNVs (P < 0.5) because the shorter alkyl chain had a higher release rate and the longer chain lasted longer. Since Cremophor-Rh40 attained a shorter alkyl chain length, it revealed a greater drug release percentage [65]. Additionally, a greater surface area in the dissolution media and SNVs based on Cremophor-Rh 40 having less size could enhance the LP's release.

Ex-vivo Permeability Study

Ex-vivo penetration tests reveal substantial data on in vivo capability. In the study of nasal permeation, the developed LP-SNVs made LP permeate better than the LP solution. As shown in Table II, the total amount of LP that permeated from the SNV formulations was between 194.1 ± 4.90 and 435.3 ± 13.53 µg/cm2. However, over 24 h, only 149.5 ± 13.5 µg/cm2 of LP permeated through the nasal mucosa from the LP solution. (Fig. 3). Table S1 summarizes the results of the calculated permeation parameters of the developed LP-SNVs. The developed SNVs showed a steady intranasal flux (Jss) that ranged from 3.52 ± 0.29 to 24.23 ± 2.21 µg/cm2/h. This was different from the LP solution, which had a Jss value of 3.48 ± 0.39 µg/cm2/h. So, the results showed that SNVs were important for improving LP permeation because they diffused through the mucosa 1.01 to 7.45 times faster than the LP solution. To make sure the model was correct, residual analysis and ANOVA were used. The results showed that the model was accurate with a precision of 16.15, an adjusted R2 of 0.9008, and a predicted R2 of 0.8145.

Fig. 3
figure 3

Ex-vivo permeation plots of generated SNVs formulations and the control solution

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$$Q24=+318.33-59.53A+26.59B-46.43C1+1.98C2$$
(8)

Significant effects were observed regarding the independent variables on the Q24h of LP-SNVs (p < 0.05). Figure 3; This better delivery of LP through the nose might be because LP-SNVs have both Span® and edge activators, which could make SNVs more flexible [66, 71]. The changes seen in nasal permeation parameters could be linked to changes in the particle sizes of the developed SNVs, since vesicles with smaller sizes had better permeation, and the opposite was also true. These outcomes may be correlated with the broader surface area associated with the smaller VS. Notably, SNVs formulations that contained Span20® and Cremophore Rh40 were more easily permeated than those containing Span 60® and P-407; this could be referred to as the small VS of fabricated SNVs, including Span 20® and Rh40. Furthermore, the smaller VS promotes a larger surface area, allowing released LP to contact the nasal mucosa surface [72]. These findings agree with those of Q8h mentioned earlier. Analysis of the variance of the final models for measured responses was illustrated in Table S2.

Selection of the Optimum Formulation

Based on a successful material selection, the optimal LP- SNVs formulation with the best physicochemical characteristics was developed. The Design Expert® software recommended multiple configurations of the independent variables. The optimal formulation was developed from 55 mg of Span60® and Cremophor-Rh40 as EA. EE% was 58.6 ± 2.51% LP, VS was 212.3 ± 8.5 nm, Q8h was 48.9% LP, Q24h was 328.7 ± 5.75µg/cm2, and Zeta potential was -31.2 mV with a desirability of 0.66. As illustrated in Table S3, the optimal LP-SNVs' actual and predicted values were comparable, with no noticeable changes (p > 0.05). This illustrated the equations' predictable nature and reliability in light of data variability.

Morphological Characterization of SNVs

The morphological investigation of optimal LP-SNVs formulation was explored via TEM examination. So, the suggested SNVs were evenly spread out, spherical, and uniformly structured, with mostly smooth surfaces and no signs of gathering (Fig. 4). In addition, morphological analysis supported the previous DLS-determined VS values.

Fig. 4
figure 4

TEM of the optimal LP-SNVs formulation

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Physical Stability of the Optimal LP-SNVs

Based on EE%, VS, % of LP release, and zeta potential, the stability of optimized LP-SNVs upon being stored at 4°C for 3 months was examined. After this time interval, no noticeable changes were seen in the evaluated characteristics. (p < 0.05), where EE% at 0 day was 58.6 ± 2.51, Q8h of 48.9% ± 3.8 of LP released, VS was 212.3 ± 8.5 nm and zeta potential was -31.2 ± 2.6 mV, while EE% after three months was 56.6 ± 3.4, Q8h of 44.2% ± 4.8 of LP released, VS was219.2 ± 7.1 nm and zeta size was-30.3 ± 3.6 mV. (Figure S1) presents the parameters studied during the investigation period.

Evaluation of In-situ Gel Containing Optimal SNVs

The findings demonstrated that the T-sol temperatures were 32.87 ± 0.08 and 30.72 ± 0.10°C and gelation time 40 s and 36 s for plain LP in-situ gel and LP-SNVs in-situ gel, respectively, which is considered suitable for the nasal application, as these temperature values facilitate nasal administration and dosage estimation [73], furthermore, LP-SNVs in situ gel's bioadhesive force was determined to be 8761 ± 5.36 dyne/cm2, a value that was sufficient to prevent the formulation from rapid drainage and hence lengthens its nasal residence time. These results may be attributed to Carbopol 934 P, a polyacrylate with several carboxylic groups, was utilized in this investigation. It is able to generate hydrogen bonds with nasal mucous membranes, increasing the mucoadhesive strength of the material [74]. In addition, the viscosities of LP-SNVs in-situ gel were 874.72 cp at 25°C and 7633.92 cp at 37°C, with Farrow’s constant of 6.5, which confirmed the pseudo-plastic properties of the formulation. Along with that, the viscosities of LP in-situ gel at 25 and 32°C were 636.16 and 6997.76 cp, respectively, with Farrow's constant of 2.05. It was also found that the hysteresis loop areas for LP-SNVs in-situ gel were 1766.38 dyne/cm2.sec and 1515.90 dyne/cm2.sec for plain LP in-situ gel, which shows a good thixotropic behavior [75]. Their findings demonstrated that, while only 132.8 µg/cm2 of LP leaked out of the plain LP in-situ gel, 312.5 µg/cm2 of LP leaked out of the LP-SNVs in-situ gel (Figure S2). Furthermore, LP-SNVs in-situ gel released about 42.89 ± 1.25% while plain LP in-situ gel released about 83.4% over 8 h; this confirmed that SNVs could capture drug and sustain their release compared to free drug solution [56].

Pharmacodynamics Studies

Throughout the 14 days, no signs of mortality in rats were seen. The pharmacodynamic results for mean systolic blood pressure before and after treatment are shown in Table III. A one-way ANOVA was utilized for statistical analysis as compared with the control group. The ANOVA analysis showed a significant difference between the groups. Following dexamethasone administration, all rats' mean blood systolic levels increased (Figure S3). Within the 14 days of therapy, a statistically notable variation was seen in the systolic blood pressure degrees that varied according to the treatment formulations, with a p-value of less than 0.05. It was observed that Group 5, which received LP-SNVs intranasal gel as treatment, significantly reduced systolic blood pressure levels to a normal value as compared to Group 3 (received LP solution as treatment) and Group 2 (diseased group). According to these findings, the LP performed better when given as LP-SNVs intranasal gel. These findings show that LP-SNVs intranasal gel can better preserve the effects of losartan at a dose that is the same as the standard LP solution. Compared to the oral formulation of LP solution, the test formulation significantly affects the systolic blood pressure profiles of experimental animals. As a result, the test formulation significantly outperformed the reference formulation in vivo in terms of pharmacodynamic characteristics.

Tolerability and Toxicity Studies

The in vivo studies were done with permission number NUB-023–011 from the Animal Ethics Committee at the Nahda University for Experimental Animals in Beni-Suef, Egypt. The study fulfilled the ARRIVE Declaration and the regulations for the use and care of animals. A histological analysis of the mucosal membrane was done to evaluate the formulation's safety Fig. 5 displays light photomicrographs taken from cross-sections of the front noses of rats that were exposed to in-situ gel formulations for two weeks. Examination revealed the absence of irritation-related symptoms, including sub-epithelial edema and vascular congestion. Furthermore, none of the rats exhibited any severe symptoms, such as the development of necrosis, the shedding of epithelial cells, or bleeding. Figure 5b shows a more or less typical histological structure. Similar outcomes were described when sheep were given an in-situ gel based on poloxamer [31].

Fig. 5
figure 5

histopathological sections of a control (un-treated group) and b Rat nasal mucosa treated with LP- SNVs in-situ gel (X400 H&E stain), where average intact epithelial lining (black arrow), average submucosa with average blood vessels (blue arrow) and average cellularity (red arrow) were shown

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Pharmacokinetics Analysis

The pharmacokinetic variables are displayed in Table IV. The highest amount of LP in the blood (Cmax) was found in LP-SNVs in-situ gel, plain LP in-situ gel, and oral solution. It was 0.78 ± 0.03, 0.42 ± 0.06, and 0.19 ± 0.02 µg/ml, respectively, at Tmax of 1 h for LP-SNVs in-situ gel and plain LP in-situ gel, and at Tmax of 1.5 h for oral LP-solution (Fig. 6). The difference in how serum opsonins and the SNVs membrane interact may have led to the shorter Tmax for LP-SNVs in-situ gel by speeding up the burst of the vesicles and the release of the LP into the bloodstream [76]. According to the collected findings, intranasal delivery of the SNVs-LP in situ gel influenced the Cmax of LP in plasma to be 4.1 times greater than that of oral solution (p 0.05). This great increase in Cmax value in the case of SNVs-LP in situ gel could be ascribed to the nanovesicles’ role as a penetration enhancer, which boosts drug permeation and reduces the first-pass effect. These findings are consistent with those that have already been disclosed [30, 77]. The AUC (0–24) values were 4.69 ± 0.9, 1.75 ± 0.46, and 0.737 ± 0.15 µg/ml.h2 for LP-SNVs in-situ gel, plain LP in-situ gel, and oral solution, respectively. The clear increase in the AUC (0–24) of LP-SNVs in-situ gel may be linked to the SNVs' flexibility and lipophilicity, which helped particles stick to nasal epithelial cells and pass through the nose, as well as better systemic absorption [26]. These results agreed with those of [78]. Consequently, it was found that delivering LP–SNVs in-situ gel via the intranasal route improved pharmacokinetic characteristics by reducing the time needed for LP to reach peak plasma concentration and enhancing its bioavailability by 6.3 times in contrast to oral drug solution.

Table IV Following the Administration of an LP Oral Solution, LP-SENVs Loaded In-situ Gel, and Plain LP Loaded In-situ Gel, the Pharmacokinetic Parameters of LP were Determined in Rat Plasma
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Fig. 6
figure 6

Rat plasma LP levels after oral LP-solution, intranasal delivery of plain LP in-situ gel, and intranasal delivery of LP-SNVs in-situ gel

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Conclusion

The optimal LP-SNVs formulation with maximum EE% (58.60%), minimum VS (202.3 nm), maximum Q8h (48.9%), and maximum Q24h (328.7µg/cm2) was incorporated in in-situ gel to prolong residence time in the nasal cavity. According to the histopathological investigation, the developed LP-SNVs in situ gel posed no threat to the nostrils and was well tolerated. Moreover, the intranasal administration of LP- SNVs in-situ gel increased the invasion of LP through the nasal cavity and increased the bioavailability and pharmacodynamic characteristics of LP compared with oral solution. Therefore, according to our hypothesis, supported by the observed data, the intranasal administration of LP-SNVs in situ gel may be a good carrier for managing hypertension to maximize the therapeutic effect of LP.