Mizolastine-solid lipid nanoparticles loaded hydrogel | International News Network

2021-11-24 01:57:49 By : Ms. Kate Lau

Javascript is currently disabled in your browser. When javascript is disabled, some functions of this website will not work.

Open access for scientific and medical research

From submission to the first editing decision.

From editor acceptance to publication.

The above percentage of manuscripts have been rejected in the past 12 months.

Open access to peer-reviewed scientific and medical journals.

Dove Medical Press is a member of OAI.

Batch reprints for the pharmaceutical industry.

We provide authors with real benefits, including fast processing of papers.

Register your specific details and specific drugs of interest, and we will match the information you provide with articles in our extensive database and send you a PDF copy via email in a timely manner.

Back to Journal »International Journal of Nanomedicine» Volume 16

Mizolastine-solid lipid nanoparticles-loaded ocular hydrogel formulation and histopathological study

Author El-Emam GA, Girgis GNS, Hamed MF, El-Azeem Soliman OA, Abd El Gawad AEGH

Published on November 24, 2021, the 2021 volume: 16 pages 7775-7799

DOI https://doi.org/10.2147/IJN.S335482

Single anonymous peer review

Editor approved for publication: Dr. Yan Shen

Ghada Ahmed El-Emam,1 Germeen NS Girgis,1 Mohammed Fawzy Hamed,2 Osama Abd El-Azeem Soliman,1 Abd El Gawad H Abd El Gawad1 1 Department of Pharmacy, School of Pharmacy, Mansoura University, Mansoura, 35516, Egypt ; 2 Department of Pathology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt. Communication: Ghada Ahmed El-Emam Department of Pharmacy, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt Telephone 20 10-9059-0989 Protection [email protected]] Background: Mizolastine (MZL) is a dual-acting non-sedating topical antihistamine anti-inflammatory drug used to relieve allergic diseases such as rhinitis and conjunctivitis. Solid lipid nanoparticles (SLN) is an advanced ophthalmology delivery system, which has the advantage of increasing corneal drug absorption, thereby improving bioavailability and achieving ocular drug targeting. Method: First, using a 32 full factorial design, MZL was formulated into MZL-SLN by thermal homogenization/ultrasound. Solid state characterization, in vitro release and stability studies have been performed. Then, use 1.5% w/v sodium alginate and 5% w/v polyvinylpyrrolidone K90 to incorporate the optimized MZL-SLNs formula into the eye hydrogel. The gel was evaluated by in vitro release and in vivo studies by applying hyperemia of allergic conjunctivitis in a rabbit eye model. Results: The optimized formula (F4) has the highest encapsulation efficiency (86​.5±1.47%), the smallest average particle size (202.3±13.59 nm) and reasonable zeta potential (-22.03±3.65 mV). Solid-state characterization of the MZL package in SLN was carried out. The in vitro results showed that under the non-Fickian Higuchi kinetic model, the sustained release curve of MZL-SLN was as long as 30 hours. Stability studies confirmed the invariance of freeze-dried MZL-SLN (F4) after 6 months of storage. Finally, the hydrogel preparation containing MZL-SLN proved that the eye congestion disappeared after 24 hours, and the conjunctiva was completely repaired. In addition, compared with the post-treatment of the same formula, the pretreatment with the hydrogel loaded with MZL-SLNs significantly reduced the expression levels of TNF-α and VEGF in the rabbit conjunctiva. Conclusion: MZL-SLNs can be considered as a promising stable and sustained-release nanoparticle system for preparing ocular hydrogels as an effective anti-allergic ocular drug delivery system. Keywords: Mizolastine, solid lipid nanoparticles, 32 full factorial design, sustained release, in vivo study

The current existing topical anti-allergic drugs are members of many pharmacological categories, such as: antihistamines, non-steroidal anti-inflammatory drugs, mast cell stabilizers, dual-acting agents (mast cell stabilizers with antihistamine effects) , Vasoconstrictors, corticosteroids and calcineurin inhibitors.

Mizolastine (MZL) is a new type of benzimidazole derivative non-sedating antihistamine with additional anti-inflammatory properties used to relieve seasonal and perennial allergic rhinitis. It is a selective H1 receptor antagonist for peripheral action, recruitment of granulocytes, vascular endothelial growth factor (VEGF), tissue necrosis factor (TNF-α), 5-lipoxygenase. 5,18 It limits the effect of histamine release from activated mast cells, the chemotaxis of inflammatory cells, and the expression of intercellular adhesion molecule-1 (ICAM1) during allergic reactions. 61 MZL is rapidly absorbed from the gastrointestinal tract, reaching a peak plasma concentration of 0.3 mg/L after about 1.5 hours. The plasma protein binding rate is about 98%. The average elimination half-life is about 13 hours. Insoluble in water (0.01 mg/mL), soluble in DMSO, slightly soluble in methanol and chloroform when heated. It has pKa values ​​of 9.99, 5.99, and 3.2, and is classified as Class II (low solubility, high permeability) according to the Biopharmaceutical Drug Disposal Classification System BDDCS. However, there is no external dosage form containing MZL on the market, and the only available form is (Lastlerge) tablets, 10 mg once a day.

MZL is one of the dual-acting topical antihistamines. It is currently the main effective treatment against benign allergic conjunctivitis. The advantage of these drugs is that they can quickly relieve symptoms by combining the effects of histamine receptor antagonists with the long-term benefits of mast cell stabilization (multimodal drugs). These combined approaches can provide immediate and sustained relief in early and late ocular allergic reactions. 1 A study conducted by Bansal et al. 5 showed that MZL controlled-release ocular inserts using Eudragit RL100 and RS100 can release drugs at a controlled rate for 5 consecutive days.

Solid lipid nanoparticles (SLN) are flexible nanocarriers used for drug delivery in almost all routes of administration, including; eye, parenteral, oral cavity and skin. They can maintain the drug delivery profile, thereby reducing the frequency of dosing and improving the therapeutic effect. In addition, they can improve drug bioavailability and achieve targeting. 32

SLNs are an incentive method for ophthalmic drug delivery because they can increase the corneal absorption of the drug, thereby increasing its bioavailability. 43 In addition, their biocompatibility and mucosal adhesion properties can improve ocular mucosal contact, prolong the drug corneal residence time and objective ocular drugs 23

Allergic conjunctivitis is an inflammatory disease that affects the ocular surface: eyelids, conjunctiva, and cornea. It is triggered by an abnormal immune hypersensitivity reaction to environmental allergens. The immune mechanism of allergic conjunctivitis is characterized by IgE-mediated mast cell degranulation and/or T lymphocyte-mediated immune hypersensitivity. 12,34,58

The purpose of this work is to formulate and evaluate the MZL in SLN using different lipid content and surfactant percentages. In addition, a 32 random full factorial design was used to study the main effects and interactions of independent variables on the physical and chemical properties of the prepared MZL-SLNs, namely the average particle size (MPS), encapsulation efficiency% (EE%), polydispersity index (PDI) and zeta potential (ZP). In addition, the solid-state characterization, in vitro release characteristics and kinetics of the optimized formula, and the stability of the lyophilized powder of the improved formula at room temperature and refrigerated temperature were also studied. Finally, the application of optimized MZL SLN-based ophthalmic hydrogel on the effect of non-infectious allergic conjunctivitis in a rabbit model.

MZL is provided by Medizen Pharmaceutical Industries of Borg El Arab, Alexandria, Egypt. Glycerol monostearate (GMS) granules (melting point 57°–65°C), compritol ATO 888 (glyceryl behenate) (melting point 65°–77°C) and precirol ATO 5 (palmitic acid glyceride) ) (Melting point 50°–60° C) was obtained as a gift from Gattefoseé Co., Saint-Priest, Cedex, France. D-trehalose dihydrate was purchased from Sisco Research Pvt, LTD, Mumbai, India. Histidine phosphate was obtained from Universal Fine Chemicals, India. Sodium chloride, Tween 80 (polysorbate 80), propylene glycol and stearic acid were kindly provided by Adwic Pharmaceutical Chemicals of El Nasr, Egypt. Sodium lauryl sulfate (SLS) was provided by Merck, Germany, and 3,3-diaminobenzidine tetrahydrochloride (DAB) was purchased from Dako, Glostrup, Denmark. HPLC grade methanol was purchased from Fisher Scientific, Germany. Sodium alginate was purchased from BDH Chemical, Liverpool, England). Methyl paraben and propyl paraben were purchased from Clariant Chemicals, Switzerland. Polyvinylpyrrolidone (PVP K90) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

The partitioning of the drug between the lipid and aqueous phases is at a temperature above the melting point of each selected lipid (ie, ATO 5, glycerol monostearate (GMS), compritol ATO 888 and stearic acid) conduct. The mixture of lipid and water in a ratio of 1:1 (w/w) was stirred for one day to fully saturate, and then the drug was added. Then, the mixture was stirred for 3 days at each specified temperature (75°±3°C). After cooling the mixture, separate the water phase, centrifuge at 6,000 rpm (high-speed benchtop centrifuge model H1650-W, Ray Wild, Germany) for 30 minutes and filter. An ultraviolet-visible spectrophotometer (model UV-1601 PC, Shimadzu, Kyoto, Japan) was used to spectrophotometrically determine the drug concentration in the water phase and lipids. 33 Lipids with high drug distribution have been selected. The distribution coefficient is calculated using Nernst equation (1)

Where; PC is the partition coefficient, Co and Caq. are the drug concentration in the lipid and aqueous phases, respectively.

SLNs are prepared using a thermal homogenization method. 24,37,38,60 Adjust the temperature to about 5°C higher than the melting point of the GMS lipid at 70°C to ensure that the lipid is completely melted. Then, MZL (10 mg) was dissolved in 2 mL of methanol and added to the lipid melt at the same temperature. 20 mL (0.1 M) of sodium chloride (NaCl) aqueous solution containing Tween 80 (surfactant) at the same temperature was added, and a hot pre-emulsion was formed by high-speed stirring on a magnetic stirrer (Heidolph, USA, Table 1). The hot pre-emulsion is then processed in a temperature-controlled ultrasonic homogenizer (Model VC505, Sonics & Materials, Inc., Newtown, CT, USA) and adjusted for 5 minutes at its maximum amplitude (100%) (one pulse is turned on and One pulse leaves). Table 1 Independent formula variables and their levels used in Design Expert

Table 1 Independent formula variables and their levels used in Design Expert

The obtained nanoemulsion was cooled to room temperature for 2 hours and then recrystallized by continuously stirring and evaporating the organic solvent. The SLN was collected by cooling centrifugation (CE16-4X100RD, Acculab, USA) at 13,000 rpm for 90 minutes, and washed once with deionized water. All batches of SLN are produced at least three times.

For the lyophilization of MZL-SLN, the collected nanoparticles were resuspended in deionized water containing 5% (w/v) D-trehalose anhydride. After that, they were pre-frozen overnight in a deep freezer at -8°C and then transferred to a lyophilizer (Labconco Lyph-Lock 4.5, USA) for 48 hours. The same program variables were used to prepare ordinary nanoparticles to be used as blanks. All samples were prepared in triplicate.

The design of experiment (DOE) method is used to provide an effective method to optimize the thermal homogenization process, and to discover the causal relationship between process variables and their results, to explore the mathematical correlation between factors and parameters.

Carry out preliminary optimization to study the influence of process parameters, such as sonication time, lipid concentration, and surfactant concentration. Thereafter, ordinary SLN and MZL-SLN were prepared according to 32 random full factorial designs to study the main influence and interaction of independent variables on the physicochemical properties of the prepared MZL-SLN. Therefore, nine possible experimental combinations were prepared, and each combination was run 3 times.

The use of three-level and two-variable screening of MZL-SLNs dispersions with 32 full factorial designs is a suitable condition for the preparation of SLNs.

For this design, three levels (300 mg, 400 mg and 600 mg) of lipid concentration A and three levels (0.25%, 0.5% and 1% w/w) of emulsifier concentration B were selected as the two key Process parameters (CPPs) [independent variables], (Table 1). Four responses were selected to study their effects on particle size (PS), particle distribution, EE %, and ZP.

In order to understand the result that depends on the size of the coefficient and the addition of positive (synergistic effect) or negative sign (antagonistic effect) 41, the following complete polynomial regression equation is carried out: (2)

Where; b0 is the intercept corresponding to the arithmetic mean of the quantitative results of nine runs, b1 to b5 represent the coefficients calculated based on the experimental detection value of Y. In addition, A and B correspond to the coding level of the independent variable. The term "AB" stands for an interactive item, which means the modification of the response parameter when two factors change at the same time. The main effects A and B represent the average results of changing one factor from a low value to a high value at a time. Use the polynomial terms A2 and B2 to further check the nonlinearity in the model.

Use analysis of variance (Design Expert 12 (Stat-Ease, Minneapolis, MN, USA)) to evaluate the model in the expression of statistical significance. Analyze surface response plots and etc. by keeping each factor at its low, medium, and high levels High-line graph, and change other factors within the scope used in the research.

All nine MZL-SLN formulas have been evaluated by PS, PDI, EE and ZP. The key quality attributes (CQA) results of various formulations have been recorded. The impact of CPPs on EE%, PS and PDI is quantified by polynomial coding equations.

After 1:10 dilution with deionized water, use the Malvern Zetasizer (Zetasizer, Nano-ZS 90, Malvern, UK) to measure the PS and PDI values ​​of all freshly prepared batches by photon correlation spectroscopy (PCS) to generate suitable Scattering intensity. Each measurement is repeated 3 times.

Laser Doppler electrophoresis is used to evaluate particle electrophoretic motion by indirectly measuring the thickness of the diffusion layer using Nano-ZS 90. Each sample is diluted at 25°C and the measurement is repeated three times. The refractive index of water is fixed at 1.33.29

Each lyophilized MZL-SLNs preparation weighing 100 mg was dissolved in 50 mL methanol in a 50 mL volumetric flask, sonicated for 15 minutes to release the drug, heated to 80°C, and then diluted appropriately with methanol.

Filter the sample. Use a spectrophotometer (λmax=230 nm) to estimate the EE of MZL-SLN. For each formula, the results are expressed as the average of triplicate samples. EE% is determined using the following formula: (3)

Morphological examination of suspended F4-SLN was performed using TEM (JEM-2000EX II, JEOL, Tokyo, Japan). After 1 mL of prepared F4-SLN was diluted ten times with deionized water, it was run at 80 kV. After that, ultrasonic treatment was performed for 10 minutes in an ultrasonic bath. A drop of the diluted sample was dropped on the surface of the carbon-coated copper grid and dried at room temperature for 5 minutes. Finally, the image capture and analysis using Digital Micrograph and Soft Imaging Viewer software are studied.

SEM (X-MaxN, JSM-6510LV, Oxford Instruments, UK) was used to study the surface morphology, shape and uniformity of drugs, GMS lipids and optimized SLN. Before using a DC sputter coater for inspection, fix the sample with gold under low vacuum to ensure the surface conductivity of the particles. 25

The spectra of MZL, GMS, Tween 80, their physical mixtures corresponding to optimized formulations, freeze-dried F4 and their ordinary SLN are obtained by FT-IR spectroscopy (Thermo Fisher Scientific iS10 Nicolet). A small sample (2 mg) is mixed with potassium bromide. Then, they were ground into fine powder and pressed into KBr discs with a hydrostatic press. The scanning range is 500 to 4,000 cm-1.

Approximately 10 mg sample of each of MZl, GMS, and the physical mixture of the selected formulation in the same proportions. The freeze-dried F4 and its ordinary SLN were placed in a standard aluminum pan (Shimadzu DSC 50, Tokyo, Japan) and heated at a constant temperature at a heating rate of 10°C/min (35°–300°C) . Dry nitrogen atmosphere, purge 62 at a flow rate of 20 mL/min

(Rigaku Rint-2500VL, Tokyo, Japan) was used to analyze any changes in the crystallinity of the compound before and after formulation by XRD analysis. The X-ray diffraction pattern of MZL, GMS, the physical mixture corresponding to the optimized formula, as well as the X-ray diffraction pattern of the freeze-dried SLN of optimized F4 and its ordinary SLN are measured by an X-ray diffractometer equipped with Cu-Kα radiation at 3°–50 °C, 2θ angle, 45 kV voltage and 9 mA current. 51

MZL-SLN (F4) is composed of a lipid (GMS) mixture with a weight of 400 mg (0.08%) and 0.5% w/v Tween 80 as a stabilizer to obtain small particles, low PDI and high ZP and EE. Best formula%.

The hydrogel is introduced to obtain a viscosity level suitable for topical eye applications. In short, the gelling mixture (1.5% w/v sodium alginate and 5% w/v PVP K90) was fully dispersed in distilled water, and propylene glycol (10% w/v) was added as a plasticizer. Then, the appropriate weight of freeze-dried MZL-SLN equivalent to 1 mg MZL/g hydrogel and the same weight of ordinary freeze-dried SLN are mixed with a homogenizer to obtain medicated hydrogel and ordinary hydrogel, respectively.

The dispersion is neutralized dropwise with NaOH solution, and the pH value is adjusted to 6.5-7 under gentle stirring. Methyl paraben and propyl paraben (1:1 ratio) are used as preservatives (0.1% w/v). The final prepared hydrogel containing 0.1% w/v MZL was left overnight without stirring to remove any bubbles. The drug-loaded SLNs hydrogel formulation is stored at 2°-8°C until use. The same protocol was used to prepare a blank hydrogel as a negative (no treatment) control.

The in vitro release of MZL from freshly lyophilized drug-containing SLN (F4) was studied. In addition to the aqueous suspension (as a control), a hydrogel prepared with a specific weight (1g) was evenly distributed on a modified vertical Franz diffusion cell with a diameter of 2.5 cm. The in vitro release of blank hydrogels containing free drugs is also considered. A dialysis membrane with a molecular weight cut-off between 12,000-14,000 Daltons is used and is tightly sandwiched between the donor and acceptor compartments.

The receptor compartment contains 70 mL of release medium, composed of phosphate buffer pH 6.8, which contains 0.5% w/v SLS. The dialysis membrane is soaked in the release medium overnight before being installed in the diffusion cell. Franz diffusion cells were placed in a GFL shaking incubator (Gesellschaft fur Labortechnik, Burgwedel, Germany) maintained at 37°±0.5°C, and continuously stirred at 100 rpm during the entire experiment. The lyophilized ordinary SLN and the medicated F4-SLN containing 1±0.12 mg MZL were suspended in distilled water and sonicated, and then placed in the donor compartment. At different time intervals, aliquots (2 mL) were taken up to 36 hours, filtered through a 0.45 μm microporous filter, and then supplemented with fresh media to maintain tank conditions throughout the experiment. In addition, the free MZL suspension was subjected to the same procedure to serve as a control.

The drug concentration of the sample was additionally analyzed by a spectrophotometer at 230 nm. Each experiment was completed in triplicate, and the cumulative MZL release percentage was calculated at each time interval.

In order to determine the drug release kinetics from SLN and its prepared hydrogels, kinetic equations were used to mathematically fit the in vitro release data, such as zero-order, first-order, Higuchi diffusion and Korsmeyer-Peppas semi-empirical models. The choice of advanced mathematical model depends on using GraphPad Prism software version 6 to deliver the kinetic release curve with the highest coefficient of determination (R2). Each experiment was repeated three times, and the average value was used.

The physical stability of the optimized formula (F4) was evaluated under different storage conditions using the International Conference on Harmonization (ICH) guidelines. 27

The SLNs aqueous dispersion (F4) loaded with MZL is freshly prepared, lyophilized and stored in a completely sealed amber glass vial, wrapped in aluminum foil, and kept in a cold storage (4°±1°C) and environment (25°± 2 °C, 60%±5% relative humidity) without any movement within 6 months at the temperature. Evaluate the stability of the selected formulation after the freeze-dried F4 powder is redispersed. The measured parameters are the physical appearance, PS, distribution, surface charge and EE%, 5 after time 0 (initial) and 1, 2, 3, and 4 And 6 months of storage. A common SLN was prepared and used as a control.

The Draize test is the most reliable way to determine the eye irritation of ordinary and MZL-SLN containing sodium alginate/PVP K90 hydrogel. 20 This optimized ophthalmic formulation was selected based on in vitro drug release and stability data. According to the guidelines outlined in the "Guidelines for the Care and Use of Laboratory Animals" (NIH Publication 85-23, revised in 1985), all animal work was approved by the Ethics Committee of the School of Pharmacy, Mansoura University, Egypt. Seven New Zealand male albino rabbits received the optimized formula to evaluate the degree of irritation. The Draize test uses a scoring system from 0 (no irritation) to 3 (highest irritation and redness) for the cornea, iris, and conjunctiva. The test preparation was smeared into the conjunctival sac of the right eye, and physiological saline was instilled in the left eye as a control. Check the cornea, iris, and conjunctiva for any signs of irritation or congestion caused by the preparation. The eye irritation score test was performed within 1, 2, 5, 8 and 24 hours after administration. 50

Induces rabbit conjunctivitis by instilling a 1% (w/v) histamine solution into the eye. 45 Put two drops of the solution into both eyes of each rabbit. One eye uses the tested ophthalmic preparation as a test, and the other eye uses saline drops as a control. Check the eyes every 5 minutes until the maximum congestion occurs 30 minutes after instillation of the histamine solution.

The experimental procedures followed the ethical principles of the use of laboratory animals by the Scientific Committee of the School of Pharmacy, Mansoura University, Egypt. Four groups of male albino rabbits (six in each group) were used, weighing 2.0-2.5 kg. Rabbits receive normal feeding, ventilation and light. For each animal, one inflamed eye was used as a test and the second as a control. Appropriate weight of each formula equivalent to 0.1 mg MZL was applied to the tested eye, and 100 μL saline drops were used in the other eye. 30 minutes from the instillation of the histamine solution (maximum redness), it is treated with a gel formulation loaded with MZL.

The four groups (n = 6 each) are divided into the following: Group I: normal untreated negative control (phosphate buffered saline (PBS drops)) Group II: positive control (histamine solution) Group III: with alginic acid Sodium free MZL treatment/ PVP K90 hydrogel compared with ordinary hydrogel Group IV: Compared with ordinary hydrogel, treated with MZL-SLN containing sodium alginate/PVP K90 hydrogel

During the entire study, observations were made every 5 minutes to completely eliminate eye redness after application of the selected formulation. Check the cornea with a magnifying glass. In addition, photograph the general appearance of the control and test eyes.

The purpose of this study is to evaluate the effect of local ocular MZL on the animal model of albino New Zealand rabbits with allergic conjunctivitis. New Zealand rabbits are divided into six groups:

Group I: Normal untreated negative control (PBS drops) Group II: Positive control (histamine solution) Group III: 1.5% w/w sodium alginate/5% w/w PVP loaded with free MZLــ K90 hydrogel post-treatment group IV: post-treatment with 1.5% w/w sodium alginate/5% w/w PVP K90 hydrogel loaded with MZL-SLNs: group V: post-treatment with 1.5% w/ loaded with free MZLــ w Sodium alginate/5% w/w PVP K90 hydrogel VI group pretreatment: 1.5% w/w sodium alginate/5% w/w PVP K90 hydrogel pretreated with MZL-SLNs

Animals in each group were sacrificed, conjunctival specimens were dissected, and immediately immersed in 10% formalin neutral buffer for fixation. After 24 hours, the sample was washed, dehydrated and clarified. The paraffin-embedded tissue was sliced ​​to 5 μm using a microtome, and stained with hematoxylin and eosin (H&E) for further inspection using an optical microscope. Count eosinophils, plasma cells, and apoptotic cells in four squares with a diameter of 1 μm in each high-power field. A Java-based image processing program (ImageJ analysis) was used to measure the thickness of the eyelid covering epithelium.

The paraffin block that separates the conjunctiva is sliced ​​and immersed in a detergent, and then passed through a continuous concentration of alcohol. After that, the immunohistochemical protocol of VEGF and TNF-α paraffin blocks was studied by antigen retrieval heated in acetic acid (pH 6.0). Then block the endogenous peroxidase with H2O2 (3%) for 10 minutes.

Use anti-TNF-α primary antibody and VEGF rabbit polyclonal antibody, ready-to-use, and incubate overnight at 4°C. Then, the tissue sample was washed 3 times with PBS, and the anti-rabbit secondary antibody was added for 1 hour at the same time. Observe the markings by incubating with DAB chromogen for 5 minutes at room temperature. This is used for visualization of peroxidase activity in many applications. In the peroxidase reaction, DAB acts as a hydrogen donor in the presence of peroxide. The oxidized DAB forms an insoluble brown end product, which is used in immunohistology and immunoblotting procedures. Finally, these sections were subsequently counterstained with H&E. Use ImageJ.19 to count every 1,000 immunopositive cells

The statistical analysis used one-way analysis of variance, followed by the Tukey-Kramer multiple comparison test, and the Student's t test for stability studies and comparison of different groups. Student's t test was also used to compare different groups using GraphPad Prism software version 6 at P<0.05. All results are given as mean ± standard deviation.

According to the measurement of the partition coefficient of the drug, GMS (log P = 0.55±0.11) was selected as the lipid basis for the preparation of MZL-SLNs (Figure 1). Its high biocompatibility and slow-release characteristics make it an excellent excipient for nano-formulations. Figure 1 Using different lipids to determine the drug partition coefficient. Abbreviations: MZL, mizolastine; GMS, glyceryl monostearate; P, partition coefficient.

Figure 1 Using different lipids to determine the drug partition coefficient.

Abbreviations: MZL, mizolastine; GMS, glyceryl monostearate; P, partition coefficient.

Hot homogenization is the preferred method for preparing sentinel lymph nodes overloaded with hydrophobic drugs because of its relative simplicity, high efficiency, and enhanced EE, so it is used in about 50% of reported studies. 27

We observed that after adding sodium chloride, the growth of PS during centrifugation increased, so the efficiency of centrifugation increased, and the drug was trapped in the SLN. This is because NaCl has a significant effect on vesicles, provided that its concentration is high enough (>50 mM), which is expected to be mainly related to osmotic pressure. 53

SLNs have several characteristics, such as good drug-carrying capacity and the ability to capture hydrophilic and hydrophobic substances with multiple characteristics, and deliver drugs at a prescribed rate, thereby enhancing their intracellular absorption. Through lipids, vesicles are used as simplified models of cells and biological membranes. Their similarity with biofilms makes them an ideal structure, not only for studying existing biological systems, but also for studying the emergence, function and evolution of primitive cells. 13

Minimum PS, minimum PDI, maximum EE %, and reasonable ZP are critical requirements, which should be considered to improve drug absorption and subsequent bioavailability.

In terms of full factorial design, the dependent variables MPS, PDI, EE and ZP are regarded as indicators of the repeatability and efficiency of the processing technology. Based on the data and parameters of the F1-F9 factorial design, we proposed and discussed the polynomial equations of our four dependent variables (Table 2). Table 2 Coding independent variables and characteristics of MZL-loaded SLN prepared according to 32 full factorial design

Table 2 Coding independent variables and characteristics of MZL-loaded SLN prepared according to 32 full factorial design

Not only the average PS (nm) of SLNs is the main one, but their PDI value is also a measure of the PS distribution. Therefore, these two basic criteria of NPs affect drug release rate, biodistribution, and bioavailability. Because the lipid carrier larger than 100-150 nm can be absorbed by phagocytes or stay in the tissue for a long time. 13 Danaei et al. 2018 emphasized the importance of size and PDI in the successful design, formulation, and development of nanosystems for drugs, and other applications.

The PS of MZL-SLN ranges from 172.53±1.77 nm to 500.07±10.6 nm, which is mainly suitable for ocular delivery, while the PDI value ranges from 0.087±0.04 to 0.36±0.04, as described in (Table 2). The small value of PDI describes the narrow distribution of PS and provides a uniform suspension. In addition, they hope to maintain colloidal dispersion stability and avoid the generation of particles or precipitates.

In the model fitting of PS, the P value is recommended for the "quadratic model" of MPS analysis to maximize the adjusted R2 and predicted R2. However, the linear model is recommended for PDI. It seems that there are many unimportant model items. Model reduction may improve the model.

Equations 4 and 5 in terms of MPS (Y1) and PDI (Y2) are as follows: (4) (5)

The coding equation is useful for detecting the relative influence of the factors by comparing the coefficients of the factors. The actual factor equations can be used to predict the response of each factor at a given level. Coefficients with positive signs represent positive consequences. It has been noted that by increasing the concentration of lipid (A) in the dispersion, larger particle sizes can generally be obtained. The maximum MPS (500.07±10.6 nm) obtained at the high level A (1) and the low level B (-1) of batch F3.

Lipid concentration is the main factor that has a positive effect on the above two reactions. When GMS (A) is increased from 300 mg to 600 mg and B remains unchanged, PS and PDI increase (Table 2). Similarly, the interaction term AB, that is, when lipids and surfactants are increased together, cause a moderate increase in MPS, which may be due to the relatively high amount of lipids present, indicating a synergistic effect on PS.

The increase in PS with the increase in the amount of lipid (A) may be due to the aggregation of more particles, resulting in larger PS, because they increase the viscosity and reduce the rate of diffusion into the water phase, resulting in nanoparticle Formation of larger size or lipid aggregates. 15

On the other hand, the concentration of Tw 80 (B) has a negative impact on PS, but the positive impact of experts on PDI is negligible. As the surfactant concentration increases from 0.25% to 1% w/v, the interfacial tension between the organic phase and the water phase is reduced by forming a space barrier on the particle surface, thereby better stabilizing the smaller lipid droplets, thereby Protect the smaller lipid droplet particles and prevent them from coalescing into larger particles. Therefore, a stable emulsion of smaller and uniform droplet size nanoparticles with low polydispersity can be effectively formed. 22,31,36

The use of response surface and contour plots further clarified the correlation between the dependent variable and the independent variable. Figure 2 shows the contour plots and 3D response surface plots of MPS and PDI, respectively, and illustrates the influence of the independent variables A and B on the two response parameters. Figure 2 The profile (A and, B) and three-dimensional surface (C and, D) diagrams show the effect of the interaction between lipid mass (A) and surfactant concentration (B) on particle size and PDI.

Figure 2 The profile (A and, B) and three-dimensional surface (C and, D) diagrams show the effect of the interaction between lipid mass (A) and surfactant concentration (B) on particle size and PDI.

This critical quality attribute (CQA) is an appropriate method for judging the effectiveness and reproducibility of processing technology. The EE% of the prepared SLN ranges from 29.2±6.28 to 88.5±0.71%. The obtained EE (%) polynomial equation is expressed as follows:

After carefully examining the previous equation, we concluded that the concentration of lipid (A) and the concentration of surfactant (B) have a negative effect on EE%. At low level (-1) A (300 mg) and low level B (0.25%), the retention of MZL increased significantly, as in batch F9 (75.8±2.46%).

Crystallization is closely related to drug incorporation. The higher the crystallinity, the smaller the amount of drug retained in the sentinel lymph node, and vice versa. This can be explained by the effect of GMS on EE%, because this lipid type is a lipid that can form highly crystalline particles with a perfect crystal lattice, leading to drug discharge, especially at higher concentrations, so the crystal of GMS The grid prevents more effective drug retention 22 Another explanation is that increasing the lipid concentration has a positive effect on the solubility of the drug in the lipid core, and the viscosity of the mixture increases significantly. 30

In order to more easily explain the impact of different CPPs on CQAs, several graphs are created using the model equations of different CQAs. Figures 3A and B show contour plots and three-dimensional (3D) response surface plots of EE. Figure 3 The contour (A and, C) and three-dimensional surface (B and, D) diagrams show the effect of the interaction between lipid quality and surfactant concentration on the retention efficiency and zeta potential, respectively.

Figure 3 The contour (A and, C) and three-dimensional surface (B and, D) diagrams show the effect of the interaction between lipid quality and surfactant concentration on the retention efficiency and zeta potential, respectively.

The model diagram shows that the gradual increase of the surfactant concentration from 0.25% w/v to 1% w/v leads to a continuous decrease in the EE of the SLN formulation (Figure 3A). The higher value of the B factor (17.12) indicates that the surfactant concentration is the main aspect that affects EE%. This observed decrease in EE can be clarified by the distribution phenomenon. As the dissolution of the drug in the outer aqueous phase increases, the high surfactant level in the outer phase may increase the distribution of the drug from the inner phase to the outer phase, so additional drugs can diffuse and dissolve therein. 36

This is confirmed in our design, because the formula contains a relatively large amount of surfactants, for example (F1 and F5) show better leakage, so the EE% is reduced, which may be caused by the increase in surfactant concentration The interfacial tension is reduced.

Among all tested batches, the best PS with the largest% EE was achieved in batch F4, which was formulated with a mixture of medium level A (400 mg) and medium level (B) (0.5%).

The chemical nature of the particles and the degree of repulsion between similarly charged particles in the nanodispersion have a major impact on polarity. In general, SLN requires a higher positive or negative ZP value, because similar charges will cause electrostatic repulsion, thereby preventing particle aggregation. Such standards are widely helpful in predicting the stability of colloidal suspensions. 54

In this design, SLN surrounded by non-ionic surfactants (such as Tw 80) tends to remain stable regardless of the relatively low ZP value. The SLN shield with surfactant reduces the electrophoretic mobility of the particles through spatial stabilization, thereby reducing the ZP. 48 In the case of F4, ZP is about -22 mV, which can still satisfy a completely stable system.

The obtained polynomial equation representing the regression of the ZP model is as follows:

The ZP of all the prepared formulations showed regular negative values, ranging from – 10.8 ± 0.46 to – 26.03 ± 3.35 mV (F7 and F2, Table 2). The polynomial equation shows that the lipid mass has a negative coefficient for ZP, and the value is slightly lower, while the surfactant concentration has a positive effect.

Contour lines and three-dimensional (3D) response surface plots (Figure 3C and D) show that when medium levels of surfactant (B) are used, the ZP of high levels of lipids (A) is the highest. In addition, the lowest ZP values ​​are obtained at higher levels of A and B.

Table 3 summarizes the regression analysis results for all four responses (Y1, Y2, Y3, and Y4). It is found that the "predicted R2" values ​​of all dependent variables are reasonably consistent with the "adjusted R2" values. Table 3 ANOVA results of the response

Table 3 ANOVA results of the response

The regression model equation proves that the response Y1 (MPS) is significantly affected by the positive effects of the quadratic contributions of all model terms (A, B, AB, A2, and B2). At the same time, Y2 (PDI) is negatively affected by the linear contributions of factors (A) and (B). Except that A, B, and B2 are significant model terms, the response Y3 (EE%) is significantly affected by the positive effect of the secondary influence of factor B, and the response Y4 (ZP) is affected by the positive effect of the secondary participation of factor B Significantly affect the terms A, AB and B2.

The response surface model is tested with analysis of variance. The model P value is ˂0.05 significance level, indicating that the predictive model can describe the relationship between the independent variable and the dependent variable well.

Sufficient accuracy to measure the signal-to-noise ratio. A ratio greater than 4 is desirable. This model can be used to navigate the design space.

Based on design expert version 12.0.9.0, response surface (research type) user-defined design type and 88.2% desirability factor, it is used to fix the prediction target at minimization (PS and PDI) and maximization (EE% and ZP) In addition to low PS and PDI, the optimized formulas F4 and (A [0] and B [0]) represent the highest EE%. The optimized MZL-SLN was prepared using the optimized level of components and process variables summarized in Table 4. Table 4 The adjusted levels of independent variables and the predicted and observed responses of the optimized formula

Table 4 The adjusted levels of independent variables and the predicted and observed responses of the optimized formula

The research values ​​of PS, PDI, EE and ZP of optimized F4 are 185.36±2.56 nm, 0.26±0.01, 86.5±1.47% and -22.03±3.65 mV, respectively. This SLN formula will be further evaluated later. Figure 4 shows the PS distribution and ZP distribution of the optimized MZL-SLN. Figure 4 Representative (A) particle size distribution map and (B) zeta potential distribution of optimized MZL-SLN.

Figure 4 Representative (A) particle size distribution map and (B) zeta potential distribution of optimized MZL-SLN.

The size, shape and surface morphology of the nanoparticles were visualized by transmission electron microscopy (TEM) (Figure 5A). The size and shape of MZL-SLNs are uniform, with nanometer diameter and small spherical particles, and the lipid core is rich in drugs and surfactant shells. 9 Figure 5 F4-SLNs (A) transmission electron microscope and scanning electron microscope (I) MZL, (II) GMS lipid and (III) F4-SLN microscope (B) images.

Figure 5 F4-SLN transmission electron microscope (A) and (I) MZL, (II) GMS lipid and (III) F4-SLN scanning electron microscope (B) images.

It can be clearly seen from Figure 5B that the particles of F4 are spherical, regular in shape, smooth in surface and uniform in distribution. Due to the lipid nature of the carrier and the sample preparation before SEM analysis, the particles will be dispersed far away. 6

FT-IR helps us confirm the identity of MZL and detect the interaction of drugs with other ingredients.

The infrared spectrum of MZL is specified in (Figure 6A (I)), which shows that at 3054 cm−1 (C–H arom.), 2845–2933 cm−1 (C–H aliph.), 1511–1559 cm− The unique peak 1 (aromatic ring), 1011 cm-1 (CN), (3316-3417 cm-1) corresponds to the peak of NH stretching vibration and 1678 cm-1 depicts C=O stretching. Figure 6 Solid characterization. Abbreviations: GMS, glyceryl monostearate; FTIR, Fourier transform infrared spectroscopy; DSC, differential scanning calorimetry; XRD, X-ray diffraction.

Note: (A) FTIR spectrum, (B) XRD pattern and (C) DSC curve (I) MZL, (II) GMS, (III) Tween 80, (IV) physical mixture, (V) ordinary F4-SLN, and (VI) Drug-loaded F4-SLN.

Abbreviations: GMS, glyceryl monostearate; FTIR, Fourier transform infrared spectroscopy; DSC, differential scanning calorimetry; XRD, X-ray diffraction.

Note: (A) FTIR spectrum, (B) XRD pattern and (C) DSC curve (I) MZL, (II) GMS, (III) Tween 80, (IV) physical mixture, (V) ordinary F4-SLN, and (VI) Drug-loaded F4-SLN.

The FT-IR spectrum of GMS (II) shows that there is a characteristic IR peak at 2851-2919 cm-1, which may be related to the carbon-hydrogen stretching in the -CH2 alkane group present in the fatty acid acyl chain. However, the beak at 1179 cm-1 is related to the CC stretch coupled to CH2 and the peak at 1438 cm-1 (C=C). The wave number at 1735 cm-1 is due to the C=O stretching vibration associated with the carboxyl group. 46

The Tw 80 spectrum (III) shows the absorption band of methyl (-CH3) at 2920 cm-1, while the absorption band at 2864 cm-1 is due to -CH2 stretching. The band at 1735 cm-1 can be attributed to C=O.

The spectrum of the physical mixture of F4 (IV) shows that the total band of the drug and other components is reduced in intensity due to the dilution effect. The IR spectrum of the freeze-dried drug-loaded SLN (VI) showed no unique absorption band of MZL. This may be a reflection of drug retention in the lipid matrix. These results are consistent with 51, and they believe that the absence of drug peaks is because isoniazid is more likely to be effectively incorporated into the free space created between the broken fatty acid chains.

According to FTIR results, there is no significant interaction between the drug and lipids in the SLN formulation, and MZL is compatible with the applied FTIR excipients.

PXRD analysis is a unique method that can determine the crystalline or amorphous state of a drug and its crystal changes. 28 The XRD patterns of MZL, GMS lipids, their physical mixtures, ordinary F4-SLN, and drugs are shown in Figure 6B.

The XRD spectrum of MZL (I) shows the angle of the main peak; (2θ) angles are 17.9°, 18.36° and 34.5°, which represent the crystalline nature of the drug. The decrease in GMS crystallinity was also evaluated by XRD analysis (II), which has obvious peaks at angles of 19.5°, 22.9° and 36.7° (2θ). The physical mixture (III) retains the characteristic peaks of the drug and GMS.

Figure 6B (IV and V) illustrates the sharp decrease in lipid peak intensity, indicating that the less ordered GMS crystals present in the formulations of common and drug-loaded SLNs have decreased crystallinity.

The loss of crystallinity in the XRD pattern of SLN (F4) (V) loaded with lyophilized MZL is distinguished by the disappearance of the sharp peak and the loss of the most unique peak of the drug. These findings indicate that MZL is trapped in lipid defects in the amorphous state. It is expected that changes in lipid and drug crystallinity will affect the drug release profile from nanoparticles. The amorphous form is believed to have several characteristics, such as higher energy and larger surface area, and subsequently, greater solubility and dissolution rate, and therefore improved bioavailability. 28

Since the crystallinity of the drug turns into a molecularly dispersed amorphous or disordered crystalline phase and reduces the crystalline state of lipids, we have deeply rooted the results of incorporating the appropriate drug into the sentinel lymph node. Therefore, a less ordered lipid matrix facilitates an increase in the number of voids in its structure. Therefore, it can hold a larger amount of drugs. This explanation is consistent with what 8 reveals.

The DSC results indicate the possible changes in lipid crystallinity after adding drugs and preparations as SLN.

Figure 6C shows the DSC curves of drugs, GMS lipids, their physical mixtures, ordinary F4-SLN and drug-containing F4-SLN.

The DSC thermogram of MZL (Figure 6C(I)) shows a melting endothermic peak at 223.5°C, which indicates the crystalline nature of pure Mzl; this also confirms the XRD results. Figure 6C II shows the unique sharp endothermic peak of GMS at 60.8°C. The sharp peaks of a large number of lipids illustrate its crystalline nature.

The thermogram of their physical mixture (III) only experienced a sharp melting point corresponding to the bulk lipid (GMS), and the intensity of the drug endothermic peak decreased as it moved to a lower temperature of 203°C, which It is because part of the molecules of MZL are dispersed in GMS as a mixture of drugs and excipients, which reduces the purity of each component in the mixture. This has also been explained in a similar way.16 He studied the decomposition of some drugs in well-defined thermal events, which transformed the applicability of thermal techniques to the characterization of drug/excipient interactions.

It is worth noting that in freeze-dried ordinary SLN (56.6°C) (IV) and MZL-loaded SLN (56.1°C) (V), the melting point of a large amount of lipids (GMS) decreases after being formulated as SLN. This decrease can be explained by the fact that the phase transition temperature of the colloidal dispersion is always much lower than the phase transition temperature of anhydrous lipids. 2 Another explanation for the decrease in melting point is that the small PS of SLN leads to high surface energy, which creates a suboptimal energy state, resulting in a decrease in the melting peak39.

The melting peak of MZL disappeared in the thermal analysis chart of the MZL-SLN preparation, indicating that the drug was completely dissolved or uniformly dispersed in the lipid matrix after heating the lipid. Because the crystals are more organized, the space available for different molecules is smaller. These molecules interfere with the thermodynamically required crystal ordering and are responsible for higher drug retention. twenty one

From the DSC thermogram, the interaction between Mzl and GMS is not distinguished.

A 48-hour in vitro release study was completed with a modified Franz diffusion cell to illustrate the mechanism of MZL release from SLN. The in vitro release of MZL from separate drug suspensions (CL) and selected MZL-SLNs F4 formulations was performed in phosphate buffer pH 6.8 containing 0.5% w/v SLS at 37°C to maintain The sedimentation conditions are shown in Figure 7. Figure 7 Control Mizolastine suspension (CL), optimized F4-SLN, 0.1% w/v MZL-hydrogel and F4 hydrogel formula at pH 6.8 phosphate buffer The in vitro release profile in liquid containing 0.5% w/v SLS. Note: The data are expressed as mean ± standard deviation (n=3); SLS, sodium lauryl sulfate; SLN, solid lipid nanoparticles.

Figure 7 The in vitro release curve of control mizolastine suspension (CL), optimized F4-SLN, 0.1% w/v MZL-hydrogel and F4 hydrogel formulation in pH 6.8 phosphate buffer, which contains 0.5% w/v SLS.

Note: The data are expressed as mean ± standard deviation (n=3); SLS, sodium lauryl sulfate; SLN, solid lipid nanoparticles.

Generally speaking, the release of drugs from lipid-based colloidal systems is affected by several factors, including: the temperature and properties of the release medium, the drug load and the location of the drug in the particle, the size and contour of the particle, and the size and contour of the drug. The nature of the crystal arrangement and matrix lipids, stabilizers, and the organization around the particles, as well as the manufacturing method of nanoparticles. 26

The diffusion of free MZL reached 66.5±2.5% at approximately 8 hours, and approximately complete drug release within 24 hours. On the contrary, the release profile of MZL from SLN has a biphasic mode, including burst and sustained drug release, which is essential for prolonging the retention time of the drug and ensuring good efficacy. It is rapid, releasing approximately 43.7±1.3% in 8 hours, monitored by slow and continuous release reaching 71.3±2.3% in the entire 30 hours.

This biphasic release behavior may be related to the fact that the presence of hydrophobic long-chain fatty acids in lipid-forming SLN hinders drug release, because the drug must be released from the core-shell nanostructure and directly exposed to the release medium to obtain a more sustained Release mode. 40

The initial burst effect may be related to the short diffusion path of the part of the drug located on the outer shell of the sentinel lymph node. These results are closely related to 49. They proposed that the burst effect of ciprofloxacin is due to the rapid dissolution of drug molecules on the surface of the SLN.

The second stage of sustained release can be explained as that MZL is dispersed and evenly embedded in the lipid matrix, and can only be released from it through slow dissolution and diffusion.

According to a research report of 14, PS has a direct effect on the drug release profile, because smaller particles have a larger surface area and are exposed to the release medium, which is mainly due to the high initial burst effect.

At the same time, the MZL-SLN dispersed in the gel produces the smallest drug burst. The minimization of this initial burst and the slower long-term drug release may be attributed to the diffusion resistance of the gel in the semi-solid matrix and the adhesive properties of the polymer gel structure. In addition, the thickness of the diffusion boundary layer limits the release of drug molecules into the aqueous buffer. 63 However, the drug release percentages of MZL- and F4-hydrogels within 48 hours were 89.13±1.03% and 70.07±2.1%, respectively. This may be due to the diffusion of the drug from the surface of the SLN and then from the core.

The drug release data is suitable for different kinetic models. Table 5 shows the fitting parameters such as the correlation coefficient (R2) and index "n" of the Korsmeyer-Peppas equation. Table 5 Free Mizolastine (CL) and SLN-F4 formulations containing 0.5% w/ kinetic release model v SLS at pH 6.8

Table 5 Kinetic release model of free mizolastine (CL) and SLN-F4 formulations containing 0.5% w/v SLS at pH 6.8

The data is more suitable for the Higuchi model, because the R2 value is relatively larger than the values ​​of other kinetic models, CL and SLN-F4 are 0.9164 and 0.9680, respectively.

Korsmeyer-Peppas proved to be the most suitable model for further analysis of matrix-based drug dosage form release. This method needs to be used when the release mechanism is not obvious or may involve multiple release phenomena. 33

It is found that the value of the release index n observed using the Peppas model is 0.5 ≤ n ≤ 0.89, which indicates that the release curve is a pairing of diffusion and erosion mechanisms (abnormal non-Fickian transmission). 42

In addition, the drug points to an "n" value close to 0.5, which mainly shows the diffusion release of the drug. In short, drug release from the polymer matrix is ​​a diffusion control process rather than polymer erosion. These results were found to be consistent with those obtained by 49.

Finding that these results are consistent with those obtained by 55, they reported that the kinetic model of the in vitro release profile of loratadine from SLN can be described by abnormal transport or non-Fickian diffusion mechanisms.

By checking the physical appearance, PS, PDI, ZP and EE of MZL during the entire storage period of 6 months at refrigerated temperature and room temperature, the stability of the optimized SLN formulation (F4) was emphasized. During the entire storage period, we did not observe any signs of drug crystallization, phase separation or appearance changes, such as the color and odor of the SLNs preparation.

Compared with other drug delivery systems, the dimensional stability of NPs is more important because of their larger specific surface area. Compared with the size before freeze-drying (185.36±2.56 nm), notice a relatively slight increase in PS (202.3±13.59 nm).

Table 6 shows that the PS, size distribution, EE and ZP of F4 changed from 202.3±13.59 nm, 0.26±0.01, 86.5±1.47% and -22.03±3.65 mV to 209.77±6.57 nm and 0.290±44.74 nm after 6 months, respectively. 3.21% and -18.86±2.00 mV. Table 6 Stability data of MZL-SLN (F4) after storage at two different temperatures

Table 6 Stability data of MZL-SLN (F4) after storage at two different temperatures

Obviously, after F4 was stored at room temperature for 6 months, PS, PDI, EE% and ZP did not change significantly (P<0.05). This indicates the high stability and more electrostatic repulsion caused by the high energy barrier. The results of the analysis of variance clarified the insignificant deviations of these parameters from the initial parameters during the entire storage period at room temperature.

On the contrary, under refrigeration conditions, the PS of F4 hardly changes, maintaining a high ZP value during the course of this study, providing a high energy barrier and more electrostatic repulsion. Except for the 6th month of storage under refrigerated conditions, a significant increase in PS and PDI (P<0.05) and a decrease in EE and ZP values ​​were recorded.

The polymorphic transition of the lipid matrix from a metastable state to a stable form leads to the excretion of the drug from the lipid matrix, which results in an increase in PS and a decrease in encapsulation capacity. 21,26

Converting SLN into lyophilized powder can avoid aggregation of nanoparticles, thereby improving their stability. In addition, the hydrophobicity of lipid long-chain fatty acids forms imperfect crystals with many defects. Therefore, this may interrupt the recrystallization affinity, which may improve the physical stability of the prepared nanoparticles. 56,59

Therefore, the obtained results show that the stability of the freeze-dried MZL-loaded SLN (F4) after 6 months of storage has been strongly confirmed, especially in the long-term replenishment of its efficiency under environmental conditions.

There are no signs of irritation, redness, tearing or congestion caused by the formula. The eye irritation score test is performed at intervals after administration, and the score is zero. Therefore, these results indicate that the hydrogel formulation loaded with MZL-SLNs is non-irritating to the eyes.

Figure 8A shows the general appearance of a normal rabbit eye, indicating that the conjunctiva and cornea are normal with no mucus secretion, while Figure 8B shows the inflamed rabbit after 15 minutes (moderate redness) and (C) 30 minutes (severe redness) The general appearance of the eye) was instilled with histamine solution, respectively. Figure 8 Rabbit eyes with normal conjunctiva and cornea generally without mucus secretions (A), moderate hyperemia 15 minutes after histamine instillation (B), severe hyperemia after 30 minutes histamine instillation (C) hydrogel (D) , 24 hours after free drug-loaded hydrogel treatment (E) and 24 hours after MZL-SLNs hydrogel treatment (F).

Figure 8 Rabbit eyes with normal conjunctiva and cornea generally without mucus secretions (A), moderate hyperemia 15 minutes after histamine instillation (B), severe hyperemia after 30 minutes histamine instillation (C) hydrogel (D) , 24 hours after free drug-loaded hydrogel treatment (E) and 24 hours after MZL-SLNs hydrogel treatment (F).

Figure 8D illustrates the effect of the normal formulation of (sodium alginate/PVP K90) hydrogel, showing vascularized diffuse macular and conjunctival thickening, excessive mucus, and no curable symptoms. However, Figure 8E shows the effect of the tested hydrogel containing free drug on inflamed rabbit eyes, showing mild recovery of normal conjunctival mucosa and less edema.

For the hydrogel formulations containing MZL-SLN, it was observed that eye congestion and repaired conjunctiva completely disappeared after 24 hours (F).

Allergic conjunctivitis is a pathological change characterized by the accumulation of eosinophils, which are activated by the release of harmful mediators as the main basic protein, exotoxin and peroxidase, and also secrete pro-inflammatory cytokines such as IL-1α , IL-1β, IL-6 or IL-8, and chemokines that have an adverse effect on tissues. 52

Conjunctival exposure to a variety of allergens has a high incidence and is highly sensitive. It contains sebaceous glands and apocrine glands, which produce secretions that are important for the integrity and vitality of the eye. Since the allergic inflammatory response in the conjunctiva is harmful, its severity can be estimated by the number of eosinophil infiltration and other harmful white blood cells in the inflammatory response, such as plasma cells, macrophages, and tissue cells, as well as conjunctival mucosa and Pathological changes of related glandular tissues.

The normal group (Figure 9A) shows the normal mucosa with normal lining epithelium, with a thickness of about 5 to 6 layers. Goblet cells are scattered in the epithelium. The normal propria (matrix) is composed of fine fibrous connective tissue without inflammatory infiltration. Normal conjunctiva shows stratified epithelium (arrow) and normal submucosa and normal interstitial spindles forming fibroblasts (arrow). Figure 9B shows a model of allergic conjunctivitis induced by histamine instillation. The histamine treatment group showed strong inflammatory exudates, mainly macrophages, plasma cells (arrows) and eosinophilic epithelium (arrows), indicating squamous epithelial hyperplasia and desquamation. Fig. 9 Histopathological examination microscope of conjunctiva of allergic conjunctivitis model rabbits after local application of different gel preparations. Normal control (A), histamine (B), treated MZL gel (C), treated MZL-SLNs gel (D), pretreated MZL gel (E), and pretreated MZL-SLNs Gel (F). H&E, 400 times. Note: Normal conjunctiva shows stratified epithelium (arrow), normal submucosa and normal matrix spindles form fibrocytes (arrow). The histamine treatment group showed strong inflammatory exudates, mainly macrophages, plasma cells (arrow) and eosinophilic scatter covering the epithelium (arrow), which indicates squamous epithelial hyperplasia and desquamation.

Figure 9 Microscopic examination of histopathological examination of the conjunctiva of rabbits with allergic conjunctivitis after topical application of different gel preparations. Normal control (A), histamine (B), treated MZL gel (C), treated MZL-SLNs gel (D), pretreated MZL gel (E), and pretreated MZL-SLNs Gel (F). H&E, 400 times.

Note: Normal conjunctiva shows stratified epithelium (arrow), normal submucosa and normal matrix spindles form fibrocytes (arrow). The histamine treatment group showed strong inflammatory exudates, mainly macrophages, plasma cells (arrow) and eosinophilic scatter covering the epithelium (arrow), which indicates squamous epithelial hyperplasia and desquamation.

Histamine is considered to be an effective mediator released by mast cells after being activated by the allergen that causes allergic conjunctivitis. The conjunctiva exposed to histamine shows a large number of eosinophils, which are activated and degranulated where they invade the lamina propria of the conjunctival epithelium. In addition, plasma cell and tissue cell infiltration extensively infiltrate conjunctival mucosa as immunogenic cells. The matrix showed an excess of fibroblasts and inflammatory cells. The blood vessels are congested and dilated. In addition, compared with the normal group, significant proliferation and apoptosis were observed in stratified squamous epithelium.

Regarding, the third group was post-treated with free MZL hydrogel, eosinophils invaded the epithelial layer, causing degenerative changes and hyperplasia. At the same time, the MZL-SLNs loaded hydrogel post-treatment group (IV) had mild hyperplasia of the conjunctival epithelium, and eosinophils did not invade the epithelium, which was in contrast to the free drug treatment group III.

Among all groups, group V pretreated with free drug-loaded hydrogel showed inflammatory aggregates and moderate recruitment of eosinophils that invaded the conjunctival mucosa. There is less interstitial edema, fewer inflammatory cells, and vasodilation. After pretreatment of the rabbit model of allergic conjunctivitis with hydrogel loaded with MZL-SLNs (group VI), the normal conjunctival mucosa returned to normal, the thickness was close to normal, the lamina propria was normal, and there was mild eosinophil and plasma cell infiltration.

Generally, compared with the post-treatment group, the allergic conjunctivitis model rabbits pretreated with the hydrogel loaded with MZL-SLN showed improvement in ocular inflammation in both clinical and histopathological examinations, with obvious inflammatory infiltration. This is detected by a significant drop in clinical scores. The histological scores of eosinophils, plasma cells, apoptosis and mucosal epithelial thickness are shown in Table 7. Table 7 Histological scores of eosinophils, plasma cells, apoptosis and mucosal epithelial thickness after topical application in the rabbit model of allergic conjunctivitis

Table 7 Histological scores of eosinophils, plasma cells, apoptosis and mucosal epithelial thickness after topical application in the rabbit model of allergic conjunctivitis

The histological scores of eosinophils, plasma cells, apoptosis and mucosal epithelial thickness between the different groups were statistically different (P<0.05). Generally speaking, free MZL or SLN-loaded hydrogel for eye pretreatment can reduce pathological scores and alleviate allergic symptoms.

The statistical results showed that the positive control and free drug-loaded hydrogel post-treatment rabbit conjunctival eosinophil count, plasma cell count, epithelial thickness and apoptosis count increased significantly (P<0.05). Or the pretreatment group showed that the mucosal epithelium and submucosa had harmful changes due to hypersensitivity reactions.

At the same time, the post-treatment of the eye with MZL-SLNs hydrogel in the IV group showed pathological changes relative to the free drug treatment group. Over time, the eosinophil count, plasma cell recruitment and rabbit conjunctival epithelial damage decreased. Relatively reduced. Apoptosis compared with positive control.

Among them, group V and group VI were pretreated with free Mzl-loaded hydrogel and MZL-SLNs-loaded hydrogel, respectively, which were close to the normal group, with the least infiltration of eosinophils and plasma cells (Figure 10 (I and II)), except for relatively Less apoptosis and normal conjunctival thickness (Figure 10 (III&IV)). The significant difference in the severity of all pathological changes between normal control and rabbit conjunctival hydrogels loaded with MZL-SLNs may be attributed to the fact that the hydrogels containing MZL-SLNs significantly enhance the effect of MZL on rabbit histamine-induced conjunctivitis The anti-allergic activity' model. These results may be due to their nano-size range that leads to mutual enhancement of corneal absorption, improves ocular bioavailability of the ocular surface and conjunctival sac, prolongs the retention time of the eye, and provides a sustained drug release profile. Therefore, SLN can be an effective ocular drug delivery system. 4,47 Figure 10 Mizolastine pre-treatment and post-treatment affect histamine-induced eosinophil count (A), plasma cell count (B), epithelial thickness (C) increase) and apoptosis count (D) ). Note: P<0.05 *vs normal negative control group, #vs histamine positive control group, @vs post-treatment with free drug hydrogel, and $vs pre-treatment with free drug using Student's t test (unpaired).

Figure 10 The effect of mizolastine pretreatment and posttreatment on histamine-induced increase in eosinophil count (A), plasma cell count (B), epithelial thickness (C) and apoptosis count (D).

Note: P<0.05 *vs normal negative control group, #vs histamine positive control group, @vs uses free drug hydrogel post-treatment, and $vs uses Student's t test (unpaired) free drug pretreatment.

Regarding the free MZL after treatment or before treatment, these pathological changes were significantly reduced only after pretreatment with hydrogel loaded with MZL (P<0.05).

Once eosinophils migrate to the tear film, they will attach to the activated conjunctival epithelial cells through β2-integrin expressed on the surface of eosinophils. Specific up-regulated adhesion receptors, such as intercellular adhesion molecule-1 . 3

A recent study showed that conjunctival infection can cause systemic inflammation, including the induction of inflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α. It has been previously established that IL-1β is an important inflammatory cytokine and is involved in the inflammatory response to injury and autoimmune diseases. 10

In addition, it has been found that epithelial cells, inflammatory cells (eosinophils, monocytes/macrophages) and conjunctival fibroblasts produce VEGF after stimulation. 10,57 Therefore, two important biomarkers were selected in our study, namely TNF-α and VEGF.

Earlier, it was reported that MZL is effective and well-tolerated for long-term treatment of perennial allergic rhinoconjunctivitis because it has the effect of selectively blocking H1 receptors. In animal models, it has been shown to have anti-inflammatory properties. By inhibiting the release of soluble intercellular adhesion molecules 1 in the early and late stages, the effect lasts for more than 24 hours after a single administration. However, some anti-allergic effects of MZL have been observed in animal models, such as inhibiting the release of histamine from mast cells and inhibiting cell migration. 17,44

This was also reported in another study, which emphasized the fact that MZL inhibits the release of histamine from rodent mast cells and inhibits the release of soluble intercellular adhesion molecule 1, thereby preventing the chemotaxis of inflammatory cells . 35

A study investigated by 11 showed that MZL has a significant effect on early events, symptom relief, and pro-inflammatory cytokines.

Figures 11A and B demonstrate the immunolabeling against TNF-α and VEGF, respectively. Immunolabeling for VEGF and TNF-α showed that the expression of fibroblasts, lamina propria inflammatory cells and stratified squamous epithelium increased in the histamine-treated group (II), while immunostaining was negative in the normal control group (I). Figure 11 TNF-α (A) and VEGF (B) in rabbit conjunctiva of allergic conjunctivitis model after local application of different gel preparations by immunolabeling microscope: normal control (I), histamine (II), MZL after treatment Gel (III), treated MZL-SLNs gel (IV), pretreated MZL gel (V), and pretreated MZL-SLNs gel (VI). Note: Immunolabeling against TNF-α and VEGF indicates an increase or decrease in the expression of stratified squamous epithelial cells (black arrows). The blue arrow shows the expression of fibroblasts and inflammatory cells in the lamina propria.

Figure 11 The immunolabeling microscopy of TNF-α (A) and VEGF (B) in the conjunctiva of rabbits with allergic conjunctivitis after topical application of different gel preparations: normal control (I), histamine (II), treated MZL gel (III), post-treated MZL-SLNs gel (IV), pre-treated MZL gel (V), and pre-treated MZL-SLNs gel (VI).

Note: Immunolabeling against TNF-α and VEGF indicates an increase or decrease in the expression of stratified squamous epithelial cells (black arrows). The blue arrow shows the expression of fibroblasts and inflammatory cells in the lamina propria.

Figures 11A and B (III and IV) show that compared with the free MZL hydrogel and the control group, post-treatment with the Mzl-SLN-loaded hydrogel inhibited the increase in TNF-α and VEGF protein expression.

Compared with the vi group that received free MZL hydrogel (V&VI), the V group pretreated with the promising MZL-SLN-loaded hydrogel showed a significant decrease in the expression of the two biomarkers.

The statistical analysis of the Student's t-test (unpaired t-test) using the positive signal expression of TNF-α and VEGF in the two treatment regimens clarified that compared with the positive control or free MZL treatment, the eyes of rabbits receiving MZL-SLNs hydrogel It significantly reduces Figure 12A and B. Figure 12 The effect of mizolastine pretreatment and post-treatment on the increased expression of TNF-α (A) and VEGF (B) in the rabbit conjunctiva induced by histamine compared with the normal and positive control groups. Note: P<0.05 *vs normal negative control group, #vs histamine positive control group, @vs free drug hydrogel post-treatment, $vs free drug pre-treatment using Student's t test (unpaired).

Figure 12 Compared with the normal group and the positive control group, the effect of mizolastine pretreatment and post-treatment on the increased expression of TNF-α (A) and VEGF (B) in the rabbit conjunctiva induced by histamine.

Note: P<0.05 *vs normal negative control group, #vs histamine positive control group, @vs free drug hydrogel post-treatment, $vs free drug pre-treatment using Student's t test (unpaired).

Post-treatment of the eye with hydrogel loaded with free MZL has no significant effect on immunoreactivity. On the other hand, compared with the positive control, in the pre-treatment or post-treatment group, the expression of TNF-α and VEGF after MZL-SLNs hydrogel was significantly reduced (P<0.05).

In this study, it was proved that MZL significantly reduced the expression levels of TNF-α and VEGF protein in the rabbit conjunctivitis model. The results indicate that the comprehensive ability of MZL to down-regulate these proteins explains the remarkable effect of MZL-SLNs hydrogels.

Consistent with histopathological examination, the pretreatment of eyes with hydrogels loaded with MZL-SLNs is better than hydrogels loaded with free drugs, which can significantly reduce the expression of TNF-α and VEGF protein (P<0.05) than any One method produced a significant reduction (P<0.05) of free drug or post-treatment with MZL-SLNs loaded hydrogel and no significant difference from that recorded in the normal group. This advantage may be attributed to the increase in protein expression and absorption in cells and the improvement of mucosal adhesion and eye retention of such hydrogels.

A study conducted by 7 showed that sentinel lymph nodes increase the ocular bioavailability of tobramycin by increasing the residence time on the corneal surface and conjunctiva compared with the equivalent dose of tobramycin in water.

MZL-SLNs were successfully prepared by thermal homogenization and ultrasonic technology. Follow the full factorial design paradigm to adjust CQA. The optimized MZL-SLN (F4) has the highest EE% (86.5±1.47%), the smallest MPS (202.3±13.59 nm), and a reasonable ZP of -22.03±3.65 mV. Solid state characterization confirmed that MZL is well encapsulated in SLN and the drug is in an amorphous state. TEM and SEM images show the formation of spherical particles in the nanometer size range. The in vivo results of this study confirmed the ideal potential of the hydrogel loaded with MZL-SLNs to successfully reduce the symptoms of histamine-induced conjunctivitis in a rabbit eye model. In addition, compared with the post-treatment of the same formula, in rabbits with conjunctivitis, pretreatment with hydrogel loaded with MZL-SLN can reverse the abnormal regulation of inflammation and significantly reduce the expression of TNF-α and VEGF. level. In fact, the hydrogels loaded with MZL-SLNs deserve further consideration because they serve as a promising nanoparticle system for the treatment of severe non-infectious allergic conjunctivitis.

This work was funded by the author of this article.

The author reports no conflicts of interest in this work and declares that there are no competing financial or non-financial interests.

1. Ackerman S, Smith LM, Gomez PJ. Eye itching associated with allergic conjunctivitis: the latest evidence and clinical management. The Adv Chronic disease. 2016; 7(1): 52–67. doi:10.1177/2040622315612745

2. Aman RM, Abu Hashim II, Meshali MM. New chitosan-based solid lipid nanoparticles can enhance the biological persistence of the magical phytochemical "oleandrin". Eur J Pharm Sci. 2018; 124: 304-318. doi:10.1016/j.ejps.2018.09.001

3. Amin K. The role of mast cells in allergic inflammation. Respiratory medicine. 2012;106(1):9-14. doi:10.1016/j.rmed.2011.09.007

4. Bachu RD, Chowdhury P, Al-Saedi ZHF, Karla PK, Boddu SHS. Ocular drug delivery barrier-the role of nanocarriers in the treatment of anterior segment ophthalmopathy. pharmaceutics. 2018;10(28):1-31. doi:10.3390/pharmaceutics10010028

5. Bansal H, Khatry S, Arora S. Mizolastine procedure release ocular insert formulation and evaluation. Int J Pharm Sci Res. 2013; 4(1): 497–501.

6. Butani D, Yewale C, Misra A. Topical amphotericin B solid lipid nanoparticles: design and development. Colloidal surfing B Biological interface. 2016;139:17-24. doi:10.1016/j.colsurfb.2015.07.032

7. Cavalli R, Gasco MR, Chetoni P, Burgalassi S, Saettone MF. Solid lipid nanoparticles (SLN) serve as an ocular delivery system for tobramycin. Int J Pharm. 2002;238(1–2):241–245. doi:10.1016/S0378-5173(02)00080-7

8. Cavendish M, Nalone L, Barbosa T, etc. Research on the prefabrication and development of solid lipid nanoparticles containing perillyl alcohol. J Therm anal heat. 2019;141(2):767–774. doi:10.1007/s10973-019-09080-0

9. Chen R, Wang S, Zhang J, Chen M, Wang Y. Aloe-emodin-loaded solid lipid nanoparticles: formulation design and in vitro anticancer research. Drug delivery. 2015;22(5):666–674. doi:10.3109/10717544.2014.882446

10. Chen Y, Hong X. Carvedilol reduces the effect of conjunctivitis through changes in inflammation, Ngf and Vegf levels in a rat model. Exp Ther Med. 2016;11(5):1987-1992. doi:10.3892/etm.2016.3140

11. Ciplandi G, Cirillo I, Vizzacaro A. Mizolastine and fexofenadine regulate cytokine patterns after nasal allergen challenge. Eur Ann Allergy Clinical Immunology. 2004;36(4):146–150.

12. Córdova C, Gutiérrez B, Martínez-García C, etc. Oleanolic acid controls allergic and inflammatory reactions in experimental allergic conjunctivitis. Public Science Library One. 2014; 9(4): e91282. doi:10.1371/journal.pone.0091282

13. Danaei M, Dehghankhold M, Ataei S, etc. The influence of particle size and polydispersity index on the clinical application of lipid nanocarrier system. pharmaceutics. 2018;10(2):57. doi:10.3390/pharmaceutics10020057

14. Dandagi PM, Dessai GA, Gadad AP, Desai VB. Preparation and evaluation of Lornoxicam nanostructured lipid carrier (NLC). Int J Pharm Pharm Sci. 2014;6(2):73-77.

15. Date AA, Nagarsenker MS. A single-step and low-energy method for preparing solid lipid nanoparticles and nanostructured lipid carriers using biocompatible solvents. Eur J Pharm Res. 2019;1(1):12-19. doi:10.34154/2019-EJPR.01(01).pp-12-19/euraass

16. De Oliveira GGG, Feitosa A, Loureiro K, Fernandes AR, Souto EB, Severino P. Study on the compatibility of paracetamol, chlorpheniramine maleate and phenylephrine hydrochloride in physical mixtures. Saudi Journal of Medicine, 2017; 25(1): 99-103. doi:10.1016/j.jsps.2016.05.001

17. Del Cuvillo A, Sastre J, Montoro J, etc. Allergic conjunctivitis and H. J Investig Allergol Clin Immunol. 2009;19(1):11-18.

18. Deza G, Giménez-Arnau AM. Management and treatment of contact urticaria syndrome. In: Contact urticaria syndrome: diagnosis and management. Giménez-Arnau AM, Maibach HI, editor. Cham: Springer International Press; 2018:161-170.

19. Dong S, Wu X, Xu Y, Yang G, Yan M. (Immunohistochemical study of Stat3, Hif-1α and Vegf in pterygium and normal conjunctiva: experimental research and literature review). Moore Vision. 2020; 26:510-516.

20. Draize JH, Woodard G, Calvery HO. Methods to study the irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharmacol Exp Ther. 1944; 82: 377-390.

21. Dudhipala N, Veerabrahma K. Candesartan cilexetil loaded solid lipid nanoparticles for oral administration: characterization, pharmacokinetics and pharmacodynamic evaluation. Drug delivery. 2016; 23: 395-404. doi:10.3109/10717544.2014.914986

22. Emami J, Yousefian H, Sadeghi H. Targeted nanostructured lipid carrier for artemisinin brain delivery: design, preparation, characterization, optimization and cytotoxicity. J Pharm Pharm Sci. 2018; 21:225s–241s. doi:10.18433/jpps30117

23. Garud A, Singh D, Garud N. Solid lipid nanoparticles (SLN): methods, characterization and applications. Int Curr Pharm. 2012;1(11):384–393. doi:10.3329/icpj.v1i11.12065

24. Gazi AS, Sailaja AK. Preparation and evaluation of paracetamol solid lipid nanoparticles prepared by thermal homogenization method[J]. J Nanomed Res. 2018; 7(2): 152–154. doi:10.15406/jnmr.2018.07.00184

25. Ghanbarzadeh S, Hariri R, Kouhsoltani M, Shokri J, Javadzadeh Y, Hamishehkar H. The use of solid lipid nanoparticles enhances the stability and skin delivery of hydroquinone. Colloidal surfing B Biological interface. 2015;136:1004-1010. doi:10.1016/j.colsurfb.2015.10.041

26. Gordillo-Galeano A, Mora-Huertas CE. Solid lipid nanoparticles and nanostructured lipid carriers: a review that emphasizes particle structure and drug release. Eur J Pharm Biopharm. 2018; 133: 285-308.

27. Gupta S, Kesarla R, Chotai N, Misra A, Omri A. Using the experimental design of brain targeting and enhancing bioavailability, through high-pressure homogenization, a systematic method for formulating and optimizing efavirenz solid lipid nanoparticles. Biomedical Res Int. 2017;18. doi:10.1155/2017/5984014

28. Kang JH, Chon J, Kim YI, etc. Preparation and evaluation of tacrolimus loaded thermosensitive solid lipid nanoparticles for improving skin distribution. International J Nanomedicine. 2019; 14: 5381–5396. doi:10.2147/IJN.S215153

29. Karn-Orachai K, Smith SM, Saesoo S, etc. The effect of surfactants on the physical and chemical properties of solid lipid nanoparticles containing Γ-oryanol. Colloidal surfing physical and chemical engineering. 2016; 488: 118-128. doi:10.1016/j.colsurfa.2015.10.011

30. Khames A, Khaleel MA, El-Badawy MF, El-Nezhawy AOH. Natamycin solid lipid nanoparticles-continuous ocular delivery system with higher corneal permeability against deep fungal keratitis: preparation and optimization. International J Nanomedicine. 2019; 14: 2515-2531. doi:10.2147/IJN.S190502

31. Kovacevic A, Savic S, Vuleta G, Müller RH, Keck CM. Polyhydroxy surfactants used in the formulation of lipid nanoparticles (SLN and NLC): effects on size, physical stability and particle matrix structure. Int J Pharm. 2011; 406: 163-172. doi:10.1016/j.ijpharm.2010.12.036

32. Kumar R, Singh A, Garg N, Siril PF. Solid lipid nanoparticles are used to control the delivery of non-steroidal anti-inflammatory drugs with poor water solubility. Ultrasonic sonochemistry. 2018; 40: 686-696. doi:10.1016/j.ultsonch.2017.08.018

33. Kumar R, Sinha VR. Lipid nanocarriers: an effective method for ocular delivery of hydrophilic drugs (Valacyclovir). AAPS Pharmaceutical Technology. 2017; 18: 884-894. doi:10.1208/s12249-016-0575-2

34. Mandola A, Nozawa A, Eiwegger T. Histamine, histamine receptors and antihistamines in allergic reactions. Lymphatic Signs J. 2019; 6:35-51. doi:10.14785/lymphosign-2018-0016

35. Lebrun-Vignes B, Diquet B, Chosidow O. Clinical pharmacokinetics of mizolastine. Clinical pharmacokinetics. 2001;40(7):501–507. doi:10.2165/00003088-200140070-00002

36. Moghddam SMM, Ahad A, Aqil M, Imam SS, Sultana Y. The box-behnken design method was used to optimize the nanostructured lipid carrier for topical administration of nimesulide. Artif Cell Nanomedicine Biotechnology. 2017;45(3):617–624. doi:10.3109/21691401.2016.1167699

37. Müller RH, Maèder K, Gohla S. Solid Lipid Nanoparticles (SLN) for Controlled Drug Delivery-A Review of the Prior Art. Eur J Pharm Biopharm. 2000;50:161-177. doi:10.1016/S0939-6411(00)00087-4

38. Müller RH, Radtke M, Wissing SA. 'Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are used in cosmetic and dermatological preparations. Adv Drug Deliv Rev. 2002;54:S131–S55. doi:10.1016/S0169-409X(02)00118-7

39. Natarajan J, Baskaran M, Humtsoe LC, Vadivlan R, Justin A. Enhance the brain-targeting effect of olanzapine through solid lipid nanoparticles. Artif Cell Nanomedicine Biotechnology. 2017; 45: 364-371. doi:10.3109/21691401.2016.1160402

40. Niu Z, Conejos-Sánchez I, Griffin BT, O'Driscoll CM, Alonso MJ. Lipid-based nanocarriers for oral peptide delivery. Adv Drug Deliv Rev. 2016; 106:337-354. doi:10.1016/j.addr.2016.04.001

41. Petkar KC, Chavhan S, Kunda N, etc. Development of novel octanoyl chitosan nanoparticles for improving the pulmonary administration of rifampicin: optimization by factorial design. AAPS Pharmaceutical Technology. 2018;19:1758-1772. doi:10.1208/s12249-018-0972-9

42. Ritger PL, Pepas NA. A simple equation to describe solute release I. Fickian and non-Fickian releases from non-expandable devices in the form of plates, spheres, cylinders, or discs. J Control release. 1987; 5: 23-36. doi: 10.1016/0168-3659(87)90034-4

43. Sánchez-López E, Espina M, Doktorovova S, Souto EB, García ML. Lipid Nanoparticles (SLN, NLC): Overcoming the anatomical and physiological obstacles of the eye-Part II-Drug-loaded lipid nanoparticles in the eye. Eur J Pharm Biopharm. 2017; 110: 58-69. doi:10.1016/j.ejpb.2016.10.013

44. Scadding GK, Tasman AJ, Murrieta-Aguttes M, Bachert C; Riperex Research Group. Mizolastine is effective and well tolerated for long-term treatment of perennial allergic rhinoconjunctivitis. J International Medical Research. 1999;27:273-285. doi:10.1177/030006059902700603

45. Sebbag L, Allbaugh RA, Weaver A, Seo YJ, Mochel JP. Histamine-induced conjunctivitis and blood-tear barrier destruction in dogs: a model for ocular pharmacology and therapeutics. Former pharmacist. 2019;10(752):1-11. doi:10.3389/fphar.2019.00752

46. ​​Seyed YA, Shahidi F, Mohebbi M, Varidi M, Golmohammadzadeh S. The effect of different lipids on the physical and chemical properties and stability of phycocyanin-loaded solid lipid nanoparticles. Iran J Food Science and Technology. 2017;14(67):83-93.

47. Seyfoddin A, Shaw J, Al-Kassas R. Solid lipid nanoparticles for ocular drug delivery. Drug delivery. 2010;17:467-489. doi:10.3109/10717544.2010.483257

48. Shah R, Eldridge D, Palombo E, Harding I. The particle size and zeta potential are used to optimize and evaluate the stability of solid lipid nanoparticles. J Physical Science. 2014;25(1):59–75.

49. Shazly GA. Ciprofloxacin controlled solid lipid nanoparticles: characterization, in vitro release and evaluation of antibacterial activity. Biomedical Res Int. 2017; 9. doi:10.1155/2017/2120734

50. Shokry M, Hathout RM, Mansour S. Explore gelatin nanoparticles as a new nanocarrier of timolol maleate: enhanced in vivo efficacy and safe histological characteristics. Int J Pharm. 2018; 545: 229-239. doi:10.1016/j.ijpharm.2018.04.059

51. Singh M, Guzman-Aranguez A, Hussain A, Srinivas CS, Kaur IP. Solid lipid nanoparticles for ocular delivery of isoniazid: evaluation, proof-of-concept, and in vivo safety and kinetics. Nanomedicine. 2019; 14: 465-491. doi:10.2217/nnm-2018-0278

52. Stojković N, Cekić S, Ristov M, etc. Histamine and antihistamine/histamine I antihistamine. Acta Facultatis Medicae Naissensis. 2015; 32: 7-22. doi:10.1515/afmnai-2015-0001

53. Sut TN, Jackman JA, Yoon BK. The effect of NaCl concentration on bicelle-mediated SLB formation. Langmuir. 2019;35(32):10658–10666. doi:10.1021/acs.langmuir.9b01644

54. Sznitowska M, Wolska E, Baranska H, ​​Cal K, Pietkiewicz J. The influence of lipid components and surfactants on the properties of solid lipid microspheres and nanospheres (SLM and SLN). Eur J Pharm Biopharm. 2017; 110:24-30. doi:10.1016/j.ejpb.2016.10.023

55. Üner M, Karaman EF, Aydoğmuş Z. Loratadine solid lipid nanoparticles and nanostructured lipid carriers for topical application: physicochemical stability and drug penetration through rat skin. Trop J Pharm Res. 2014; 13: 653-660. doi:10.4314/tjpr.v13i5.1

56. Vivek K, Reddy H, Murthy RS. Study on the effect of lipid matrix on drug encapsulation, in vitro release and physical stability of olanzapine-loaded solid lipid nanoparticles. AAPS Pharmaceutical Technology. 2007; 8:16-24. doi:10.1208/pt0804083

57. Wang F, Xu P, Xie KC, Chen XF, Li CY, Huang Q. The influence of tumor microenvironmental factors on the expression of VEGF. Biomedical representative. 2013; 1: 539-544. doi:10.3892/br.2013.115

58. Wei CC, Kung YJ, Chen CS, et al. Retinal inflammation caused by allergic conjunctivitis promotes the progression of myopia. Biomedical Science. 2018; 28: 274-286. doi:10.1016/j.ebiom.2018.01.024

59. Westesen K, Bunjes H, Koch MHJ. The physicochemical characterization of lipid nanoparticles and the evaluation of their drug-carrying capacity and sustained-release potential. J Control release. 1997; 48: 223-236. doi:10.1016/S0168-3659(97)00046-1

60. Wissing SA, Kayser O, Müller RH. Solid lipid nanoparticles for parenteral administration. Adv Drug Deliv Rev. 2004;56:1257-1272. doi:10.1016/j.addr.2003.12.002

61. Xiong X, Song L, Chen F, Ma X. The effect of the combination of mizolastine and proteoglycan on chronic urticaria: a randomized controlled trial. Arch Dermatol Res. 2019; 311: 801-805. doi:10.1007/s00403-019-01967-0

62. Yang Ming, Chen X, Wang Yao, et al. Comparative evaluation of thermal decomposition behavior and thermal stability of powdered ammonium nitrate under different atmosphere conditions J Hazardous materials. 2017; 337: 10-19. doi:10.1016/j.jhazmat.2017.04.063

63. Yang X, Trinh HM, Agrahari V, Sheng Y, Pal D, Mitra AK. Nanoparticle-based topical ophthalmic gel formulation for sustained release of hydrocortisone butyrate. AAPS Pharmaceutical Technology. 2016;17:294-306. doi:10.1208/s12249-015-0354-5

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and include the Creative Commons Attribution-Non-commercial (unported, v3.0) license. By accessing the work, you hereby accept the terms. The use of the work for non-commercial purposes is permitted without any further permission from Dove Medical Press Limited, provided that the work has an appropriate attribution. For permission to use this work for commercial purposes, please refer to paragraphs 4.2 and 5 of our terms.

Contact Us• Privacy Policy• Associations and Partners• Testimonials• Terms and Conditions• Recommend this site• Top

Contact Us• Privacy Policy

© Copyright 2021 • Dove Press Ltd • Software development of maffey.com • Web design of Adhesion

The views expressed in all articles published here are those of specific authors and do not necessarily reflect the views of Dove Medical Press Ltd or any of its employees.

Dove Medical Press is part of Taylor & Francis Group, the academic publishing department of Informa PLC. Copyright 2017 Informa PLC. all rights reserved. This website is owned and operated by Informa PLC ("Informa"), and its registered office address is 5 Howick Place, London SW1P 1WG. Registered in England and Wales. Number 3099067. UK VAT group: GB 365 4626 36

In order to provide our website visitors and registered users with services that suit their personal preferences, we use cookies to analyze visitor traffic and personalize content. You can understand our use of cookies by reading our privacy policy. We also retain data about visitors and registered users for internal purposes and to share information with our business partners. By reading our privacy policy, you can understand what data we retain, how we process it, who we share it with, and your right to delete data.

If you agree to our use of cookies and the content of our privacy policy, please click "Accept".