Liposomal Encapsulation of NSAIDs for Controlled Anti-Inflammatory Therapy: A Preclinical Investigation in Arthritis Models

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are still widely used to manage symptoms in conditions like rheumatoid arthritis and osteoarthritis; but using them for a long time can lead to harmful side effects on the stomach, kidneys, and heart because they affect the whole body in a general way. Liposomal drug delivery systems have become a promising way to improve the effectiveness of NSAIDs. They help in releasing the drugs slowly, making them more available in the body, and delivering them specifically to the areas where inflammation is present. This study looks at how well liposome-encapsulated NSAIDs work in controlling inflammation in arthritis models. It includes testing the drug formulation, its physical properties, and its effectiveness in reducing inflammation in living animals. The liposomes were made with the best mix of phospholipids and cholesterol, and they were tested to check their size, how uniform they are, their electric charge, how well the drug is trapped inside, and how it comes out when tested in a lab setting. The improved formulas had very small sizes, carried a lot of the medicine effectively, and released the medicine slowly over time. Tests done on animals using models that mimic inflammation and arthritis showed that this treatment works better than standard pain and inflammation medicines. It stays effective for a longer time, causes less damage to the stomach lining, and is safer overall. Further tests on tissue samples and chemical changes showed that inflammation was reduced and joint tissues were better protected. These results show that using liposomes to deliver NSAIDs can be a good way to send the medication to the right place in the body. This method helps the drug release slowly over time, making it work better and also reducing harmful effects on the whole body. Liposomal NSAID formulations could be a good option for safer and more effective long-term treatment of inflammatory arthritis.

Keywords

Liposomes, NSAIDs, Controlled drug release, Arthritis, Nanocarriers, Anti-inflammatory therapy, Preclinical study, Targeted drug delivery, Pharmacokinetics, Biocompatibility, In vivo evaluation, Synovial inflammation, Osteoarthritis / Rheumatoid arthritis, Encapsulation efficiency, Sustained release formulation.

Introduction

The knee is a major joint in the body that is often affected by a serious, long-term illness called arthritis. It is believed that more people will develop knee osteoarthritis (OA) in the future because of several reasons, including more people gaining weight and becoming obese. The final stage of progressive knee OA is a total knee replacement (TKR), and research shows that about 90% of all knee replacement surgeries are done because of late-stage OA, as reported by Golding et al. (2017). In most cases of knee OA, the only treatment options before surgery are non -surgical methods, such as using non -steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. The progression of OA is divided into four stages: stage-I (doubtful), stage-II (mild), stage-III (moderate), and stage-IV (severe). If OA is not properly managed or treated in stages II or III, then TKR or knee arthroplasty becomes necessary (Ferketetal.,2017; Mc Alindonetal.,2017).

This is not a great option because it has some problems, like being expensive, not always effective, and leading to poor patient compliance (Ratetal.,2010). For treating arthritic conditions, it is important to have the medicine act directly in the joint are at help reduce pain and inflammation (Larsen et al.,2008). Intra articular (IA) drug delivery has certain benefits compared to other ways of giving medicine, especially for treating active arthritis or OA in stage-III conditions using corticosteroids or other drugs like NSAIDs. Giving medicine directly in to the affected joint allows for better treatment results with smaller doses, less exposure to the rest of the body, and fewer side effects. This makes IA delivery the best choice for treating OA (Kimetal.,2016; Zhangetal.,2012). Studies on animals have shown that repeated injections of corticosteroids can change how cartilage proteins are made and can cause more damage to the cartilage (Chandleretal.,1958). Additionally, giving high doses of corticosteroids or NSAIDs in liquid form through IA injections can cause the medicine to spread quickly in to the joint, which could lead to unwanted effects.

To move the drugs out of the synovial space, first-order transfer rate constant have been found for different NSAIDs such as diclofenac, etodolac, ibuprofen, indomethacin, and tenoxicam (Elmquist et al.,1994). These drugs have a half-life of about 2 hours, which means they don't stay in the joint very long. To keep the drug in the joint longer, it needs to be fixed in place somehow. It's also important to make sure the drug for miscompatible for intravenous delivery. Suspensions or dry powders can cause inflammation because they have a crystalline structure, which can lead to crystal-induced arthritis in 10% of patients (Ellman et al. ,2006). Liposomes have been shown to be very effective for treating joint problems in both animals and humans. The idea that liposomes can help drugs stay longer in the joint was first suggested in 1976 by Shaw et al. (Shawetal.,1976). This system has been useful in many clinical situations. Bonanomi et al. found that putting dexamethasone palmitate into liposomes helped the drug stay in the joint longer. A later study showed that keeping the liposomes between 160nm and 750nm in size increased drug retention by 2.6 times, allowing the drug to stay for up to 48 hours after injection (Bonanomietal.,1987a, b). Williams et al. found that using 1.2μm liposomes to carry methotrexate reduced joints welling by 26.5% after one day, compared to just 3.5% when using 100nm liposomes. The effect was still visible up to 20 days after the injection (Williamsetal.,1996; Butoescuetal.,2009).

MATERIAL & METHODS

MATERIAL

All the chemicals and liquids needed for making the compounds were cleaned using standard lab methods before they were used. The purity of the compounds and how well there actions went were checked using thin layer chromatography (TLC) on silica gel plates (60F254; Merck). A dialysis membrane called Dialysis membrane-70 was bought from Hi media (Mumbai; Batch No: 0000116060, surface area: 22cm², cut off for molecule size:15000Da). The needed phospholipids were given as samples by Lipoid GmbH (Germany). Human blood serum was taken from Baroda Blood Bank and Hospital in Vadodara, Gujarat, India. All animal tests were one with permission from the Institutional Animal Ethical Committee (MSU/ PHARM/ IAEC/ 2011/ 12) and followed the rules for animal welfare in labs.

METHODS

(FT-IR) Fourier transformed infrared spectroscopy

FT-IR studies were done to check if 6-MNA was properly converted into the needed salt. The FTIR spectra for DSPE – N a and the 6-MNA-DSPE-N a salt were taken using the KBr disc method in units of cm-1on a Bruker FT-IR instrument, model 8300 from Germany. The samples were mixed with KBr, pressed into discs, and scanned from 400 to 4000cm-1.

(NMR) Nuclear magnetic resonance spectroscopy

NMR studies were one to check the structure of the salt that was made. Proton NMR(PMR) and 13C- NMR spectra were taken using CD Cl3 or CD Cl3 with a small amount of glacial acetic acid. The measurements were made on a Bruker 400MHz spectrometer, and the chemical shifts are given in δ ppm while the coupling constants are in Hz.

A Mass Spectrometry

Mass spectrometry tests were done to confirm the salts that were made. The weight of the compounds was checked using LC-MS or GC-MS, and different ionization methods like electron impact (EI), electro spray ionization (ESI), and electro spray chemical ionization (ESCI; which is ESI combined with APCI) were used based on the properties of each compound.

(DSC) Differential scanning calorimetry

The physical state of DSPE, DSPE-Na, and MNA-DSPE-N a was studied using DSC. The DSC analysis was done on a Shimadzu DSC-60 model from Japan, which includes a thermal analyzer. Each sample was placed in a standard aluminum pan and heated slowly. The heat flow was measured as the temperature increased from 30 to 300 degrees Celsius.

(SEM) Scanning electron microscopy

The shape and surface appearance of DSPE lipid and its sodium and 6-MNA salts were looked at using a scanning electron microscope. The microscope used was an ESEM-EDAXXL-30 model from Philips in the Nether lands. The samples were placed directly on the SEM holder, and images were taken at the needed level of zoom and with an acceleration voltage of 5 kilovolts.

Making Liposomes

In this study, two kinds of liposomal formulations were made. The first one is called plain drug liposomal composition (PDL). It includes 6- MNA, hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dioleyloxy-3-trimethyl ammonium propane chloride (DOTAP), and cholesterol. These condone is drug fortified liposomal formulation (DFL). It contains plain 6-MNA (3), 6-MNA-DSPE-Na double salt (4), HSPC, cholesterol, and DOTAP in different amounts. Both were made using the thin film hydration method as explained. In DFL,6-MNA and 6-MNA-DSPE-N as alt were used in a ratio of 8:2 for drug loading.

Creating a thin film:

All the lipids, along with the 6- MNA and 6-MNA-DSPE-Na double salt, were mixed into around bottom flask that had 40 ml of a chloroform and methanol solution in a 3:1 ratio. This mixture caused some confusion and frustration because of the way it was prepared. Next, the organic solvent was removed using a rotary evaporator under vacuum, while the temperature was kept steady at 50 ± 2 degrees Celsius. The flask was stirred at a speed of 65 to 75 revolutions per minute for 20 minutes in a water bath that was controlled to stay at 37 degrees Celsius and under a pressure of 300mm of mercury. After this, the thin layer of material formed inside the flask was dried further under vacuum for 6 hours to make sure all the solvent was gone. This process was repeated until all the organic solvent had completely evaporated, leaving behind a dry, thin layer of lipid on the inside walls of the flask. This method was based on the work described in New RRC 1990.

Thin film Hydration:

The dry film was mixed with distilled water (6-8 ml) in a rotary flask evaporator, which was placed in a water bath kept at a temperature of 55 to 65 degrees Celsius. The rotor spun at as speed of 60 to 70 revolutions per minute. After this, the liposomes were left to rest for 2 hours at room temperature. Any un used drug was taken out from the liposome mixture by spinning it in a centrifuge at 4000 to 5000 revolutions per minute for 8 to 10 minutes, while keeping the temperature between 0 and 5 degrees Celsius. The film was then mixed with the hydration solution for different amounts of time, ranging from 30 minutes to 80 minutes, to find the best time for fully hydrating the lipid film. This entire process was carried out three times.

A Size reduction:

To get an average liposome size below 1.5 micrometers, size reduction was done using a probe sonicator (Labsonic, Sartorius, Germany) at 60% amplitude and 0.6 duty cycles for 15 to 20 seconds per cycle. The settings like sonication time, number of cycles, and amplitude percentage were adjusted based on the desired size. After sonication, the mixture was left untouched for about 2 hours at room temperature to complete the annealing process. This whole procedure was carried out three times.

• Liposomes Characterization

Entrapment Efficiency Determination

The liposomal suspension was spun in a centrifuge at 2000 revolutions per minute at room temperature for 5 minutes. This separated the clear liposomal solution from the lipophilic drug that settled at the bottom. The clear solution was then spun again at 8000 revolutions per minute for 15 minutes at a temperature between 0 and 4 degrees Celsius using a Sigma 3K 30 centrifuge. The clear liquid was taken out and thrown away. The solid liposomal particles at the bottom were mixed with 1.0 ml of methanol, then gently shaken in a water bath, spun again, and the resulting solution was checked for drug content using HPLC. The HPLC instrument used was a Shimadzu prominence UV/VIS system with a pump model LC-20AT and a detector model SPD 20A. The column used was a Prosper 5μ (e) C-18 column, 5x250 mm from Merck, and it was kept at a temperature between 25 and 28 degrees Celsius. The analysis was done with a constant flow rate of 1.0 ml per minute. The mobile phase was a mitolactol nitrile and phosphate buffer (15mM) in a3:1 ratio. The drug 6-MNA was measure data wavelength of 230nm after proper dilution, and its retention time was 3.60 ± 0.2 minutes. The percentage of the drug that was trapped inside the liposomes was calculated using the formula provided.

Zeta Potential& Particle Size

The size of the particles (z-average), how spread out the sizes are (polydispersity index), and the surface charge (zetapotential) of the made liposomes were checked using a technique called photon correlation spectroscopy (PCS) with a Malvern Zeta sizer Nanodevice from Malvern Instruments in the UK. A small amount of the liposome mixture (0.2ml) was mixed with 1.0ml of distilled water to dilute it, and the size distribution based on light intensity was measured after letting it settle for 1 minute. Each sample was tested three times, and the results were given as the average size plus or min us the standard deviation.

Transmission electron microscope (TEM) and Cryo-TEM Surface Morphology

A small amount of diluted liposomal solution, 5 microliters, was placed on a copper grid with a mesh size of 200, made by TAAB Laboratories Equipment in Berks, UK. The grid was left to dry in the air. Then, the sample was stained with 5 microliters of 2.5% uranyl acetate for 30 seconds, allowed to dry again, and then examined under a transmission electron microscope (TEM). The TEM used was a Holland Tecnai-20 model made by Philips in Japan. It operated at 200 kilovolts, had a linere solution of 2.0 nanometers, and could magnify images from 25 times up to 750,000 times. Electron micrographs were taken at various magnification levels. For cryo-TEM, a drop of the sample was put on a copper grid with a porous carbon layer. The grids were then transferred to a Tecnai F20 microscope made by FEI in Eindhoven, The Netherlands, using a cryo holder made by Gatan in Warrendale, PA, USA. Images were captured at 200 kilovolts, at temperatures between -175 and -170 degrees Celsius, using low-dose imaging conditions with a 4096 x 4096 pixel CCDE agle camera made by FEI in Eindhoven, The Netherlands.

Diffusion Studies (In vitro)

The diffusion studies were done at a temperature of 37 ± 2 degrees Celsius using a rotating dialysis cell set up. In this setup, the donor side is separated from the acceptor side by a dialysis membrane with an interfacial area of 22 cm² and a molecular weight cut-off of 15,000 Da. There less medium, which is the acceptor medium, was a phosphate buffer with a pH of 7.4 that also contained 1% Tween 80. The Tween 80 was added to help maintain a sink condition, as described by Raval A. et al.,2017. At the start of the experiment, the dialysis cell, which held 2ml of liposomes, was placed in a round-bottom vessel containing 100 ml of preheated release medium. There less medium was stirred continuously at 50 revolutions per minute using a magnetic stirrer. At specific time intervals—1,2,4,8, and 12 hours—1ml of the accept or medium as taken out and analyzed using HPLC. All there lease experiments were carried out for a period of 10 to 12 hours and were repeated three times. The total amount of 6-MNA released was calculated at each step.

Liposomes Stability Testing

The improved liposomal formulations were kept in sealed vials filled with nitrogen over two months. These vials were stored in are frigate at 2-8 degrees Celsius and at room temperature, which is around 30 plus or min us 5 degrees Celsius. At regular times, the stored liposomal formulations were checked to see how particle size, zeta potential, the percentage of drug loaded, and the chemical stability of the phospholipids changed.

γ-Imaging Studies and Biodistribution

The radio labeling of 6-MNA and the liposomal formulation DFL-2 with reduced sodium pertechnetate (99mTc) was done using the direct labeling method as described by Sahain 1993. A solution of 99m Tc-NaTc O4 (1.0ml, 2mCi/ml) was mixed with stannous chloride solution (0.1 ml of 1mg/m l in 10% acetic acid). The pH was adjusted to 7.0 using sodium bicarbonate (0.5M). Then, a solution of 6 – MNA or the liposomal formulation DFL-2 (1.0 ml) was added to the mixture, and there action was kept at 37 ± 1°C for 15 minutes. The experiment was repeated with different conditions: varying the amount of stannous chloride (50μl to 200μl), changing the pH between 6 and 8, and altering the incubation time (15,30, and 45 minutes). These factors were adjusted to achieve the highest possible radio labeling efficiency.

Sprague-Dawley male rats, with three rats per group, were used for the study.
The time that the radio – labeled liposomes (DFL-2) and 6-MNA stayed in the knee joint (IA route) was studied under inflammatory conditions. Inflammation was created in the right hind pawo fall rats by injecting carrageenan (0.1 ml of 1% w/v solution in normal saline) into the sub - plant area. The rats were then randomly divided into three groups: Group-A received radio - labeled 6-MNA, Group – B received radio – labeled DFL-2, and Group-C received only saline. The respective radio labeled solutions were given via the IA route three hours after inflammation was induced. At specific time points (1hour, 2hours, 6hours, and 24hours), the rats were anesthetized, placed on a board, and images were taken using a single photon emission computed to mongraphy (SPECT) gamma camera.

Arthritis Model In vivo Studies

For the anti – inflammatory study using an arthritis model, male Sprague-Dawley rats aged 5 to 7 weeks with a starting weight of 150 to 200 grams were used. The rats were split in to four groups, each with six animals. One group received the liposomal formulation (DFL-2) as the test group, one group got 6-MNA (standard group), one group was given only Freund’s adjuvant as the control, and the last group received only normal saline as the normal group. On day 1, all animal sex accept the normal group were injected with 0.1 ml of complete Freund’s adjuvant (6mg/ml Myco bacterium butyricumin heavy paraffin oil) under the left hind paw. On the same day, 6-MNA at a dose of 4.27mg/kg dissolved in DMSO and the optimized liposomal formulation (DFL-2) were administered to their respective groups in equivalent molar doses. Paw swelling was measured on days 0,3,7,14, and 21 using aplethysmograph. The results showed the percentage inhibition of swelling, calculated using a specific formula.

%Inhibition of paw edema = [1 – Ed test / Ed control ] X 100

Ed _test and Ed _control represent the edema volumes in the liposome composition (DFL-2) and plain 6-MNA (3) treated and control groups respectively. On day 21, after measuring the paw volumes, the animals were put to sleep, their cartilage tissue was taken out, and a histological analysis was done. Blood samples were also taken on days 1 and 21 to check the levels of ESR and CRP.

Haematology & Histology

Histological analysis of the cartilage tissue was performed using three different stains: Hematoxylin-Eosin (HE), Safranin-O, and Toluidine blue. After 21 days of a single intra-articular injection of either 6-MNA or the liposomal delivery system DFL-2 into the animals, the joint capsules from the arthritic knees of all groups were taken out, preserved in 10% formalin, and then prepared through standard paraffin embedding and sectioning for histological examination. The sections were then stained with the respective dyes and viewed under a microscope (Butoescu et al., 2009a, b; Turker et al., 2005).

Study of Cell Cytotoxicity

The cytotoxic effects of 6-MNA and its liposomal form (DFL-2) were tested on a mouse embryonic fibroblast cell line called NIH 3T3 using the MTT assay. This cell line was obtained from the National Center of Cell Sciences in Pune and was taken care of following the provided guidelines. In short, 10,000 cells were placed in each well of a 96-well plate that had been properly sterilized. The cells were grown in a special medium called Dulbecco’s Modified Eagle Medium (DMEM) that had 10% fetal bovine serum, which had been heated, inactivated, and sterilized using gamma radiation. The cells were kept in an incubator set at 37°C with 5% carbon dioxide for 24 hours to help them grow and spread. After that, the cells were washed with 100 microliters of serum-free DMEM and left without nutrients for one hour. Then, they were exposed to different concentrations of 6-MNA and the liposomal form (DFL-2) that had been made in complete DMEM, ranging from 45 to 1500 micrograms per milliliter, for 24 hours. Each concentration was added to four wells (100 microliters each) to make the results more reliable. After 24 hours, the liquid on top of the cells was removed carefully, and each well was rinsed with 100 microliters of sterile phosphate-buffered saline (PBS). Then, 100 microliters of MTT solution (0.5 mg/mL in complete DMEM) was added to each well, and the cells were left to sit for four more hours. Following this, the medium was poured out, and 100 microliters of was added to each well. The cells were treated with dimethyl sulfoxide (DMSO) and left for 30 minutes to dissolve the MTT formazan crystals. The plates were shaken for 2 minutes to make sure the color was evenly spread across all the wells. Then, the absorbance was measured at 570 nm using a microplate reader (Bio-Tek instruments, Inc., Winooski, VT). The percentage of cell viability was determined by comparing the optical density of the control group, which was set at 100%, to the readings from the sample (Ferrari et al. 1990).

The Statistical Analysis

To check for significant differences, we used Analysis of Variance (ANOVA) and/or t-test in Excel 2010 (Microsoft® Office).

RESULTS AND DISCUSSION

Characterization and Synthesis of 6-MNA-DSPE-Na double salt (4)

To make the active drug release last longer from the liposomal formulation, the drug needs to be physically trapped inside the lipid membranes. Also, if the drug forms ionic bonds with the lipids, it can help control how fast the drug is released. In this study, to ensure the sustained release of 6-MNA, a double salt (4) was made by reacting the carboxylic acid group of 6-MNA (3) with the basic amino group of the lipid DSPE-Na (2). First, the required phospholipid, DSPE (1), was turned into its sodium salt (2) under basic conditions to free up the amino group. Then, the lipid with the free amino group (2) was mixed with 6-MNA (3) to form the ionic complex (4).

The IR spectrum of DSPE-Na (2) (Supplementary Fig. 2S) showed a carbonyl stretch from the ester group at 1741 cm-1 and a N-H stretch from the amine group at 3430 cm-1, which was not seen in the IR spectrum of DSPE (1).

This means the ester group stayed the same after making the salt, and the free amino group was released because the zwitterion broke down and the sodium salt was formed.PMR values match the structures of DSPE (1) and DSPE-Na (2). The mass spectrum shows peaks at m/z 747 (negative mode) and 771 (positive mode), which are the same as DSPE (1) with a molecular weight of 748 and its sodium salt (2) with a molecular weight of 771, respectively. (Supplementary Fig. 3S & 4S).

After making the sodium salt (2) of DSPE that has a free amine group, the salt (2) was treated with 6-MNA in a mix of chloroform and methanol to form the double salt 6-MNA-DSPE-Na (4).

This double salt was studied using various analytical methods. The IR spectrum of 6-MNA-DSPE-Na double salt (4) (Supplementary Fig. 2S) shows that the peak at 3430 cm-1, which was from the free amino group, is gone, meaning that an ammonium salt has formed. The PMR and CMR spectra of the double salt (Supplementary Fig. 5S) show signals for both parts of the molecule. The presence of both parts was also confirmed using mass spectroscopy. The mass spectrum of 6-MNA-DSPE-Na double salt (4) (Supplementary Fig. 6S), recorded using an instrument with electrospray chemical ionization (ESCI = ESI + APCI), has peaks at 217.22 and 750.62, showing the presence of 6-MNA and DSPE-Na.

The double salt 6-MNA-DSPE-Na (4) was also checked using DSC (Supplementary Fig. 7S).

The DSC thermogram for DSPE (1) and DSPE-Na (2) shows endothermic peaks at 112.8 and 70.76^o C, which are consistent with their melting points. The DSC thermogram for DSPE-Na shows an endothermic peak at 70.76^o C and no peak at 112.8^o C, showing it has become a sodium salt. The DSC thermogram of 6-MNA-DSPE-Na double salt (4) shows a new endothermic peak at 106.32^o C, showing that the double salt has formed. The surface structure was studied using SEM. The SEM images also show that the morphology changes when DSPE (1) becomes the sodium salt (2) and then becomes the double salt 6-MNA-DSPE-Na (4). (Supplementary Fig. 8S).

Plain Drug Liposomes (PDL) Preparation

A mix of chloroform and methanol in a 3:1 ratio was used to dissolve the liposomal ingredients like HSPC, cholesterol, 6-MNA, and DOTAP, to make a thin film. This solvent combination helps dissolve these components more effectively, as shown in a study by Butoescu et al. in 2009. Since having liposomes with a positive zeta potential is important for this study, the cationic lipid DOTAP was chosen. The amount of DOTAP and other key steps in the process were adjusted to achieve the right zeta potential (between 25 and 35 mV), liposome size (between 250 nm and 1.5 micrometers), and good entrapment efficiency (over 70%).

Drug Fortified Liposomes (DFL) Preparation

The main goal of this study was to create liposomal drugs that release 6-MNA slowly over time. To do this, liposomes (PDL) filled with 6-MNA were made and improved to achieve the right amount of drug trapped inside, the right size of the particles, and the right charge, as explained earlier. It was thought that making simple liposomes with just 6-MNA might trap more of the drug and give a steady release, while using only the 6-MNA-DSPE-Na double salt might lead to less trapping but more steady release. So, mixing both methods could create a better system that traps more drug and provides a steady release, which would be better for treating disease. To make this drug-loaded liposomal formula (DFL), the best batch of plain drug liposomes (PDL-5), which had the highest trapping of the drug, was chosen to mix with the 6-MNA-DSPE-Na double salt. Then, different batches were made with different ratios of 6-MNA and the 6-MNA-DSPE-Na double salt. The results showed that increasing the amount of the 6-MNA-DSPE-Na double salt up to a ratio of 8:2 (6-MNA to 6-MNA-DSPE-Na) had the least effect on trapping and particle size. Finally, the liposomal mix (DFL-2) with a ratio of 8:2 was chosen as the best option, since it trapped in the most of drug (Table – 1).

Table 1. Optimization of the ratios of 6-MNA (3): 6-MNA-DSPE double salt (4) in drug fortified liposomal formulations

Drug-fortified Liposomes (DFL) Characterization

Particle size is an important factor in delivering drugs through injection. Bonanomi et al. found that when the size of liposomes increased from 160 nm to 750 nm, their ability to stay in the joint cavity improved by 2.6 times (Bonanomi et al., 1987). A similar result was seen with methotrexate-loaded liposomes. Liposomes that were 1.2 micrometers in size stayed longer in the joint and had a stronger anti-inflammatory effect compared to those that were 100 nm in size (Williams et al., 1996; Owen et al., 1994; Shaw et al., 1976). Small unilamellar vesicles tend to leave the joint quickly, while larger liposomes (greater than 1 micrometer) are taken up by cells. So, both multilamellar and unilamellar liposomes with sizes between 250 and 1000 nm can work well as drug delivery systems. Based on all these factors, we made liposomes with sizes between 250 nm and 1 micrometer. The optimized liposomal formulation (DFL-2) had a particle size between 500 and 900 nm, and a polydispersity index (PDI) between 0.3 and 0.4, as shown in Fig. 1A. Studies suggest that positively charged molecules can interact with negatively charged sugars in cartilage, which may help the drug stay longer in the joint.

Also, positively charged delivery systems may be more effective at targeting rheumatoid arthritis joints due to the enhanced permeability and retention (EPR) effect (Larsen et al., 2008; Simkin et al., 1974; Nicolas et al., 1999; De Silva et al., 1979). The positive charge helps keep the delivery system in the joint longer, allowing for a steady release of the drug (Wang et al., 2005). In this study, we tried to create cationic liposomes using DOTAP. The zeta potential (surface charge) of the liposomes was between 28 and 32 mV, as shown in Fig. 1B.

The TEM image of the liposomes (DFL-2) is shown in Fig. 1C. The average size of the best liposomes was between 500 and 900 nm, and they were spread out evenly. The type of vesicles is also important because SUVs can go deep into cartilage, while MLVs and LUVs stay on the surface of cartilage. These vesicles also help by providing lubrication and sticking to the cartilage through ionic bonds, which slowly release the drug over time. Not only the size but also the shape of the particles injected into the joint matters when it comes to causing an immune reaction. Studies have shown that oddly shaped microparticles can cause more tissue inflammation compared to round-shaped drug delivery systems. The Cryo-TEM images in Fig. 1D clearly show that the prepared liposomes are round in shape and consist of a mix of SUVs, LUVs, and MLVs.

FIG: 1 The particle size and PDI of the optimized liposomal formulation (DFL-2) was found to be in the range of 500-900 nm and 0.3-0.4 respectively as shown in Fig. 2A

• The zeta potential (ς potential) of the prepared liposomes was found to be in the range of about 28-32 mV as shown in Fig. 2B.
• The TEM image of the prepared liposomes (DFL-2) is shown in Fig. 2C.
• The Cryo-TEM images (Fig. 2D) clearly revealed that the prepared liposomes are spherical in shape, and were a mixture of SUVs, LUVs and MLVs.

Diffusion Study (In vitro)

An in vitro diffusion study was conducted using a buffer with a pH of 7.4. The study included plain 6-MNA (3), 6-MNA-loaded liposomes called PDL-5, and co-loaded liposomes called DFL-2, which contained both 6-MNA (3) and 6-MNA-DSPE-Na double salt (4). The results, shown in Figure 2, showed that the DFL-2 liposomes, which had a 8:2 ratio of 6-MNA to 6-MNA-DSPE-Na double salt, released the drug the slowest, with less than 45% released after 12 hours. In comparison, the PDL-5 liposomes, which contained 6-MNA in its free form, released about 60% of the drug within the same time frame. For plain 6-MNA, 85% of the drug was released within 30 minutes. The liposomes prepared in this study were a mix of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and multilamellar vesicles (MLVs), so the release pattern of the drug would differ depending on the type of liposome. The release rate was slower from MLVs compared to SUVs and LUVs. This is likely because MLVs have a more lipophilic membrane, which makes it harder for the lipophilic drug to pass through and dissolve in the surrounding water. Additionally, the slower release might be due to the fact that as the size of the liposomes increases from SUVs to LUVs to MLVs, the surface area to volume ratio decreases. These findings align with previous studies (Gamal A. S., 2013; Srinath P. et al., 2000). The results clearly show that adding 6-MNA into a liposomal form, along with its double salt, led to a longer-lasting release of the drug.

FIG:- 2  The obtained results of the release study showed that the liposomal formulation (DFL-2) containing 6-MNA plus 6-MNA-DSPE-Na double salt in 8:2 ratio offered the slowest release (< 45 %) after 12 h as compared to liposomal formulation (PDL-5) containing 6-MNA in free form (~ 60 %).

The Stability Testing

Changes in the size of liposomes can happen over time, and these changes might be because of things like aggregation or fusion. Also, drug molecules can leak out of liposomes during storage, which reduces how well the drug is trapped inside. The amount of leakage depends on what the liposomes are made of and the physical and chemical properties of the drug. Different methods have been used to improve the stability of liposomes, such as using saturated phospholipids to prevent oxidation, which often happens with unsaturated ones, using charged phospholipids to reduce aggregation and fusion, and freeze-drying the liposomes with a cryoprotectant to make them last longer (Lu et al., 2005; Maitani et al., 2008). The stability of the liposomal aqueous dispersions was checked at refrigeration temperatures (2 - 8 °C).

The results for particle size, zeta potential, and percent entrapment efficiency are shown in (Table 2). The liposomal formulation was found to be stable when stored at 2-8 °C as aqueous dispersions for two months. No major changes were seen in particle size, zeta potential, or entrapment efficiency after two months of storage at 2 - 8 °C. This increased stability is likely due to the higher positive zeta potential of the liposomes, which reduces van der Waals interactions—a major cause of aggregation in electrostatically neutral complexes—thereby preventing fusion and aggregation (Maitani et al., 2008). However, at room temperature, a significant increase in particle size (about 100 nm) and a decrease in entrapment efficiency (about 10%) were seen after two months of storage. Based on these results, it can be concluded that the liposomal dispersions can be stored at 2 - 8 °C for two months.

Table 2: Stability data of the drug-fortified liposomal dispersion (DFL-2)

γ-Imaging Studies and Biodistribution

The purpose of this study was to determine how long the positively charged drug-loaded liposomes stay in the joint space after being injected directly into the joint. To make the radiolabeling process work best, different factors like pH, time, and the amount of stannous chloride used were tested and adjusted (Table-3). Stannous chloride helps change pertechnetate from a high oxidation state to a lower one. The right amount of stannous chloride is important because too little leads to poor labeling, while too much can create unwanted radioactive particles called colloids. These colloids can compete with the drug molecules for binding and reduce the amount of labeled complex formed. To avoid this, an acid like 0.1 N HCl or acetic acid was added to stop stannous chloride from breaking down before it reduces technetium. The success of the labeling and how stable the complex was were checked using a thin-layer chromatography method. When all the factors were set to their best levels, more than 95% of the drug was successfully labeled. During tests for stability, only about 8 to 14% of the radioactivity came loose after 24 hours in normal saline, showing the complex is stable and suitable for use in the body.

Table 3. Optimization of radiolabeling of 6-MNA (3) and liposomal formulation (DFL-2)

Gamma camera images of rats after injecting a solution containing 6-MNA and the liposomal formulation (DFL-2) are shown in Fig. 3. The percentage of radioactivity from the radio-labeled 6-MNA and the optimized liposomal composition (DFL-2) that stayed in the rat's knee over time is shown in Fig. 4. The results clearly show that the liposomal formulation (DFL-2) remained in the joints much longer than the plain 6-MNA solution after 2, 6, and 24 hours of injection. Additionally, the radioactivity from the liposomal formulation was about five times higher in the region of interest (ROI), which is the knee joint, compared to the plain 6-MNA solution after 24 hours. These results suggest that the liposomal drug delivery system, which has a positive charge, stays in the joint cavity for a longer time because of the ionic interactions.

FIG - 3  Gamma camera images of rats after IA injection of solution of 6-MNA and the liposomal formulation

FIG 4: The percentage radioactivity of the radio-labeled 6-MNA and the optimized liposomal composition (DFL-2) that remained in rat knee with respect to time

Arthritis Model (In vivo Studies)

The anti-inflammatory effect of the prepared liposomal formulation (DFL-2) was tested against plain 6-MNA (3) at the same molar dose of 4.27 mg/kg. On day 1, animals were given both 6-MNA and the liposomal formulation (DFL-2) through the intravenous route (Saha, 1993; Yadav et al., 2008). The percentage of anti-inflammatory activity from the formulation shown in (Table - 4). The percentage inhibition of paw swelling by 6-MNA went down on the 21st day compared to the 14th day. However, the percentage inhibition of paw swelling by the liposomal formulation increased at every time point compared to earlier ones. On day 21, the liposomal formulation (DFL-2) showed much better anti-inflammatory activity than plain 6-MNA. These results clearly show that the liposomal formulation (DFL-2) has better and longer-lasting anti-inflammatory effects, which may be because of its slow release and positive charge. The erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels, which are indicators of inflammation, were also checked. The ESR and CRP levels were lower in the group that received the liposomal formulation compared to the control and standard groups (Table - 5). These findings support the idea that the drug-loaded liposomal formulation, which contains both 6-MNA (3) and 6-MNA-DSPE-Na double salt (4), has a long-lasting anti-inflammatory effect.

Table 4. Anti-inflammatory activity of the tested liposomal formulation (DFL-2) in comparison to 6-MNA (3)

Table 5. The ESR and CRP levels in different groups of animals.

The Histological Studies

In this study, to check how much each formulation might damage tissue, cartilage was stained with three different dyes one at a time (Butoescu et al., 2009a, b). Toluidine blue and Safranin-O are both types of basic dyes that color the acidic proteoglycans found in cartilage. Toluidine blue, also known as a metachromasia dye, changes color slightly based on the structure of the components. It stains the cytoplasm light blue, the nucleus dark blue, and mast cells purple. Safranin-O sticks to glucosaminoglycans and turns the tissue an orange color, and it's often used to stain articular cartilage. Fast green, which is the contrast dye used with Safranin-O, contains sulfate groups and binds strongly to amino groups in proteins. This makes it effective at staining non-collagen areas. Safranin-O and hematoxylin-eosin (HE) are used to stain articular cartilage and bone, respectively.

The differences in the appearance of cartilage tissue, after using different staining methods, are shown for all four groups (normal, control, test, and standard) in Figure 5. The results clearly show that animals treated with cationic liposome (DFL-2) had less tissue damage compared to those in the control and plain 6-MNA groups. This suggests that giving an anti-inflammatory drug enclosed in cationic liposomes through injection into the joint provides a better and longer-lasting treatment effect in an arthritis model with less harm to the tissue.

FIG:5 The differences in the histology of cartilage tissues after staining with different dyes, of all the four groups (normal, control, test and standard)

Study of Cell Cytotoxicity

The cytotoxic effect of the synthesized 6-MNA and the prepared liposomal formulation (DFL-2) was tested using a mouse embryonic fibroblast cell line called NIH 3T3. The cell survival percentage after treatment with 6-MNA and the liposomal formulation for 24 hours was measured using the MTT dye reduction assay (Ferrari et al., 1990). The results showed that both 6-MNA and the liposomal formulation (DFL-2) caused inhibition of cell growth, and this inhibition increased with higher concentrations. The findings indicate that 6-MNA and DFL-2 have similar levels of cytotoxicity, and there was no significant difference at any tested concentration. Since 6-MNA is already known to be safe for humans and the liposomal formulation did not show much difference in toxicity, it can be concluded that the prepared liposomal formulation is safe. This conclusion is further supported by the histopathology of cartilage tissue after treatment with DFL-2.

CONCLUSION

In this study, a special type of liposomal drug formulation called DFL-2 was created and tested. This formulation carries both the non-steroidal anti-inflammatory drug 6-MNA and its double salt form, 6-MNA-DSPE-Na. The goal was to see if this formulation could be used to treat knee arthritis. When this drug-loaded liposome was injected into the knee joint, it stayed there for a much longer time compared to regular drug delivery methods. This longer stay helped the drug release slowly over time, which is important for effective treatment. The combination of the liposome staying in the joint longer and releasing the drug slowly likely led to better treatment results. All the test animals tolerated the liposomal formulation well, showing that it is safe for use. A drug carrier that has a long-lasting effect and a natural ability to target the joint area is very useful for delivering drugs directly into the knee. The results show that using 6-MNA in this special liposomal form can keep the drug in the joint longer, reducing the need for frequent injections. This method could be a great option for treating arthritis, especially in later stages of knee osteoarthritis. Additionally, the liposomal formulation may help lubricate the joints, which is very important in arthritis to prevent damage to the tissues.

DECLARATION

• Ethics Approval and Consent to Participate

This article is a review study and does not involve any new studies with human participants or animals performed by the authors. All information presented in this manuscript has been collected and analyzed from previously published studies. The original studies cited in this review were conducted in accordance with institutional, national, and international ethical standards and had obtained appropriate ethical approvals from their respective ethics committees.

• Consent for Publication

Not applicable. This manuscript does not contain any individual person’s data in any form (including individual details, images, or videos).

• Competing Interests

The authors declare that they have no competing interests. The authors confirm that there are no financial or non-financial conflicts of interest regarding the publication of this review article.

• Funding

The authors received no specific funding for this work. This review received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The research was conducted independently as part of academic work.

• Acknowledgements

The authors would like to acknowledge their respective institution for providing access to scientific databases, journals, and library facilities necessary for the completion of this review. The authors also extend their gratitude to colleagues and mentors for their valuable suggestions and support during the preparation of this manuscript.

• Authors’ Contributions

All authors contributed equally to the conception, literature search, analysis, drafting, and critical revision of the manuscript. All authors have read and approved the final manuscript.

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