Assessment of Capsaicin Liposomes in a Rat Model of Diabetes-induced Peripheral Neuropathy

Assessment of Capsaicin Liposomes in a Rat Model of Diabetes-induced Peripheral Neuropathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of Capsaicin Liposomes in a Rat Model of Diabetes-induced Peripheral Neuropathy Simran saini, Rama Devi, Soumyajit Panda, kashish wilson, Nidhi Gupta, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8011851/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Diabetic peripheral neuropathy is a major complication arising from diabetes mellitus. It is accelerated by oxidative stress, inflammation, ER stress, and mitochondrial dysfunction. Capsaicin possesses antioxidant, anti-inflammatory, antidiabetic, and neuroprotective properties but exhibits limited bioavailability. The present study aimed to develop an oral capsaicin liposomal formulation and investigate its potential against the diabetic peripheral neuropathy rat model. Methods Three formulations, namely capsaicin-loaded liposomes, capsaicin-loaded chitosan-coated liposomes, and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin, were formulated and characterized. Rats were divided into nine groups in order to determine the liposomal formulation's capability by conducting several experiments. These included blood glucose level, lipid profile estimation, behavioural parameters, nerve conduction velocity, oxidative stress biomarkers, and histological examinations of tissues. Results The particle size of capsaicin-loaded liposomes, capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin was 183.45 nm, 252.90 nm and 291.4 nm, respectively. The entrapment efficiency of capsaicin in formulations varied between 69–80%. Liposomal formulations of capsaicin reduced the levels of blood glucose and lipid profile. Additionally, significant improvement was observed in nerve conduction velocity, behavioural parameters, and oxidative stress biomarkers. Histological findings demonstrated that capsaicin liposomal formulations alleviated the sciatic nerve degeneration and showed improvement in the islet cells of the pancreas. Conclusion Formulated capsaicin exhibits sciatic nerve regeneration, antidiabetic, and antioxidant properties as evidenced by increased nerve conduction velocity and effective regulation of metabolic parameters. The best outcomes were observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin. These findings support that an oral formulation of capsaicin-loaded liposomes has great potential against diabetic peripheral neuropathy. Capsaicin-loaded liposomes diabetic neuropathy behavioural analysis nerve conduction velocity oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Introduction The incidence of diabetes mellitus (DM) is gaining more attention day by day, and it frequently causes serious complications and substantial metabolic disorders [ 1 , 2 ]. Diabetic peripheral neuropathy (DPN) is a major complication of DM [ 3 ]. The global prevalence of DPN is 46.7% [ 4 ]. Peripheral neuropathy is a result of the degradation of myelin in myelinated nerves, damage to unmyelinated neurons, axonal atrophy, and other causes, leading to altered nerve conduction velocity and aberrant sensory function [ 5 ]. Hyperglycemia and hyperlipidemia impair the regulatory functions of Schwann cells, including autophagy and metabolism, and disrupt mitochondrial function through multiple signaling pathways. In the absence of the protection and support provided by glial cells, such as Schwann cells, sensory neurones are more susceptible to harm than motor neurones, particularly those located in the dorsal root ganglia [ 6 , 7 ]. On the other hand, several mechanisms are involved in the progression of diabetic neuropathy from hyperglycemia, such as advanced glycation end product formation, activation of protein kinase C pathway, mitogen-activated protein kinase activation, which leads to the reactive oxygen species (ROS) generation and inflammation. Activation of NF-кB triggers the expression of proinflammatory mediators (TNF-α, IL-1, IL-6), resulting in dysfunction of Schwann cells, a decrease in neurotrophic factor, nerve blood flow, nerve conduction velocity, myelinated fibre density, and further axonal degradation and neuronal damage [ 8 – 11 ]. DPN presents a new problem for clinicians since its symptoms have a significant impact on patients' mental health and quality of life. Due to several limitations, including major side effects and ineffectiveness, there are currently relatively few therapeutic options for diabetic neuropathy. For this reason, we still need to discover an effective cure for various neuropathy-related issues [ 12 ]. Capsaicin is the most abundant pungent bioactive compound obtained from Capsicum annuum . It exerts analgesic, antioxidant, and other pharmacological properties by binding to the TRPV1 receptor [ 13 ]. Capsaicin exhibits anti-inflammatory effects by inhibiting NF-кB and mitogen-activated protein kinase signalling pathway [ 14 ] and through the activation of the AMPK pathway, it influences lipid accumulation [ 15 ]. Capsaicin has great potential to treat neuropathic pain. Topical formulations of capsaicin are available for neuropathic pain, such as gels, creams, lotions, and ointments [ 16 ]. Despite the extensive evidence on capsaicin’s therapeutic efficacy, the utility of oral formulation of capsaicin in DPN remains unexplored. At present, liposomes have demonstrated as a promising carriers for oral delivery as they encapsulate the highly lipophilic substance within their phospholipid bilayers, increasing its water dispersibility and protecting it from deterioration in the gastrointestinal environment [ 17 ]. The liposomal encapsulation of capsaicin may also improve its low oral bioavailability owing to its potential to improve solubility, stability, and absorption. The liposome polymer surface modification, specifically chitosan coating, increases vesicle durability in biological settings and can extend systemic circulation, which promotes medication accumulation at target sites [ 18 ]. Chitosan, a biocompatible and biodegradable cationic polysaccharide, can adhere to negatively charged phospholipid head groups and produce a hydrated polymer shell that increases mucoadhesion, decreases leakage, and may facilitate tissue penetration [ 19 , 20 ]. The objective of this research was to prepare chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin to assess the efficacy in diabetic neuropathy and its effect on altering nerve conduction velocity. 2. Materials and Methods 2.1. Drugs and Chemicals Capsaicin and metformin were purchased from Sigma Aldrich, St Louis, MO, USA. Gabapentin was obtained from Ind Swift Laboratory, India. Glucose and lipid profile estimation kits were used. The additional reagents used in the study were all of analytical grade. 2.2. Preparation of Capsaicin-Loaded Liposomes The traditional thin film hydration method, as previously described [ 21 ] with minor modification, was used to prepare liposomes. In brief, accurately weighed capsaicin (7 or 14 mg, based on the preliminary trials) was mixed with soya lecithin (1.2 g) and dissolved in 20 ml of ethanol, and the mixture was sonicated until a clear solution was obtained. Cholesterol (0.2 g, corresponding to a 7:3 lecithin: cholesterol molar ratio), Tween 80 (0.8 g) and isopropyl myristate (0.8 g) were dissolved in another 20 ml of ethanol and added to the lipid phase, followed by thorough mixing. The combined solution was transferred to a round-bottom flask and subjected to rotary evaporation under reduced pressure to remove the organic solvents, resulting in the formation of a thin lipid film on the flask wall. The dried lipid film was hydrated with double-distilled water under gentle agitation to produce capsaicin-loaded liposomes. To prepare capsaicin-loaded chitosan-coated liposomes, the liposome formulation was mixed with 0.5 g of chitosan and 0.5 ml of acetic acid and stirred for 2 h, and subsequently probe-sonicated for 5 min to achieve uniform coating. Similarly, chitosan-coated liposomes co-loaded liposomes were prepared by dissolving the required amount of capsaicin, gabapentin and metformin in the organic phase and followed the above protocol. Blank chitosan-coated liposomes (without drug) were prepared using the same procedure used in capsaicin-loaded chitosan-coated liposomes. 2.3. HPLC Analysis The concentration of capsaicin, gabapentin and metformin in the prepared liposome samples was quantified separately using high-performance liquid chromatography (HPLC, Agilent 1260 infinity SEMI Preparative System) equipped with DAD detector and a C18 column (4.6 × 250 mm, 5 µm) maintained at 30°C. Detection was performed at 283 nm (capsaicin), 222 nm (metformin) and 210 nm (gabapentin). The mobile phase consisted of acetonitrile and water (50:50, v/v), delivered at a flow rate of 1.0 mL min⁻¹, with a 20 µL injection volume. The calibration curves were constructed using serial dilutions of drug samples. The retention times of capsaicin, metformin, and gabapentin are 2.17 min, 2.11 min, and 1.94 min, respectively (Supplementary Figures S1 -S3). 2.4. Characterization of Liposomes Preparation 2.4.1. Particle Size The particle size and distribution of the liposomal formulations were analyzed using dynamic light scattering on a Malvern Zetasizer Nano-ZS (Westborough, MA, USA) at 25°C. Prior to measurement, the liposomes were diluted in deionized water to achieve an appropriate concentration and to minimize particle aggregation [ 22 ]. 2.4.2. Drug Content and Entrapment Efficiency The drug content was estimated by the HPLC method. About 100 mg of liposomes were dissolved in Triton X-100, diluted with mobile phase and then kept on an orbital shaker at 100 rpm and 37°C for 24 h to determine the degree of entrapment [ 23 ]. The mixture was centrifuged for 20 min at 4000 rpm. The supernatant was retrieved and measured by HPLC. The entrapment efficiency was determined by measuring the free drug in 2 ml of liposomes by centrifugation at 3080× g for 30 min using a Millipore Amicon filter (MWCO 10 kDa, Darmstadt, Germany), and the supernatant was measured by HPLC. The percentage entrapment efficiency was determined using the standard equation described [ 24 ]. 2.5. Experimental Animals Sprague Dawley rats of either sex (male/female) having 150–250 g body weight and 6–8 weeks old were procured from Rodent Research Laboratory India Pvt Ltd, Haryana. The experimental protocol was approved by the Institute of the Animal Ethics Committee (CAF/MM(DU)/25/IAEC-3). The rats were housed at 12 hr day and night cycle at room temperature (24°C-28°C) and relative humidity of 60–70%. Regular food and water were provided to the animals, and all the hygiene conditions were maintained. All the experimental procedures were conducted between 9:00 a.m. and 5:00 p.m. All the experimental protocols were conducted according to the ethical guidelines, CCSEA for the investigation of laboratory animals and handling of experimental animals. 2.5 Experimental Groups All the animals were weighed and grouped into nine groups. Group 1 (Normal control): received normal saline (5 ml/kg). Group 2 (Positive control; SZSI): induced diabetes by streptozotocin 45 mg/kg) and on the 5th day, induction of sciatic nerve injury. Group 3 (SZSI-G): received gabapentin (30 mg/kg, 5 ml/kg). Group 4 (SZSI-M): received metformin (60 mg/kg, 5 ml/kg). Group 5 (SZSI-CH): received blank chitosan-coated liposomes (5 ml/kg). Group 6 (SZSI-CS): received low dose of capsaicin-loaded liposomes (250 µg/kg or 54 mg of liposomes, 5 ml/kg). Group 7 (SZSI-CHCSL): received low dose of capsaicin-loaded chitosan-coated liposomes (250 µg/kg, 5 ml/kg). Group 8 (SZSI-CHCSH): received high dose of capsaicin-loaded chitosan-coated liposomes (250 µg/kg, 5 ml/kg). Group 9 (SZSI- CHCSGM): received chitosan-coated liposomes co-loaded with capsaicin (250 µg/kg), gabapentin (30 mg/kg), and metformin (60 mg/kg, 5 ml/kg). 2.6 Induction of Diabetes Mellitus by STZ-NA DM was induced in experimental overnight fasted rats by single intraperitoneal injection of STZ (45 mg/kg) prepared in 0.1 M citrate buffer (pH 4.5) after i.p injection of nicotinamide (110 mg/kg) prepared in normal saline. Diabetes was confirmed on day 4 by measuring the blood glucose levels and those rats that were in the hyperglycemia stage (greater than 200 mg/dl) were selected for further study. Selected diabetic animals were subjected to induced sciatic nerve injury on day 5. 2.7 Sciatic Nerve Injury Model Selected diabetic animals were anesthetized with intraperitoneal injections of ketamine (60 mg/kg) and xylazine (10 mg/kg), and fur was removed from the left thigh. Using a sterilized surgical blade, a small cut was created along the left lateral thigh. To find the sciatic nerve, blunt dissection was done via the biceps femoris and gluteus superficialis muscles. Saline solution was used to clean the exposed sciatic nerve. From a 1 cm section of the sciatic nerve that was exposed, 0.5 cm was crushed twice at the mid-thigh level, resulting in the disruption of tissue organization in this area. After the surgery, the cut was stitched up using surgical thread and a surgical needle, and then betadine was applied to it. The animals were kept in separate cages with good ventilation and unlimited access to food and water. For a period of 6–7 days, betadine and antiseptics were topically administered to the sutured area in order to prevent infection. The surgery was performed using the procedure indicated earlier [ 25 ]. 2.8 Treatment Protocol The drug treatment was started from the 5th day onwards and this was considered as first day of treatment and continued for further 30 days. All drugs were given orally through intragastric tube. Behavioural parameters were assessed on the 4th, 15th, 24th, and 34th day of the experiment. Measurements of nerve conduction rate were taken on the 15th, 24th, and 34th days. At the end of the study, assessment of oxidative biomarkers in rat tissues was performed. 2.9 Body Weight The body weight of each animal was monitored at 0th, 4th, 15th, 24th, and 34th day using a digital weighing balance. The changes in body weight throughout the research experiment were carefully recorded. The initial body weight was measured before administering STZ, referred to as the 0th day body weight. After the diabetes confirmation, referred to as the 4th day, and during treatment, body weight was measured at different intervals, referred to as the 15th, 24th, and 34th day [ 26 ]. 2.10 Estimation of Blood Glucose Level and Lipid Profile Rats were fasted overnight, and their blood samples were withdrawn (under anesthesia) by retro-orbital puncture method at different intervals on 0th, 4th, 15th, 24th, and 34th day. The serum was separated by centrifugation at 4000 rpm for 5 min. The isolated serum was used to analyse blood glucose, triglycerides, and cholesterol, LDL, and HDL levels. 2.11 Behavioral Parameters 2.10.1. Assessment of Locomotor Activity An actophotometer test was conducted to evaluate the spontaneous motor behaviour of the rats. Every animal was monitored for a period of ten minutes in a confined square activity enclosure. The equipment automatically kept track of how many times the light beams were interrupted by animals. The beam of light was cut off as the rat started to explore. The digital recorder in the instrument automatically picked up and tallied each interruption. The digital counter stopped recording as the animal became immobile. When the animal started to walk, the action was recorded again [ 27 ]. 2.11.1 Evaluation of Mechanical Hyperalgesia The Randall–Selitto test assesses allodynia or neuropathic pain in animals by measuring the paw withdrawal threshold. Allodynia, a heightened sensitivity to pain stimuli, is caused by nerve injury. Prior to the test, all animals were subjected to 5 min of handling to become used to being handled. The test entailed applying more and more mechanical pressure on the animal's experimental limb. Pressure was exerted on the medial region of the plantar surface of the left hind paw of each rat until a withdrawal reaction was elicited. The pressure or weight given to elicit the withdrawal reaction was recorded for each animal [ 25 , 28 ]. 2.11.2 Assessment of Thermal Hyperalgesia Eddy’s hot plate was used for thermal hyperalgesia assessment. A heated plate maintained at a temperature of 53 ± 1°C was employed for animal placement. An electronic timer was utilized to record the latency of reaction, either by hind-paw licking or jumping. A time limit of 30 s was implemented to safeguard tissue against harm. Every rat had triple measurements at 10 min intervals [ 29 ]. 2.12 Nerve Conduction Velocity Measurements The nerve conduction velocity (NCV) was assessed in the sciatic nerve-injured rats. The NCV evaluation utilized an AD Instrument's 8-channel Power Lab, together with an animal nerve-stimulating electrode and needle electrodes. Animals were anaesthetized with Ketamine (90 mg/kg i.p.) and Xylazine (5 mg/kg i.p.) and secured to a board. Following the cleaning of the proximal and distal ends of the lower leg with alcohol, the electrodes were positioned. An action potential was created by attaching a stimulating electrode at the proximal end and recording from the distal end. The test was performed under the methods specified in references [ 30 ]. 2.13 Histopathological Analysis At the termination of the study, the animals were sacrificed by cervical dislocation for tissue isolation (nerve, pancreas, and brain). Flanks measuring 1.5 cm in length from the crushed location on the sciatic nerves were collected and preserved in 10% neutral-buffered formalin. Furthermore, all the sciatic nerve samples for histopathological examination were processed for H and E staining [ 25 ]. The pancreas was carefully removed, washed in ice-cold saline, and embedded in 10% formalin for histological examination [ 31 ]. The brain was harvested for oxidative stress biomarker assessment. 2.14 Assessment of Oxidative Stress Biomarkers Analysis for malondialdehyde (MDA), catalase, reduced glutathione (GSH), and superoxide dismutase (SOD) was performed using homogenized sciatic nerve, pancreas, and brain tissues. These assays were performed using the procedure indicated earlier [ 32 , 33 ]. 2.15 Statistical Analysis All the results were expressed as mean ± standard deviation (SD). The data of lipid profile, behavioral, and biochemical estimations were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Instat version 3.05. 3 RESULTS 3.1. Characterization of Liposomes 3.1.1. Particle Size The results of particle-size analysis of liposomes are presented in Fig. 1 . The observed average hydrodynamic diameter varied among the tested formulations as blank chitosan-coated liposomes (129.61 nm), capsaicin-loaded liposomes (183.50 nm), capsaicin-loaded chitosan-coated liposomes (252.90 nm), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (291.40 nm). The blank chitosan-coated liposomes showed the lowest particle size, which increased with capsaicin loading, subsequent chitosan coating, as well as co-loading. 3.1.2. Entrapment Efficiency Entrapment efficiencies also varied among tested formulations and were as follows: capsaicin-loaded liposomes (78.94%), capsaicin-loaded chitosan-coated liposomes (80.20%), capsaicin in the co-loaded chitosan-coated system (68.92%), gabapentin (55.0%), and metformin (47.0%). 3.2. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Body Weight Rats in the normal control group showed no significant difference in body weight over the study period (Fig. 2 ). However, after the administration of STZ, all diabetic rats showed a significant ( p < 0.001 ) drop in body weight compared to normal control rats by day 4. Positive control rats showed a continuous decrease in body weight (153.5 ± 3.7 g to 144.5 ± 3.3 g) from day 4 to day 34 compared to normal control rats. On day 15, among all drug-treated groups, rats subjected to gabapentin (Group 3) treatment (181.6 ± 29.0 g), chitosan-coated liposomes co-loaded with capsaicin, gabapentin and metformin (Group 9) treatment (178.3 ± 1.7 g) showed significant gain ( p < 0.01 ) in percentage (20.3%) of body weight. However, rats (Group 7) received low dose of capsaicin-loaded chitosan-coated liposomes (175.8 ± 18.6 g) began to gain weight slightly ( p < 0.05 ) compared to the positive control group (148.1 ± 2.3 g). However, a highly significant ( p < 0.001 ) improvement in body weight was observed in all drug-treated groups compared to the positive control by day 24 and day 34, respectively. On day 34, treatment (Group 7) with low dose of capsaicin-loaded chitosan-coated liposomes (234.8 ± 3.2g) and chitosan-coated liposomes co-loaded with capsaicin, gabapentin and metformin (Group 9) (241.7 ± 3.4 g) showed highly significant ( p 0.05 ) compared to gabapentin (221.6 ± 1.75g) and metformin (216.6 ± 1.6g). Administration of high dose of capsaicin-loaded chitosan-coated liposomes (Group 8, 225.3 ± 2.5 g) exhibited a highly significant increase ( p 0.05 ) compared to gabapentin (Group 3). However, no significant difference was noted in the body weight of rats treated with gabapentin and metformin in comparison with each other from day 15 to day 34. Overall, the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy in Group 9 demonstrated the highest improvement in body weight over the other drug treatments, as shown in Fig. 2 . 3.3. Blood Glucose Level and Lipid Profile Estimation 3.3.1. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Blood Glucose Level Blood glucose level was increased significantly ( p < 0.001 ) in all experimental groups except the normal control after the induction of STZ at day 4, and levels were drastically increased from 301.3 ± 2.2 to 310.1 ± 2.2 mg/dl by the 34th day, as shown in the positive control group (Fig. 2 ). Continuous administration of metformin and capsaicin-loaded liposome formulations for 30 days reduced the level of blood glucose significantly ( p < 0.001 ) as compared to the positive control. Compared to metformin, the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group demonstrated the highest improvement in blood glucose level over the other treated drugs from 300.4 ± 1.8 to 130.9 ± 2.6 mg/dl during the treatment phase. On the 34th day, a more significant drop ( p < 0.001 ) in glucose level was observed from all drug-treated groups except gabapentin and the blank chitosan-coated liposomes group compared to the positive control. However, the blank chitosan-coated liposomes group (303.0 ± 1.8 mg/dl) showed less significant ( p 0.05 ) compared to the positive control. Treatment with capsaicin-loaded liposome (170.5 ± 5.0 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (141.9 ± 1.5 and 133.3 ± 2.6 mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (130.9 ± 2.6 mg/dl) showed more significant reduction ( p 0.05 ) was observed with high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin. Moreover, the metformin-treated group demonstrated a marked reduction in glucose level than the gabapentin-treated group, as shown in Fig. 2 . 3.3.2.Effect of Capsaicin-Loaded Liposome Individually and in Combination on Triglyceride Levels Figure 3 illustrates the triglyceride levels in all experimental animals throughout the study. However, after the administration of STZ, all rats demonstrated an increasing level of triglycerides with a significant difference ( p < 0.001 ) as compared to normal control rats at day 4. These increasing levels are found to decrease on continuous treatment with drugs over the study period. On 15th day, metformin (189.1 ± 4.5 mg/dl), capsaicin-loaded liposome (206.9 ± 7.1mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (185.3 ± 1.9 and 193.4 ± 3.8 mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group (182.9 ± 3.8 mg/dl) showed more significant reduction ( p < 0.001 ) in triglyceride level compared to the positive control (225.8 ± 4.3 mg/dl). However, compared to the positive control, a slight improvement ( p < 0.05 ) was observed with the blank chitosan-coated liposomes group by day 24, which became significant ( p 0.05 ) in triglyceride level over the study period. On day 34, treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes (135.9 ± 2.1 and 143.9 ± 2.4 mg/dl) exhibited no significant difference ( p > 0.05 ) and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (123 ± 1.2 mg/dl) showed highly significant ( p < 0.001 ) improvement in triglyceride level compared to metformin-treated group (139.3 ± 4.2 mg/dl). Moreover, all capsaicin-loaded liposome administered groups showed marked reduction ( p < 0.001 ) in triglyceride level compared to the gabapentin-treated group. 3.3.3. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Total Cholesterol Levels In normal control rats, cholesterol levels were at their basal range, but they were constantly increased in positive control rats over the study period. However, it was found that the total cholesterol level among all drug-treated groups had declined, whilst the positive control group showed an increment from day 4 to day 34 (192.9 ± 6.1 to 203.5 ± 7.3 mg/dl). Compared to the positive control, a highly significant ( p < 0.001 ) reduction was observed in the metformin-treated group, whereas high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin exhibited a significant ( p < 0.01 ) drop in cholesterol level on day 15, which became more significant ( p < 0.001 ) by day 24. On day 34, rats subjected to metformin (131.7 ± 5.0 mg/dl), capsaicin-loaded liposome (148.9 ± 10.4 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (137.6 ± 15.2 and 140.1 ± 2.7mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (131.3 ± 3.5 mg/dl) showed marked reduction ( p 0.05 ) and blank chitosan-coated liposomes group (186.1 ± 2.4 mg/dl) exhibited less significant ( p 0.05 ) was observed with low and high doses of capsaicin-loaded chitosan-coated liposomes, and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treated group. However, all drug-treated groups except blank chitosan-coated liposomes showed more significant ( p < 0.001 ) reduction in cholesterol level compared to the gabapentin-treated group, as shown in Fig. 3 . 3.3.4. Effect of Capsaicin-Loaded Liposome Individually and in Combination on LDL Levels As depicted in Fig. 3 , LDL levels were increased drastically in positive control rats as compared to normal control rats throughout the study period. Continuous administration of all treated drugs except gabapentin and blank chitosan-coated liposomes showed a marked reduction ( p 0.05 ) during the treatment phase, whereas the blank chitosan-coated liposomes group showed less significant (p < 0.01 ) improvement compared to the positive control on day 34. On 34th day, in contrast to metformin (106.2 ± 12.4 mg/dl), treatment with blank chitosan-coated liposomes (188.2 ± 9.3 mg/dl), capsaicin-loaded liposome (147.9 ± 10.9 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (133.7 ± 9.4 and 146.3 ± 10.8 mg/dl) showed highly significant difference ( p < 0.001 ), although chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin group (129.7 ± 2.8 mg/dl) exhibited less significant difference ( p < 0.01 ) in reducing the LDL level. Moreover, all capsaicin-loaded liposome administered groups showed more significant ( p < 0.001 ) reduction in LDL level compared to the gabapentin treated group. 3.3.5. Effect of capsaicin-loaded liposome individually and in combination on HDL levels HDL levels were drastically decreased in all experimental groups as compared to the normal control on day 4. However, positive control rats showed a continuous decrease in level from day 4 to day 34. On 15th day, metformin (26.6 ± 1.8 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (23.2 ± 2.3 and 24.4 ± 2.7 mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (25.9 ± 3.2 mg/dl) showed highly significant improvement ( p < 0.001 ), whereas capsaicin-loaded liposome (22.1 ± 1.8 mg/dl) exhibited less significant ( p < 0.01 ) increase in HDL level compared to positive control (16.3 ± 0.9 mg/dl). Continuous administration of treated drugs improved the HDL level by day 24 and day 34, respectively, although treatment with gabapentin exhibited no significant improvement during the interval of time. On the 34th day, a highly significant ( p < 0.001 ) improvement was observed in all drug-treated groups, except gabapentin, as compared to the positive control group (12.6 ± 2.0 mg/dl). Administration of capsaicin-loaded liposome (28.3 ± 2.0), low and high doses of capsaicin-loaded chitosan-coated liposomes (36.1 ± 1.6 and 39.4 ± 4.8), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (41.3 ± 2.0) showed a highly significant ( p < 0.001 ) increase in HDL levels as compared to the gabapentin-treated group (17.3 ± 3.2 mg/dl). However, in contrast to metformin (41 ± 3.4 mg/dl), low dose of capsaicin-loaded chitosan-coated liposomes showed less significant difference, while high dose of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin demonstrated no significant difference ( p > 0.05 ), as shown in Fig. 3 . 3.4. Behavioral Analysis 3.4.1 Effect of Capsaicin-Loaded Liposome Individually and in Combination on Locomotor Activity Using Actophotometer Locomotor activity in all groups of experimental rats was drastically decreased compared to the normal control group rats after the administration of STZ on day 4. Administration of sciatic nerve injury on day 5 in the STZ-induced diabetes rats showed a reduction in locomotor behavior in all drug-treated rats with a highly significant difference ( p < 0.001 ) on day 15 compared to day 4. On day 15, groups treated with gabapentin (163.4 ± 2.1), low and high doses of capsaicin-loaded chitosan-coated liposomes (172.5 ± 11.4 and 162.8 ± 8.3), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (185.6 ± 18.2) showed a highly significant increase ( p < 0.001 ) as compared to positive control group (132.2 ± 6.6), whereas metformin (140.2 ± 5.5), blank chitosan-coated liposomes (133 ± 4.8) and capsaicin-loaded liposome (145.8 ± 4.2) exhibited non-significant results. However, maximum improvement was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin as compared to individual therapy of gabapentin and metformin. Furthermore, all drug-treated groups except blank chitosan-coated liposomes exhibited the highest increment ( p < 0.001 ) in locomotor behavior compared to the positive control group on day 24 and day 34, respectively. On the 34th day, low dose of capsaicin-loaded chitosan-coated liposomes (267 ± 3.6) exhibited less significant ( p < 0.01 ) and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treated group (287.2 ± 4.1) showed highly significant ( p < 0.001 ) improvement in comparison with the gabapentin-treated group (252.2 ± 4). In comparison with the metformin-treated group (191.2 ± 3.8), a large improvement ( p < 0.001 ) was observed (79.9%) with capsaicin-loaded liposome (226 ± 5.7), low and high doses of capsaicin-loaded chitosan-coated liposomes, and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group (128.6%). Moreover, rats subjected to the gabapentin-treated group exhibited a more significant increment ( p < 0.001 ) in locomotor activity compared to the metformin-treated group, as shown in Fig. 4 . 3.4.2 Effect of Capsaicin-Loaded Liposome Individually and in Combination on Mechanical Hyperalgesia via Randall Selitto The diabetic neuropathy affected the pain threshold of the rat and exhibited allodynia; therefore, a highly significant reduction ( p < 0.001 ) was observed in paw withdrawal threshold when pressure was applied through Randall Selitto in all experimental groups as compared to the normal control on day 4. Rats subjected to the positive control group produced allodynia at a significant difference ( p < 0.001 ) compared to normal control rats from day 15 to day 34. Continuous administration of the standard drugs (gabapentin and metformin) and liposomal drug formulations, except the blank chitosan-coated liposomes group, enhanced the paw withdrawal threshold significantly during the treatment phase. On day 34, all drug-treated groups exhibited a highly significant improvement ( p < 0.001 ), whereas the blank chitosan-coated liposomes group (3.1 ± 1.2 g) showed a non-significant difference compared to the positive control group (2.2 ± 1.4 g). However, chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (22.4 ± 1.3 g) showed a significant ( p < 0.01 ) increment in paw withdrawal threshold than the gabapentin-treated group (18 ± 2.2 g). In comparison with the metformin-treated group (11.1 ± 2.1 g), treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes (19 ± 1.8 g and 17 ± 1.4 g), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin exhibited a highly significant ( p < 0.001) increase in paw withdrawal threshold. Moreover, the gabapentin-treated group attenuated allodynia more significantly ( p < 0.001 ) than the metformin-treated group, as shown in Fig. 4 . 3.4.3. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Thermal Hyperalgesia via Eddy’s Hot Plate The duration was employed for each animal to exhibit a response (paw licking or jumping) to heat, or the latency period was recorded. The animals showed a prolonged latency period due to a disrupted sciatic nerve. As depicted in Fig. 5 , on day 34, paw licking latency duration among all drug-treated groups except the blank chitosan-coated liposomes group was reduced over time when compared with the positive control group (280.4 ± 0.8 s). Treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes (10.8 ± 3.0 s and 12.8 ± 4.4 s), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (9.1 ± 0.9 s) showed no significant difference ( p > 0.05 ) as compared to the gabapentin-treated group (8.6 ± 1.0 s). However, chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin and low-dose capsaicin-loaded chitosan-coated liposomes exhibited highly significant ( p < 0.001 ) and at high dose of capsaicin-loaded chitosan-coated liposomes showed less significant ( p < 0.01 ) reduction in latency duration when compared to the metformin-treated group (21.3 ± 4.4 s). Additionally, in paw jumping latency time, positive control rats showed a higher latency period as compared to normal control rats throughout the experimental study. On 34th day, administration of gabapentin (9.5 ± 2.6 s), capsaicin-loaded liposome (15.8 ± 5.1 s), low and high doses of capsaicin-loaded chitosan-coated liposomes (13.6 ± 3.8 s, and 14.1 ± 3.1 s), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (10.4 ± 2.8 s) showed highly significant ( p < 0.001 ), whereas metformin (23 ± 1.7 s) exhibited slightly improvement ( p 0.05 ) improvement in latency duration as compared to positive control (29.3 ± 0.6 s). Treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin demonstrated non-significant results ( p > 0.05 ) as compared to gabapentin, whilst in comparison with the metformin-treated group, these groups showed highly significant ( p < 0.001 ) improvement in prolonged latency duration, as shown in Fig. 5 . Notably, marked improvement ( p < 0.001 ) in both paw licking and paw jumping latency time was recorded in gabapentin-treated rats over the metformin-treated rats. 3.5. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Nerve Conduction Velocity Nerve conduction velocity in normal control rats was 89.9 ± 3.4 m/s on day 15th, whilst 27.6 ± 1.2 m/s in positive control rats, which was decreased to 23.3 ± 0.6 m/s over the study period. Administration of gabapentin (76.6 ± 3.7 m/s), capsaicin-loaded liposome (58.2 ± 2.6 m/s), low and high doses of capsaicin-loaded chitosan-coated liposomes (67.3 ± 5.8 and 64.5 ± 3.4), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (79.1 ± 3.8 m/s) showed highly significant ( p < 0.001 ) improvement, whereas blank chitosan-coated liposomes group (30.2 ± 3.4 m/s) showed slightly improvement ( p < 0.05 ) in nerve conduction velocities as compared to the positive control group on 34th day. Treatment with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed maximum attenuation over the other drug-treated groups, and exhibited a non-significant ( p > 0.05 ) result when compared with the gabapentin-treated group. Moreover, no significant improvement ( p > 0.05 ) was observed in the metformin-treated group in comparison with the positive control throughout the experimental study, as shown in Figs. 6 and 7 . 3.6 Assessment of Oxidative Stress Biomarkers 3.6.1. Effect of Capsaicin-Loaded Liposome Individually and in Combination on MDA of Rat Organs (Sciatic nerve, Pancreas, and Brain) MDA level in the sciatic nerve (6.9 ± 0.3 nM/mg protein), pancreas (6.5 ± 0.09 nM/mg protein), and brain (9.0 ± 0.9 nM/mg protein) of positive control rats increased significantly ( p < 0.001 ) compared to normal control rats. Continuous administration of treated drugs except gabapentin and blank chitosan-coated liposomes attenuated these increasing levels more significantly ( p < 0.001 ) in the sciatic nerve, pancreas, and brain compared to the positive control. On the other hand, blank chitosan-coated liposomes group showed highly significant decrease ( p < 0.001 ) in sciatic nerve (6.3 ± 0.1 nM/mg protein) and exhibited less significant decrease ( p < 0.01 ) in both pancreas (5.0 ± 0.5 nM/mg protein) and brain (7.7 ± 0.4 nM/mg protein), whereas gabapentin treated group showed a slight reduction ( p < 0.05 ) in MDA level in rat organs (nerve, pancreas, and brain). In contrast to metformin, rats subjected to chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group showed less significant difference ( p 0.05 ) in pancreas and brain, but showed significant difference ( p < 0.01 ) in sciatic nerve MDA level. Moreover, a highly significant reduction ( p < 0.001 ) in MDA level was observed in the metformin-treated group compared to the gabapentin-treated group in rat organs. Overall, the highest improvement was noted with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin group in the sciatic nerve, whereas in the pancreas and brain, low dose of capsaicin-loaded chitosan-coated liposomes showed more reduction in MDA level among all liposomal formulations, as shown in Fig. 8 . 3.6.2. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Catalase Activity in Rat Organs (Sciatic nerve, Pancreas, and Brain) From Fig. 9 , it was observed that the catalase activity in positive control rats was decreased significantly ( p < 0.001 ) in the sciatic nerve (3.7 ± 0.3 U/mg protein), pancreas (11.5 ± 1.1 U/mg protein), and brain (30.2 ± 3.7 U/mg protein), compared to normal control rats. On continuous administration of treated drugs, catalase activity was restored in rat organs (nerve, pancreas, and brain). Compared to the positive control group, treatment with metformin, and all capsaicin-loaded liposome administered groups exhibited highly significant improvement ( p < 0.001 ) in sciatic nerve, pancreas, and brain catalase activity. However, the gabapentin-treated group showed a slight increase ( p < 0.05 ) in rat organs (nerve, pancreas, and brain), whereas the blank chitosan-coated liposomes group exhibited a highly significant increase ( p < 0.001 ) in pancreas, and showed less significant improvement ( p < 0.01 ) in sciatic nerve and brain catalase activity. Compared to the gabapentin-treated group, a more significant increase was observed with the metformin-treated group in rat organs (nerve, pancreas, and brain). In comparison to the metformin-treated group, a highly significant increase ( p < 0.001 ) was noted with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin in the sciatic nerve (17.6 ± 0.7 U/mg protein), and no significant difference was observed in pancreas (32.4 ± 0.6 U/mg protein) catalase activity. Furthermore, in brain catalase activity, treatment with low dose of capsaicin-loaded chitosan-coated liposomes (51.2 ± 1.6 showed less significant difference ( p < 0.05 ) compared to the metformin-treated group (56.2 ± 4.4 U/mg protein). Overall, in the sciatic nerve and pancreas, chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed maximum improvement, whereas in the brain, the highest improvement was observed with low dose of capsaicin-loaded chitosan-coated liposomes. 3.6.3 . Effect of Capsaicin-Loaded Liposome Individually and in Combination on GSH in Rat Organs (Sciatic nerve, Pancreas, and Brain) Positive control rats demonstrated a highly significant reduction ( p < 0.001 ) in GSH activity in rat organs (nerve, pancreas, and brain) as compared with normal control rats. All drug treatment groups showed a highly significant increment ( p < 0.001 ) in GSH level, whereas the gabapentin-treated group showed a slight ( p < 0.05 ) and the blank chitosan-coated liposomes group showed lesser significant improvement ( p < 0.01 ) compared to the positive control rat organs (nerve, pancreas, and brain). Treatment with low doses of capsaicin-loaded chitosan-coated liposomes showed maximum improvement over other liposomal-treated groups with no significant difference ( p > 0.05 ) compared to the metformin-treated group in brain and pancreas GSH activity, whereas in the sciatic nerve, the highest improvement was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group among other liposomal-treated groups and showed no significant difference ( p > 0.05 ) compared to the metformin-treated group, as shown in Fig. 10 . 3.6.4 . Effect of Capsaicin-Loaded Liposome Individually and in Combination on SOD in Rat Organs (Sciatic nerve and Pancreas) Rats in the positive control group showed a marked reduction ( p < 0.001 ) in SOD activity compared to the normal control group. It was found that all drug-treated groups demonstrated highly significant ( p < 0.001 ) improvement in SOD activity, whereas the blank chitosan-coated liposomes group and gabapentin-treated group showed less improvement ( p < 0.05 ) compared to the positive control in the sciatic nerve and pancreas. However, the metformin-treated group exhibited a more significant increment in SOD activity than the gabapentin-treated group in both organs. In contrast to the metformin-treated group, treatment with low dose of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed no significant difference ( p > 0.05 ) in nerve and pancreas SOD activity. Overall, maximum increment was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group over the other liposome-treated groups in both sciatic nerve and pancreas GSH activity, as shown in Fig. 11 . 3.7 Histopathological Evaluation of Sciatic Nerve and Pancreas 3.7.1. Histopathological Evaluation of Sciatic Nerve No pathological changes were observed in the normal control group of the sciatic nerve (Fig. 12 ). The transverse section of the sciatic nerve demonstrated a collection of nerve fiber bundles, each surrounded by connective tissue, endoneurium, and encased with perineurium and epineurium. It contains axons of various sizes surrounded by myelin sheath and separated by an endoneurial connective tissue. However, in the diabetic control group, the sciatic nerve showed shrinkage, demyelination, necrosis, and degenerated nerve fibers surrounded by thin perineurium. Focal loss of endoneurium was also observed due to destructive reactions. Treatment with low dose of capsaicin-loaded chitosan-coated liposomes reduced the disturbance in morphological structure; only a few inflammatory cells were seen as compared to the high dose treated group. Blank chitosan-coated liposomes group of sciatic nerve showed axonal regrowth after treatment. The chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin formulation showed improvement in the regeneration of nerve fibers, where endoneurium was clearly seen. In addition, this formulation showed a better orderly arrangement in nerve fiber bundles. It also recovered the axons with the myelin sheet. 3.7.2. Histopathological Evaluation of Pancreas In the normal control group, the cells of the pancreas were all present in their normal proportions (Fig. 12 ). The duct was clearly seen. The islet cells are seen embedded within the acinar cells and surrounded by a fine capsule. In the positive control group, the islets were damaged, shrunken in size, and infiltration of lymphocytes was observed. The islet cells are seen to be normal in position. The size of the cell is to be back in its normal position after treatment with metformin and gabapentin. The islets of the blank chitosan-coated liposomes-treated group showed improvement after treatment. The islet cells are seen to be in normal position and more in numbers, which were also observed in the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group, as compared to their individual treatment (Fig. 12 ). 4. Discussion The incidence of diabetes is increasing more rapidly. A study reported that there are roughly 537 million individuals with diabetes globally and estimated that it will increase to 783 million by 2045 [ 2 ]. During DPN, the peripheral nerve exhibits insulin resistance, and the insulin receptors on Schwann cells and axons become unresponsive to insulin. Insulin resistance impairs Schwann cell and axonal metabolism, which leads to neuronal demyelination and neuronal damage [ 34 ]. Loss of myelin in myelinated nerves and unmyelinated nerve injury are the factors that affect nerve conduction velocity and abnormal sensory function. Hyperglycemia activates the polyol pathway, hexosamine pathway, protein kinase C pathway, and more metabolic pathways, causing detrimental effects on neurons, which result in inflammation and oxidative stress, ultimately leading to neuronal damage [ 3 ]. The infiltrating macrophages within peripheral nerve cells stimulate the production of cytokines and chemokines, hence exacerbating inflammation and causing nerve fibre injury [ 34 ]. Various natural compounds have been targeted for the management of DPN, offering effective, safe, and beneficial therapeutic strategies with fewer side effects. Polyphenols, flavonoids, and other herbal extracts play a crucial role in addressing the underlying pathophysiological mechanisms of nerve damage [ 35 – 37 ]. Novel formulations, such as nanoparticle-based delivery systems, have been explored that accelerate progress in the field by enhancing the bioavailability and therapeutic approaches [ 38 , 39 ]. Capsaicin has great potential to treat neuropathic pain and the possible mechanisms of action are depicted in Fig. 13 . Nanotechnology-based formulations such as emulsions and liposomes are the drug-delivery systems that are being explored for topically administered capsaicin [ 16 ]. The limited bioavailability of capsaicin impedes future researchers in their formulations and significantly restricts clinical uses. Consequently, it is imperative to pursue an appropriate drug delivery strategy that may enhance both the solubility and bioavailability of capsaicin concurrently. Based on previous research, our current study looked at the protective effect of an oral capsaicin-loaded liposome in diabetes-induced peripheral neuropathy. The changes in particle size and entrapment efficiency are internally consistent and compatible with the biophysical behavior that is anticipated for polymer-coated vesicles and mixed lipophilic/hydrophilic payloads. The possible explanation for the increase in particle size in capsaicin-loaded liposomes is due to the drug’s lipophilic nature, which is expected to partition into the bilayer, increasing bilayer thickness and decreasing membrane curvature. Subsequent chitosan coating of the capsaicin-loaded liposomes further increases the size to 252.90 nm due to the formation of hydrated polymer shell around the vesicles. The largest mean diameter (291.40 nm) was observed for the co-loaded chitosan-coated liposomes, because the internal aqueous volume would have increased as a result of co-loading with hydrophilic small molecules (metformin and gabapentin), which may lead to fusion events that increase the population of bigger particles, changed curvature, or swelling of the vesicle lumen. Entrapment efficiency results support the physicochemical partitioning theory. Capsaicin-loaded liposomes achieved the highest entrapment efficiency at 78.94%, indicating efficient incorporation of the highly lipophilic capsaicin into the phospholipid bilayer. The capsaicin-loaded, chitosan-coated liposomes exhibited a slightly higher entrapment efficiency (80.20%), suggesting that the positively charged chitosan coating may stabilize the lipid bilayer and reduce drug leakage during post-processing. This observation is consistent with previous findings reported [ 40 ]. However, co-loading gabapentin and metformin with capsaicin led to a decrease in capsaicin entrapment (68.92%), while gabapentin and metformin showed moderate entrapment of 55% and 47%, respectively. This reduction in entrapment efficiency could be due to the presence of two additional hydrophilic drugs that likely competed for aqueous-core and bilayer interface spaces, disrupting optimal lipid packing and reducing the hydrophobic compartment available for capsaicin. In another investigation, the particle size and entrapment efficiency of capsaicin-loaded liposomes were found to be 52.2 nm and 81.9% [ 21 ]. Diabetes was induced by intraperitoneal injection of STZ. The dose of capsaicin administered (250 µg/kg or 500 µg/kg) was based on an earlier study based on lipid multi-particulate formulation in rats, wherein the dose was 0.2 to 1 mg/kg [ 41 ]. The current study showed a significant increase ( p < 0.001 ) in body weight compared to the positive control. Low dose of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy found to increase maximum body weight and showed a highly significant difference ( p < 0.001 ) compared to gabapentin and metformin. In our study, all the capsaicin-loaded liposomal preparations showed highly significant ( p < 0.001 ) reduction in the increasing levels of glucose and triglyceride compared to the positive control from day 15 to day 34. In order to decrease glucose and triglyceride levels, the most effective outcomes were achieved with chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy, with the values of glucose (130.9 mg/dl) and triglyceride (123.0 mg/dl) levels. The glucose level from this therapy showed no significant difference ( p > 0.05 ), whereas the value of triglyceride showed 11.7% more reduction ( p < 0.001 ) than the metformin-treated group (glucose: 130.8 mg/dl and triglyceride: 139.3 mg/dl) by the 34th day. On the other hand, a highly significant improvement ( p < 0.001 ) was observed in total cholesterol, LDL, and HDL levels from all capsaicin-loaded liposome-treated groups compared to the positive control from day 24 to day 34. Treatment with chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin exhibited the highest improvement and showed a statistically significant difference ( p 0.05 ) in total cholesterol and HDL levels compared to the metformin-treated group. Blank chitosan-coated liposomes group showed less significant difference ( p < 0.01 ) in glucose, triglyceride, total cholesterol, and LDL levels, whereas it exhibited highly significant improvement ( p < 0.001 ) in HDL levels compared to the positive control on day 34. Comparable findings were documented in earlier research involving capsaicin-encapsulated nano-emulsions targeting obesity and chitosan microparticles infused with antidiabetic medication formulations for the management of DM [ 42 , 43 ]. Research indicated that chitosan has a beneficial impact on DM. The efficacy of chitosan microparticles containing antidiabetic pharmaceuticals was enhanced compared to regular medications (metformin and glibenclamide) [ 43 ]. Multiple research studies on DPN indicated that rats with sciatic nerve injury displayed various behavioural changes, including reduced locomotor activity, prolonged heat tolerance delay, development of allodynia, and diminished nerve conduction velocities. As the sciatic nerve experiences ongoing regeneration, these alterations will demonstrate a proportional response [ 25 , 44 ]. In our current study, all capsaicin-loaded liposome administered groups demonstrated highly significant improvement ( p < 0.001 ) in locomotor activity and paw withdrawal threshold compared to the positive control from day 24 to day 34. The best results were obtained with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy, which showed more improvement than the gabapentin-treated group by 13.8% in locomotor activity and 24.4% in paw withdrawal threshold. The comparable outcomes were achieved with low and high doses of capsaicin-loaded chitosan-coated liposomes in paw withdrawal threshold, whereas in locomotor activity, the low dose of capsaicin-loaded chitosan-coated liposomes showed more increase than the gabapentin-treated group, with a significant difference ( p < 0.01 ) at day 34. In heat tolerance latency, treatment with all capsaicin liposomal formulations demonstrated a significant reduction ( p < 0.001 ) in paw licking and paw jumping latency compared to the positive control. Low and high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy produced no significant difference ( p > 0.05 ) compared to the gabapentin-treated group. However, maximum improvement was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group by the 34th day. From day 15 to day 34, treatment with all capsaicin-loaded liposome formulations, as well as chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin, restored the decreased nerve conduction velocity drastically ( p < 0.001 ) compared to the positive control. Low and high doses of capsaicin-loaded chitosan-coated liposomes were found to improve nerve conduction velocity less significantly ( p 0.05 ) by day 34. The protective role of the blank chitosan-coated liposomes in nerve regeneration was not proven in behavioral analysis, except for nerve conduction velocity. In nerve conduction velocity, the blank chitosan-coated liposomes group displayed a slight improvement over the positive control at the 34th day. No research has been conducted on the sciatic nerve regeneration potential of capsaicin. A study demonstrated the transdermal administration of capsaicin nanoemulgel for diabetic neuropathy, showing superior enhancement of its antinociceptive effects compared to traditional gel [ 45 ]. On the other hand, chitosan has a beneficial effect on peripheral nerve regeneration revealed by a study that supports our current findings [ 46 ]. Evidence suggests that hyperglycemia has been shown to cause oxidative stress, which plays a key role in the evolution of DPN, by causing proteins and monosaccharides to oxidize on their own. The disparity between the production of ROS, such as free radicals, and the body’s own antioxidant enzymes leads to the development of oxidative stress, i.e., increasing MDA level and decreasing SOD, GSH, and CAT concentrations [ 47 ]. Our current study showed that all the capsaicin-loaded liposome administered groups effectively suppressed free radical activity. The study findings demonstrated a significant reduction ( p < 0.001 ) in malondialdehyde (MDA) levels with all capsaicin encapsulated liposomal formulations treatment in the sciatic nerve, pancreas, and brain compared to the positive control. Additionally, catalase activity and GSH levels showed significant ( p < 0.001 ) improvement in the sciatic nerve, pancreas, and brain. SOD concentrations were increased significantly ( p < 0.001 ) in the sciatic nerve, while in the pancreas, low dose of capsaicin-loaded chitosan-coated liposomes, as well as chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin, showed highly significant improvement ( p < 0.001 ), whereas capsaicin-loaded liposomes and high dose of capsaicin-loaded chitosan-coated liposomes exhibited less significant increment ( p < 0.01 ) in SOD concentrations compared to the positive control. Maximum reduction in MDA and improvement in GSH level in the sciatic nerve were observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treatment. In pancreas and brain, low dose of capsaicin-loaded chitosan-coated liposomes exhibited the highest attenuation in increasing MDA and showed an increase in GSH level. In sciatic nerve and pancreas catalase activity, the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treatment showed maximum improvement, whereas in the brain, low dose of capsaicin-loaded chitosan-coated liposomes showed maximum increment. Treatment with a chitosan-coated liposome co-loaded with capsaicin, gabapentin, and metformin was also found to be more effective in increasing SOD concentrations in both the sciatic nerve and pancreas. In previous studies, similar findings were reported with capsaicin-loaded nanoliposome against liver oxidative stress [ 48 ] and with capsaicin chitosan nanoparticles against rat mammary carcinogenesis [ 49 ]. The histological findings of the rat’s sciatic nerve and pancreas provided supporting evidence, revealing differences between the liposomal-treated and positive control groups. The chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed a better orderly arrangement in nerve fiber bundles and also recovered the axons with the myelin sheet. The islet cells are seen to be in normal position and more in numbers, which were also observed in the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group as compared to individual treatment. 5. Conclusion The study’s results offer strong evidence that oral nanoformulation of capsaicin-loaded liposome, capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin has the potential to alleviate diabetes-induced peripheral neuropathy. The chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed the best outcomes over the other capsaicin formulations evaluated, compared to metformin in case of blood glucose and lipid profile estimation, and with gabapentin in behavioral analysis. Current formulations are unexplored to address pharmacokinetic and toxicological studies. Furthermore, more research is needed to explore the more promising results. Abbreviations AMPK AMP-activated protein kinase DM Diabetes mellitus DPN Diabetic peripheral neuropathy EE Entrapment efficiency GSH Reduced glutathione HDL High-density lipoprotein HPLC high-performance liquid chromatography IL 1-Interleukin-1 IL 6-Interleukin-6 LDL Low-density lipoprotein MDA Malondialdehyde NCV Nerve conduction velocity NF кB-Nuclear factor kappa-B ROS Reactive oxygen species SOD Superoxide dismutase STZ Streptozotocin TNF α-Tumor necrosis factor-alpha TRPV1 Transient receptor potential vanilloid 1 receptor Declarations Competing interest The authors have no relevant financial and non-financial interests to disclose Ethical Approval The animal study was approved by the Ethics Committee of the Institutional Animal Ethical Committee (IAEC Reference No. CAF/MMDU/25/IAEC-3). Consent to participate Not Applicable Consent to Publish Not Applicable Funding The authors declare that no funds, grants, or other support were received during the preparation of the manuscript. Author Contribution Conceptualization, Simran Saini and Sumeet Gupta; Data Curation, Simran Saini, Rama Devi, Soumyajit Panda, and Anroop B Nair; Formal Analysis, Kashish Wilson, Nidhi Gupta, Sarita Sharma, Seema Bansal, Bimal K Agrawal, and Reena V Saini; Writing-Original Draft Preparation, Simran Saini, Rama Devi, Soumyajit Panda, Kashish Wilson, Nidhi Gupta, Sarita Sharma, Seema Bansal, Bimal K Agrawal, and Reena V Saini; Writing-Review and Editing, Anroop B Nair, and Sumeet Gupta. All authors have contributed significantly to the work, have read, and approved the final manuscript for publication. Acknowledgement We would like to acknowledge Maharishi Markandeshwar College of Pharmacy, M.M. (DU), Mullana, Ambala, India, for providing numerous resources, guidance, technical support, and various facilities for the completion of the study. Data Availability The data and supportive information are available within the article. References Zhang L, Wang Q, Su H, Cheng J (2021) Exosomes from adipose derived mesenchymal stem cells alleviate diabetic osteoporosis in rats through suppressing NLRP3 inflammasome activation in osteoclasts. J Biosci Bioeng 131:671–678. 10.1016/j.jbiosc.2021.02.007 Hossain MJ, Al-Mamun M, Islam MR (2024) Diabetes mellitus, the fastest growing global public health concern: Early detection should be focused. Health science reports 7 , e2004. 10.1002/hsr2.2004 Zhu J, Hu Z, Luo Y, Liu Y, Luo W, Du X, Luo Z, Hu J, Peng S (2023) Diabetic peripheral neuropathy: pathogenetic mechanisms and treatment. Front Endocrinol 14:1265372. 10.3389/fendo.2023.1265372 Tao Y, Zhang HY, MacGilchrist C, Kirwan E, McIntosh C (2025) Prevalence and risk factors of painful diabetic neuropathy: A systematic review and meta-analysis. Diabetes Res Clin Pract 222:112099. 10.1016/j.diabres.2025.112099 Previtali SC (2021) Peripheral Nerve Development and the Pathogenesis of Peripheral Neuropathy: the Sorting Point. Neurotherapeutics: J Am Soc Experimental Neurother 18:2156–2168. 10.1007/s13311-021-01080-z Moss KR, Saxena S (2025) Schwann Cells in Neuromuscular Disorders: A Spotlight on Amyotrophic Lateral Sclerosis. Cells 14. 10.3390/cells14010047 Bosch-Queralt M, Fledrich R, Stassart RM (2023) Schwann cell functions in peripheral nerve development and repair. Neurobiol Dis 176:105952. 10.1016/j.nbd.2022.105952 M SL, O P (2021) Inflammatory biomarkers as a part of diagnosis in diabetic peripheral neuropathy. J Diabetes Metab Disord 20:869–882. 10.1007/s40200-021-00734-1 Nashtahosseini Z, Eslami M, Paraandavaji E, Haraj A, Dowlat BF, Hosseinzadeh E, Oksenych V, Naderian R (2025) Cytokine Signaling in Diabetic Neuropathy: A Key Player in Peripheral Nerve Damage. Biomedicines 13. 10.3390/biomedicines13030589 Chakraborty T, Gupta S, Nair A, Chauhan S, Saini V (2021) Wound healing potential of insulin-loaded nanoemulsion with Aloe vera gel in diabetic rats. J Drug Deliv Sci Technol 64:102601 Gupta S, Sharma H, Morsy MA, Nair AB, Kaur K, Kumar S, Dhanawat M (2021) Protective effect of Plumeria Obtusa leaves extracts on dexamethasone-induced insulin-resistance diabetes mellitus in rats. Indian J Pharm Educ Res 55:S184–S192. 10.5530/ijper.55.1s.49 Leng J, Li X, Tian H, Liu C, Guo Y, Zhang S, Chu Y, Li J, Wang Y, Zhang L (2020) Neuroprotective effect of diosgenin in a mouse model of diabetic peripheral neuropathy involves the Nrf2/HO-1 pathway. BMC Complement Med Ther 20:126. 10.1186/s12906-020-02930-7 Zhang W, Zhang Y, Fan J, Feng Z, Song X (2024) Pharmacological activity of capsaicin: Mechanisms and controversies (Review). Mol Med Rep 29. 10.3892/mmr.2024.13162 Zheng Y, Chen J, Wu X, Zhang X, Hu C, Kang Y, Lin J, Li J, Huang Y, Zhang X et al (2022) Enhanced Anti-Inflammatory Effects of Silibinin and Capsaicin Combination in Lipopolysaccharide-Induced RAW264.7 Cells by Inhibiting NF-κB and MAPK Activation. Front Chem 10:934541. 10.3389/fchem.2022.934541 Liang W, Lan Y, Chen C, Song M, Xiao J, Huang Q, Cao Y, Ho CT, Lu M (2023) Modulating effects of capsaicin on glucose homeostasis and the underlying mechanism. Crit Rev Food Sci Nutr 63:3634–3652. 10.1080/10408398.2021.1991883 Hall OM, Broussard A, Range T, Carroll Turpin MA, Ellis S, Lim VM, Cornett EM, Kaye AD (2020) Novel Agents in Neuropathic Pain, the Role of Capsaicin: Pharmacology, Efficacy, Side Effects, Different Preparations. Curr Pain Headache Rep 24. 10.1007/s11916-020-00886-4 Daeihamed M, Dadashzadeh S, Haeri A, Akhlaghi MF (2017) Potential of Liposomes for Enhancement of Oral Drug Absorption. Curr Drug Deliv 14:289–303. 10.2174/1567201813666160115125756 Mohammed L, Nourddine H, Saad EF, Abdelali D, Hamid R (2021) Chitosan-covered liposomes as a promising drug transporter: nanoscale investigations. RSC Adv 11:1503–1516. 10.1039/d0ra08305d Akrawi SH, Gorain B, Nair AB, Choudhury H, Pandey M, Shah JN, Venugopala KN (2020) Development and optimization of naringenin-loaded chitosan-coated nanoemulsion for topical therapy in wound healing. Pharmaceutics 12:1–23. 10.3390/pharmaceutics12090893 Kumria R, Al-Dhubiab BE, Shah J, Nair AB (2018) Formulation and evaluation of chitosan-based buccal bioadhesive films of zolmitriptan. J Pharm Innov 13:133–143 Zhu Y, Wang M, Zhang J, Peng W, Firempong CK, Deng W, Wang Q, Wang S, Shi F, Yu J et al (2015) Improved oral bioavailability of capsaicin via liposomal nanoformulation: preparation, in vitro drug release and pharmacokinetics in rats. Arch Pharm Res 38:512–521. 10.1007/s12272-014-0481-7 Morsy MA, Nair AB (2018) Prevention of rat liver fibrosis by selective targeting of hepatic stellate cells using hesperidin carriers. Int J Pharm 552:241–250 Jahanfar S, Gahavami M, Khosravi-Darani K, Jahadi M, Mozafari MR (2021) Entrapment of rosemary extract by liposomes formulated by Mozafari method: physicochemical characterization and optimization. Heliyon 7:e08632. https://doi.org/10.1016/j.heliyon.2021.e08632 AlMulhim FM, Nair AB, Aldhubiab B, Shah H, Shah J, Mewada V, Sreeharsha N, Jacob S, Design (2023) Development, Evaluation, and In Vivo Performance of Buccal Films Embedded with Paliperidone-Loaded Nanostructured Lipid Carriers. Pharmaceutics 15. 10.3390/pharmaceutics15112530 Ghosh S, Haldar S, Gupta S, Bisht A, Chauhan S, Kumar V, Roy P, Lahiri D (2020) Anisotropically Conductive Biodegradable Scaffold with Coaxially Aligned Carbon Nanotubes for Directional Regeneration of Peripheral Nerves. ACS Appl Bio Mater 3:5796–5812. 10.1021/acsabm.0c00534 Sachan N, Saraswat N, Chandra P, Khalid M, Kabra A (2022) Isolation of Thymol from Trachyspermum ammi Fruits for Treatment of Diabetes and Diabetic Neuropathy in STZ-Induced Rats. BioMed research international 2022 , 8263999. 10.1155/2022/8263999 Gosavi DD, Kamdi AS, Kalambe SM, Bohra PN (2020) The spontaneous motor activity of aqueous extract of Withania coagulans fruits in Swiss albino mice by actophotometer. Int J Basic Clin Pharmacol 9:1440–1443. 10.18203/2319-2003.ijbcp20203633 Joshi A, Choudhury S, Baghel VS, Ghosh S, Gupta S, Lahiri D, Ananthasuresh GK, Chatterjee K (2023) 4D Printed Programmable Shape-Morphing Hydrogels as Intraoperative Self-Folding Nerve Conduits for Sutureless Neurorrhaphy. Adv Healthc Mater 12:e2300701. 10.1002/adhm.202300701 Zhang Y, Huang F, Xu Y, Xiang W, Xie C (2021) TRPV1 is involved in the antinociceptive effects of resveratrol in paclitaxel-induced neuropathic pain. All Life 14:66–74. 10.1080/26895293.2020.1861111 Shi X, Chen Y, Nadeem L, Xu G (2013) Beneficial effect of TNF-α inhibition on diabetic peripheral neuropathy. J Neuroinflamm 10. 10.1186/1742-2094-10-69 Nurdiana S, Goh YM, Ahmad H, Dom SM, Syimal'ain Azmi N, Noor M, Zin NS, Ebrahimi M (2017) Changes in pancreatic histology, insulin secretion and oxidative status in diabetic rats following treatment with Ficus deltoidea and vitexin. BMC Complement Altern Med 17:290. 10.1186/s12906-017-1762-8 Njan AA, Adenuga FO, Ajayi AM, Sotunde O, Ologe MO, Olaoye SO, Erdogan ON, Iwalewa OE (2020) Neuroprotective and memory-enhancing effects of methanolic leaf extract of Peristrophe bicalyculata in rat model of type 2 diabetes mellitus. Heliyon 6:e04011. 10.1016/j.heliyon.2020.e04011 Sandireddy R, Yerra VG, Komirishetti P, Areti A, Kumar A (2016) Fisetin Imparts Neuroprotection in Experimental Diabetic Neuropathy by Modulating Nrf2 and NF-κB Pathways. Cell Mol Neurobiol 36:883–892. 10.1007/s10571-015-0272-9 Eid SA, Rumora AE, Beirowski B, Bennett DL, Hur J, Savelieff MG, Feldman EL (2023) New perspectives in diabetic neuropathy. Neuron 111:2623–2641. 10.1016/j.neuron.2023.05.003 Merghany RM, Saleh RA, Hamed AA, El-Dessouki AM, El-Shiekh RA, Zaki ES, Mahfoz AM (2025) Natural therapy proposed for the management of diabetic peripheral neuropathy (DPN). Inflammopharmacology. 10.1007/s10787-025-01790-2 Kabir MT, Tabassum N, Uddin MS, Aziz F, Behl T, Mathew B, Rahman MH, Akter R, Rauf A, Aleya L (2021) Therapeutic Potential of Polyphenols in the Management of Diabetic Neuropathy. Evidence-based complementary and alternative medicine: eCAM 2021 , 9940169. 10.1155/2021/9940169 Pottathil S, Nain P, Morsy MA, Kaur J, Al-Dhubiab BE, Jaiswal S, Nair AB (2020) Mechanisms of antidiabetic activity of methanolic extract of Punica granatum leaves in nicotinamide/streptozotocin-induced type 2 diabetes in rats. Plants 9:1609 Khursheed R, Singh SK, Wadhwa S, Gulati M, Kapoor B, Awasthi A, Kr A, Kumar R, Pottoo FH, Kumar V et al (2021) Opening eyes to therapeutic perspectives of bioactive polyphenols and their nanoformulations against diabetic neuropathy and related complications. Expert Opin Drug Deliv 18:427–448. 10.1080/17425247.2021.1846517 Jia T, Rao J, Zou L, Zhao S, Yi Z, Wu B, Li L, Yuan H, Shi L, Zhang C et al (2017) Nanoparticle-Encapsulated Curcumin Inhibits Diabetic Neuropathic Pain Involving the P2Y12 Receptor in the Dorsal Root Ganglia. Front NeuroSci 11:755. 10.3389/fnins.2017.00755 Sebaaly C, Trifan A, Sieniawska E, Greige-Gerges H (2021) Chitosan-coating effect on the characteristics of liposomes: A focus on bioactive compounds and essential oils: A review. Processes 9:445 Sahin K, Kucuk O, Orhan C, Sahin E, Fowler K, White T, Durkee S, Bellamine A (2021) Bioavailability of a Capsaicin Lipid Multi-particulate Formulation in Rats. Eur J Drug Metab Pharmacokinet 46:645–650. 10.1007/s13318-021-00697-x Lu M, Cao Y, Ho CT, Huang Q (2017) The enhanced anti-obesity effect and reduced gastric mucosa irritation of capsaicin-loaded nanoemulsions. Food Funct 8:1803–1809. 10.1039/c7fo00173h Avram I, Confederat L, Constantin SM, Stan CI, Profire L Biological evaluation of chitosan-antidiabetic drug formulations for the treatment of diabetes mellitus Pawar SH, Gite AA, Patil RA (2025) Amelioration of Paclitaxel Induced Neuropathy by Gymnema sylvestre in Wistar Rats. Asian J Biol Sci 18:602–614 Saab M, Raafat K, El-Maradny H (2022) Transdermal Delivery of Capsaicin Nanoemulgel: Optimization, Skin Permeation and In Vivo Activity against Diabetic Neuropathy. Adv Pharm Bull 12:780–790. 10.34172/apb.2022.080 Pop NL, Mitrea D-R, Urdă-Cîmpean AE, Glossa-Athanasoula A, Toma VA, Moldovan R, Nan A, Decea N, Filip A, Clichici S (2021) Chitosan oral administration stimulates regeneration after experimentally induced peripheral nerve injury. Health, Sports & Rehabilitation Medicine 22 Alharthy KM, Balaha MF, Devi S, Altharawi A, Yusufoglu HS, Aldossari RM, di Alam A (2023) Giacomo, V. Ameliorative Effects of Isoeugenol and Eugenol against Impaired Nerve Function and Inflammatory and Oxidative Mediators in Diabetic Neuropathic Rats. Biomedicines 11. 10.3390/biomedicines11041203 Giri TK, Mukherjee P, Barman TK, Maity S (2016) Nano-encapsulation of capsaicin on lipid vesicle and evaluation of their hepatocellular protective effect. Int J Biol Macromol 88:236–243. 10.1016/j.ijbiomac.2016.03.056 Dhamodharan K, Vengaimaran M, Sankaran M (2021) Capsaicin Encapsulated Chitosan Nanoparticles Augments Anticarcinogenic and Antiproliferative Competency Against 7,12 Dimethylbenz(a)anthracene Induced Experimental Rat Mammary Carcinogenesis. J Pharm Res Int 33:126–144. 10.9734/jpri/2021/v33i41A32311 Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx floatimage1.jpeg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8011851","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":549058909,"identity":"936dd17b-7c9a-4670-a0df-b2651b142c09","order_by":0,"name":"Simran saini","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Simran","middleName":"","lastName":"saini","suffix":""},{"id":549058910,"identity":"0a8fe7d9-1c94-471f-bf4d-1898e7f37d39","order_by":1,"name":"Rama Devi","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Rama","middleName":"","lastName":"Devi","suffix":""},{"id":549058911,"identity":"e7c21fb2-4b4b-4844-9520-81ed3cb389ff","order_by":2,"name":"Soumyajit Panda","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Soumyajit","middleName":"","lastName":"Panda","suffix":""},{"id":549058913,"identity":"281b1e47-0649-4470-a91c-82b820936ea1","order_by":3,"name":"kashish wilson","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"kashish","middleName":"","lastName":"wilson","suffix":""},{"id":549058915,"identity":"537e2b8d-1e24-4c9d-8320-414a5fb37e59","order_by":4,"name":"Nidhi Gupta","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Nidhi","middleName":"","lastName":"Gupta","suffix":""},{"id":549058918,"identity":"e323d7ad-fe93-4224-b59d-e3f41365481a","order_by":5,"name":"sarita sharma","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"sarita","middleName":"","lastName":"sharma","suffix":""},{"id":549058919,"identity":"5c9ecf72-376d-48b7-a49e-cf7e5fba3f4a","order_by":6,"name":"seema bansal","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"seema","middleName":"","lastName":"bansal","suffix":""},{"id":549058920,"identity":"915f7c89-f453-49d1-a5e8-08d6bbd6b40e","order_by":7,"name":"Bimal k agrawal","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Bimal","middleName":"k","lastName":"agrawal","suffix":""},{"id":549058921,"identity":"8a58b73f-09d6-4fa8-95c8-9beb2de602d0","order_by":8,"name":"Reena V Saini","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Reena","middleName":"V","lastName":"Saini","suffix":""},{"id":549058922,"identity":"ba48b56d-14e2-44c0-b7a3-9b5ddd09ea08","order_by":9,"name":"Anroop B Nair","email":"","orcid":"","institution":"King Faisal University","correspondingAuthor":false,"prefix":"","firstName":"Anroop","middleName":"B","lastName":"Nair","suffix":""},{"id":549058923,"identity":"ec033a5b-362e-4c84-9121-bb533f18d90d","order_by":10,"name":"Sumeet Gupta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYJADxsc/KoAUM3MDEYoTgJiNgdmY4QxICyPxWtikGdvAtuHXIj8jO/HBxx+1idvlmx9IF86rjeZvB2r5UbENpxaDG7mbDWckHE/c2cZmYDxz2/HcGYcZGxh7ztzGrUUid5s0T8KxxA3HGAwSeLcdy20AamFmbMOtRX5G7vbff8Ba2D8c4J1zLHc+IS0MN3K3MTMk1AC18Bg28zbU5G4gpMXgzNvNkj1pB4x3tuUUM844diB3I1DLQXx+kW/P3fjhh02d7Hbm49t/fKipy513/vDBBz8q8DgMAg4zGMAYIHCAkHogqINpqSNC8SgYBaNgFIw0AABZfWPJt0xN6AAAAABJRU5ErkJggg==","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":true,"prefix":"","firstName":"Sumeet","middleName":"","lastName":"Gupta","suffix":""}],"badges":[],"createdAt":"2025-11-02 15:08:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8011851/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8011851/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96708437,"identity":"22fa893f-d181-4a27-83cf-ce14c64063ba","added_by":"auto","created_at":"2025-11-25 10:02:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6727739,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptFinalrevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/9b5cfc8e3f86f48b1cf07e7b.docx"},{"id":96621746,"identity":"952ac343-0d22-465d-b8d4-a8ac6bd6eaf6","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12194,"visible":true,"origin":"","legend":"","description":"","filename":"0ed6a0259ac54471a3df7e35f73d2bd8.json","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/94aa41f7251d3b94d79c800f.json"},{"id":96709939,"identity":"8a98baf6-1e01-41cc-99ed-d90b204be3b9","added_by":"auto","created_at":"2025-11-25 10:09:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89589,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/56129bd65f7d6b87a23e2da4.docx"},{"id":96709767,"identity":"d57d22d0-9ff2-4333-bf01-338e23bfc669","added_by":"auto","created_at":"2025-11-25 10:09:37","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188945,"visible":true,"origin":"","legend":"","description":"","filename":"0ed6a0259ac54471a3df7e35f73d2bd81enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/eacc541407876c7e4c92c380.xml"},{"id":96709893,"identity":"3925a873-6ed8-436c-92f5-0ee2055b1fee","added_by":"auto","created_at":"2025-11-25 10:09:46","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90615,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/a2c534c0e5760ee1d72aa3cf.png"},{"id":96621755,"identity":"6cfb25c5-3712-4b89-9e38-fc9f36791a81","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160354,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/4d789f8f08cf66b430925394.png"},{"id":96708806,"identity":"c32403c2-493d-455c-afdb-98466e2c3344","added_by":"auto","created_at":"2025-11-25 10:05:29","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109804,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/99c56059891bde13f62be421.png"},{"id":96621771,"identity":"73e269a8-5ba6-45ef-854f-a0e97186d1c1","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3388525,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/f75b50e7d00c0fb2602ea7e6.png"},{"id":96621752,"identity":"674c72f0-cf22-4e50-8f7d-79ef1b26a8c1","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":232034,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/86459ffe8e71b5129f7daa87.jpeg"},{"id":96621763,"identity":"42d5f6fc-ef4e-42ab-8608-ecc102248464","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134177,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/9f94504b1645bd4fe27673a3.png"},{"id":96621760,"identity":"1929e2f0-2e6f-47cb-8019-e7a71a235139","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178420,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/72bf7ae5aeaff222f79c9c47.png"},{"id":96621772,"identity":"c6a4e496-4c0f-40f4-bb80-b0629e94acf8","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":352293,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/8400e669066286aeadac8b35.png"},{"id":96708878,"identity":"ccf9b37c-6aaa-4a45-b5cf-6725d44415ff","added_by":"auto","created_at":"2025-11-25 10:05:49","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":396985,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/2104344b2d2a1a6ae4c438a5.png"},{"id":96708633,"identity":"d6aac466-0a54-4d03-82f1-e6f0190c5069","added_by":"auto","created_at":"2025-11-25 10:04:53","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":399242,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/c0c76963317058e871b4daab.png"},{"id":96621780,"identity":"3304c0a1-136e-44fd-955a-011a76ea9309","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":228249,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/fd259314db27f9b1b4d30b34.png"},{"id":96708573,"identity":"8fb59fc5-0fcf-4077-a38b-87cc5c214573","added_by":"auto","created_at":"2025-11-25 10:04:37","extension":"jpeg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":442401,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/42c85575747df9413d3808a3.jpeg"},{"id":96621781,"identity":"50e7b155-1425-439b-8c51-c2a2b71cd421","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159936,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/1a22bec44279a3ed7d6cb3ed.png"},{"id":96708695,"identity":"bcd40864-149f-470e-bbc0-82453d66d3e2","added_by":"auto","created_at":"2025-11-25 10:05:07","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102746,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/4cb6aa58b89d7609b62536ce.png"},{"id":96621776,"identity":"5c2c4b00-3b9f-4de1-8a00-6c56d480ed6f","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23674,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/b260521b00f1a6bbb9da428d.png"},{"id":96621778,"identity":"eb4cbebb-81da-40cb-9744-5a1ffaee13a6","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42081,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/06150fe14b2fc97122df0aaa.png"},{"id":96708420,"identity":"cba5b6c0-9644-43aa-8e23-8976a5729a81","added_by":"auto","created_at":"2025-11-25 10:01:51","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25278,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/1bac175627840af164f5839a.png"},{"id":96621775,"identity":"a9cea371-1485-4d38-8fca-af87f8364ad9","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":529810,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/9f86cf838fa79ba7c286e349.png"},{"id":96621782,"identity":"1a6052e5-3b04-4ef2-bfc5-87345df4afb6","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49326,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/afe8aaa0371fcb7c40471d92.png"},{"id":96621787,"identity":"eaafa57b-74a6-4920-ac42-32ce4378a7de","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17781,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/31460c5a7b313c3edc24d9da.png"},{"id":96621788,"identity":"fc7f5305-9260-48b7-a44f-127acb476cac","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34764,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/671c410386ecbb714dd707cb.png"},{"id":96621777,"identity":"8509fa70-a218-4caa-a286-9131ad917372","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67177,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/ff3b426210a170e8aa199b50.png"},{"id":96621789,"identity":"ec824af6-3ffa-4da5-b89b-98ce07da67c3","added_by":"auto","created_at":"2025-11-24 11:04:11","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80966,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/a766077078432419b57704ce.png"},{"id":96621785,"identity":"8b5fdfc5-a816-450c-b970-5b6fe1dee109","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82233,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/1fb38c65017d9a145738f2d3.png"},{"id":96709920,"identity":"e2b44980-99d8-4ccc-967f-9ab22628a10b","added_by":"auto","created_at":"2025-11-25 10:09:48","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48452,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/1563b922471ef929c7945856.png"},{"id":96621779,"identity":"eede6df5-49b2-41fa-b02f-9d9e4ad48db6","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96033,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/ba90077b661ef2f33a84a3b2.png"},{"id":96621783,"identity":"13c05465-d017-4f4c-a21f-73bdaa51f20b","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42981,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/646f1123e33be57d82661085.png"},{"id":96708509,"identity":"b5a39cc5-a610-485b-aa2c-940fa0d4e7d1","added_by":"auto","created_at":"2025-11-25 10:04:02","extension":"xml","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":190044,"visible":true,"origin":"","legend":"","description":"","filename":"0ed6a0259ac54471a3df7e35f73d2bd81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/59d3f1e3ead5a347cdd91fd7.xml"},{"id":96709874,"identity":"42a8cec1-4db6-47a9-a566-d834dd7238c5","added_by":"auto","created_at":"2025-11-25 10:09:44","extension":"html","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":207481,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/aecbc51d4502da7ff7e669e7.html"},{"id":96621743,"identity":"388500dc-b32d-408e-af68-7470e39c40c8","added_by":"auto","created_at":"2025-11-24 11:04:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134177,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution of blank chitosan-coated liposomes (A), capsaicin-loaded liposomes (B), capsaicin-loaded chitosan-coated liposomes (C), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (D).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/e7f5255ac39802e53ee03e97.png"},{"id":96621744,"identity":"48d5e603-1fde-4c17-8b73-021f2dc4eae6","added_by":"auto","created_at":"2025-11-24 11:04:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178420,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on the body weight and blood glucose levels of rats. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; # represents PC (Positive control; SZSI) vs all groups; ¥ represents SZSI-G vs all groups; £ represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/ab6c0f5e67979659ac0043ad.png"},{"id":96621745,"identity":"eec0568e-0c4f-4fa1-991a-16d3c095d021","added_by":"auto","created_at":"2025-11-24 11:04:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352293,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on lipid levels. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; # represents PC (Positive control; SZSI) vs all groups; ¥ represents SZSI-G vs all groups; £ represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/e27140eb70cabdf9b53b7bf4.png"},{"id":96709907,"identity":"ef0d0bb4-9991-409b-a937-da43477630f6","added_by":"auto","created_at":"2025-11-25 10:09:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":396985,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on locomotor activity and mechanical hyperalgesia via Randal Selitto of rats. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; # represents PC (Positive control; SZSI) vs all groups; ¥ represents SZSI-G vs all groups; £ represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/9b26eaf55ba6fecdc9460f76.png"},{"id":96709080,"identity":"29a64a30-6f31-4c29-a25e-7bdf8ee7c1f1","added_by":"auto","created_at":"2025-11-25 10:07:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":399242,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on paw licking latency and jumping latency via Eddy's Hot Plate. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; # represents PC (Positive control; SZSI) vs all groups; ¥ represents SZSI-G vs all groups; £ represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/20ab474e4455069a775d78ea.png"},{"id":96708811,"identity":"8953956d-1a25-4f8a-994b-be0fb52ea6e3","added_by":"auto","created_at":"2025-11-25 10:05:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":228249,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on nerve conduction velocity. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; # represents PC (Positive control; SZSI) vs all groups; ¥ represents SZSI-G vs all groups; £ represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/b8359f7940dc6490bc75d6c2.png"},{"id":96708854,"identity":"52327fec-a012-40da-9c4e-03a7e59f40e9","added_by":"auto","created_at":"2025-11-25 10:05:41","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":442401,"visible":true,"origin":"","legend":"\u003cp\u003eNerve conduction velocity, (A) Normal Control, (B) Positive Control, (C) SZSI-G (30 mg/kg), (D) SZSI-M (60 mg/kg), (E) SZSI-CH (100 μg/kg), (F) SZSI-CS (250 μg/kg), (G) SZSI-CHCS (250 μg/kg), (H) SZSI-CHCS (500 μg/kg), (I) SZSI-CHCSGM (250 μg/kg).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/c03a0a44cd332c51525be77c.jpeg"},{"id":96621758,"identity":"b063e587-a610-43b4-8b94-72987afa8d83","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":159936,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on Sciatic nerve (A), Pancreas (B), and Brain (C) MDA level. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; b represents PC (Positive control; SZSI) vs all groups; c represents SZSI-G vs all groups; d represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant. \u003csup\u003e#\u003c/sup\u003ep\u0026lt; 0.001, \u003csup\u003e$\u003c/sup\u003ep\u0026lt; 0.01, *p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/5479c5bfc459e49fc2cfd293.png"},{"id":96621750,"identity":"6903f9fa-3046-4ce6-963c-c105a436df71","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":90615,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on Sciatic nerve (A), Pancreas (B), and Brain (C) Catalase activity. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; b represents PC (Positive control; SZSI) vs all groups; c represents SZSI-G vs all groups; d represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant. \u003csup\u003e#\u003c/sup\u003ep\u0026lt; 0.001, \u003csup\u003e$\u003c/sup\u003ep\u0026lt; 0.01, *p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/2409880869f1c814ea5d44dd.png"},{"id":96708927,"identity":"9b5a840a-4ef7-4f2c-815b-2124acbcbd82","added_by":"auto","created_at":"2025-11-25 10:06:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":160354,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on Sciatic Nerve (A), Pancreas (B), and Brain (C) GSH activity. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; b represents PC (Positive control; SZSI) vs all groups; c represents SZSI-G vs all groups; d represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant. \u003csup\u003e#\u003c/sup\u003ep\u0026lt; 0.001, \u003csup\u003e$\u003c/sup\u003ep\u0026lt; 0.01, *p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/54edffd3dfdede403a521ad3.png"},{"id":96708915,"identity":"84dd3b80-d5a6-4c39-a94b-6b4fa7b3d8a4","added_by":"auto","created_at":"2025-11-25 10:06:18","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":109804,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of capsaicin-loaded liposome individually and in combination on Sciatic nerve (A) and Pancreas (B) SOD activity. All the values are expressed in the form of mean ± SD. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey’s Multiple Range Test; a represents NC (Normal control) vs all groups; b represents PC (Positive control; SZSI) vs all groups; c represents SZSI-G vs all groups; d represents SZSI-M vs all groups. A P-value less than 0.05 was considered significant. \u003csup\u003e#\u003c/sup\u003ep\u0026lt; 0.001, \u003csup\u003e$\u003c/sup\u003ep\u0026lt; 0.01, *p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/ec3cdd92d388f13a46aae1a0.png"},{"id":96709050,"identity":"90541273-e190-48cd-ba04-b2f770f5e333","added_by":"auto","created_at":"2025-11-25 10:07:20","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":3388525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological examination of the Sciatic nerve and Pancreas.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/a7f1448779cf4fbe6bc33ac9.png"},{"id":96621767,"identity":"1053ca8f-522d-4a76-a39b-099a2dfe0037","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"jpeg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":232034,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of action of capsaicin for DPN.\u003c/p\u003e","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/38e15aef5f851ff6e5072de4.jpeg"},{"id":97135407,"identity":"69794a26-0515-46b4-b1bd-7125603e53ea","added_by":"auto","created_at":"2025-12-01 09:43:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7724813,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/2893beb0-c25b-4d5e-b5aa-21f9cf97a2fe.pdf"},{"id":96621747,"identity":"5cbf9f02-f70e-43e6-a230-554d7f809201","added_by":"auto","created_at":"2025-11-24 11:04:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":89589,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/b191cb4d44daaebb1ed7cab7.docx"},{"id":96708453,"identity":"da71bc03-b369-454f-9c78-11f4a084758a","added_by":"auto","created_at":"2025-11-25 10:02:48","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":326466,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8011851/v1/907a73142033f65338857596.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Capsaicin Liposomes in a Rat Model of Diabetes-induced Peripheral Neuropathy ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe incidence of diabetes mellitus (DM) is gaining more attention day by day, and it frequently causes serious complications and substantial metabolic disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Diabetic peripheral neuropathy (DPN) is a major complication of DM [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The global prevalence of DPN is 46.7% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Peripheral neuropathy is a result of the degradation of myelin in myelinated nerves, damage to unmyelinated neurons, axonal atrophy, and other causes, leading to altered nerve conduction velocity and aberrant sensory function [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hyperglycemia and hyperlipidemia impair the regulatory functions of Schwann cells, including autophagy and metabolism, and disrupt mitochondrial function through multiple signaling pathways. In the absence of the protection and support provided by glial cells, such as Schwann cells, sensory neurones are more susceptible to harm than motor neurones, particularly those located in the dorsal root ganglia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the other hand, several mechanisms are involved in the progression of diabetic neuropathy from hyperglycemia, such as advanced glycation end product formation, activation of protein kinase C pathway, mitogen-activated protein kinase activation, which leads to the reactive oxygen species (ROS) generation and inflammation. Activation of NF-кB triggers the expression of proinflammatory mediators (TNF-α, IL-1, IL-6), resulting in dysfunction of Schwann cells, a decrease in neurotrophic factor, nerve blood flow, nerve conduction velocity, myelinated fibre density, and further axonal degradation and neuronal damage [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. DPN presents a new problem for clinicians since its symptoms have a significant impact on patients' mental health and quality of life. Due to several limitations, including major side effects and ineffectiveness, there are currently relatively few therapeutic options for diabetic neuropathy. For this reason, we still need to discover an effective cure for various neuropathy-related issues [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCapsaicin is the most abundant pungent bioactive compound obtained from \u003cem\u003eCapsicum annuum\u003c/em\u003e. It exerts analgesic, antioxidant, and other pharmacological properties by binding to the TRPV1 receptor [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Capsaicin exhibits anti-inflammatory effects by inhibiting NF-кB and mitogen-activated protein kinase signalling pathway [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and through the activation of the AMPK pathway, it influences lipid accumulation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Capsaicin has great potential to treat neuropathic pain. Topical formulations of capsaicin are available for neuropathic pain, such as gels, creams, lotions, and ointments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Despite the extensive evidence on capsaicin\u0026rsquo;s therapeutic efficacy, the utility of oral formulation of capsaicin in DPN remains unexplored. At present, liposomes have demonstrated as a promising carriers for oral delivery as they encapsulate the highly lipophilic substance within their phospholipid bilayers, increasing its water dispersibility and protecting it from deterioration in the gastrointestinal environment [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The liposomal encapsulation of capsaicin may also improve its low oral bioavailability owing to its potential to improve solubility, stability, and absorption. The liposome polymer surface modification, specifically chitosan coating, increases vesicle durability in biological settings and can extend systemic circulation, which promotes medication accumulation at target sites [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Chitosan, a biocompatible and biodegradable cationic polysaccharide, can adhere to negatively charged phospholipid head groups and produce a hydrated polymer shell that increases mucoadhesion, decreases leakage, and may facilitate tissue penetration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The objective of this research was to prepare chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin to assess the efficacy in diabetic neuropathy and its effect on altering nerve conduction velocity.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Drugs and Chemicals\u003c/h2\u003e\u003cp\u003eCapsaicin and metformin were purchased from Sigma Aldrich, St Louis, MO, USA. Gabapentin was obtained from Ind Swift Laboratory, India. Glucose and lipid profile estimation kits were used. The additional reagents used in the study were all of analytical grade.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of Capsaicin-Loaded Liposomes\u003c/h2\u003e\u003cp\u003eThe traditional thin film hydration method, as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] with minor modification, was used to prepare liposomes. In brief, accurately weighed capsaicin (7 or 14 mg, based on the preliminary trials) was mixed with soya lecithin (1.2 g) and dissolved in 20 ml of ethanol, and the mixture was sonicated until a clear solution was obtained. Cholesterol (0.2 g, corresponding to a 7:3 lecithin: cholesterol molar ratio), Tween 80 (0.8 g) and isopropyl myristate (0.8 g) were dissolved in another 20 ml of ethanol and added to the lipid phase, followed by thorough mixing. The combined solution was transferred to a round-bottom flask and subjected to rotary evaporation under reduced pressure to remove the organic solvents, resulting in the formation of a thin lipid film on the flask wall. The dried lipid film was hydrated with double-distilled water under gentle agitation to produce capsaicin-loaded liposomes. To prepare capsaicin-loaded chitosan-coated liposomes, the liposome formulation was mixed with 0.5 g of chitosan and 0.5 ml of acetic acid and stirred for 2 h, and subsequently probe-sonicated for 5 min to achieve uniform coating. Similarly, chitosan-coated liposomes co-loaded liposomes were prepared by dissolving the required amount of capsaicin, gabapentin and metformin in the organic phase and followed the above protocol. Blank chitosan-coated liposomes (without drug) were prepared using the same procedure used in capsaicin-loaded chitosan-coated liposomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. HPLC Analysis\u003c/h2\u003e\u003cp\u003eThe concentration of capsaicin, gabapentin and metformin in the prepared liposome samples was quantified separately using high-performance liquid chromatography (HPLC, Agilent 1260 infinity SEMI Preparative System) equipped with DAD detector and a C18 column (4.6 \u0026times; 250 mm, 5 \u0026micro;m) maintained at 30\u0026deg;C. Detection was performed at 283 nm (capsaicin), 222 nm (metformin) and 210 nm (gabapentin). The mobile phase consisted of acetonitrile and water (50:50, v/v), delivered at a flow rate of 1.0 mL min⁻\u0026sup1;, with a 20 \u0026micro;L injection volume. The calibration curves were constructed using serial dilutions of drug samples. The retention times of capsaicin, metformin, and gabapentin are 2.17 min, 2.11 min, and 1.94 min, respectively (Supplementary Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Characterization of Liposomes Preparation\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Particle Size\u003c/h2\u003e\u003cp\u003eThe particle size and distribution of the liposomal formulations were analyzed using dynamic light scattering on a Malvern Zetasizer Nano-ZS (Westborough, MA, USA) at 25\u0026deg;C. Prior to measurement, the liposomes were diluted in deionized water to achieve an appropriate concentration and to minimize particle aggregation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Drug Content and Entrapment Efficiency\u003c/h2\u003e\u003cp\u003eThe drug content was estimated by the HPLC method. About 100 mg of liposomes were dissolved in Triton X-100, diluted with mobile phase and then kept on an orbital shaker at 100 rpm and 37\u0026deg;C for 24 h to determine the degree of entrapment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The mixture was centrifuged for 20 min at 4000 rpm. The supernatant was retrieved and measured by HPLC. The entrapment efficiency was determined by measuring the free drug in 2 ml of liposomes by centrifugation at 3080\u0026times; g for 30 min using a Millipore Amicon filter (MWCO 10 kDa, Darmstadt, Germany), and the supernatant was measured by HPLC. The percentage entrapment efficiency was determined using the standard equation described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Experimental Animals\u003c/h2\u003e\u003cp\u003eSprague Dawley rats of either sex (male/female) having 150\u0026ndash;250 g body weight and 6\u0026ndash;8 weeks old were procured from Rodent Research Laboratory India Pvt Ltd, Haryana. The experimental protocol was approved by the Institute of the Animal Ethics Committee (CAF/MM(DU)/25/IAEC-3). The rats were housed at 12 hr day and night cycle at room temperature (24\u0026deg;C-28\u0026deg;C) and relative humidity of 60\u0026ndash;70%. Regular food and water were provided to the animals, and all the hygiene conditions were maintained. All the experimental procedures were conducted between 9:00 a.m. and 5:00 p.m. All the experimental protocols were conducted according to the ethical guidelines, CCSEA for the investigation of laboratory animals and handling of experimental animals.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Experimental Groups\u003c/h2\u003e\u003cp\u003eAll the animals were weighed and grouped into nine groups. Group 1 (Normal control): received normal saline (5 ml/kg). Group 2 (Positive control; SZSI): induced diabetes by streptozotocin 45 mg/kg) and on the 5th day, induction of sciatic nerve injury. Group 3 (SZSI-G): received gabapentin (30 mg/kg, 5 ml/kg). Group 4 (SZSI-M): received metformin (60 mg/kg, 5 ml/kg). Group 5 (SZSI-CH): received blank chitosan-coated liposomes (5 ml/kg). Group 6 (SZSI-CS): received low dose of capsaicin-loaded liposomes (250 \u0026micro;g/kg or 54 mg of liposomes, 5 ml/kg). Group 7 (SZSI-CHCSL): received low dose of capsaicin-loaded chitosan-coated liposomes (250 \u0026micro;g/kg, 5 ml/kg). Group 8 (SZSI-CHCSH): received high dose of capsaicin-loaded chitosan-coated liposomes (250 \u0026micro;g/kg, 5 ml/kg). Group 9 (SZSI- CHCSGM): received chitosan-coated liposomes co-loaded with capsaicin (250 \u0026micro;g/kg), gabapentin (30 mg/kg), and metformin (60 mg/kg, 5 ml/kg).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Induction of Diabetes Mellitus by STZ-NA\u003c/h2\u003e\u003cp\u003eDM was induced in experimental overnight fasted rats by single intraperitoneal injection of STZ (45 mg/kg) prepared in 0.1 M citrate buffer (pH 4.5) after \u003cem\u003ei.p\u003c/em\u003e injection of nicotinamide (110 mg/kg) prepared in normal saline. Diabetes was confirmed on day 4 by measuring the blood glucose levels and those rats that were in the hyperglycemia stage (greater than 200 mg/dl) were selected for further study. Selected diabetic animals were subjected to induced sciatic nerve injury on day 5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Sciatic Nerve Injury Model\u003c/h2\u003e\u003cp\u003eSelected diabetic animals were anesthetized with intraperitoneal injections of ketamine (60 mg/kg) and xylazine (10 mg/kg), and fur was removed from the left thigh. Using a sterilized surgical blade, a small cut was created along the left lateral thigh. To find the sciatic nerve, blunt dissection was done via the biceps femoris and gluteus superficialis muscles. Saline solution was used to clean the exposed sciatic nerve. From a 1 cm section of the sciatic nerve that was exposed, 0.5 cm was crushed twice at the mid-thigh level, resulting in the disruption of tissue organization in this area. After the surgery, the cut was stitched up using surgical thread and a surgical needle, and then betadine was applied to it. The animals were kept in separate cages with good ventilation and unlimited access to food and water. For a period of 6\u0026ndash;7 days, betadine and antiseptics were topically administered to the sutured area in order to prevent infection. The surgery was performed using the procedure indicated earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Treatment Protocol\u003c/h2\u003e\u003cp\u003eThe drug treatment was started from the 5th day onwards and this was considered as first day of treatment and continued for further 30 days. All drugs were given orally through intragastric tube. Behavioural parameters were assessed on the 4th, 15th, 24th, and 34th day of the experiment. Measurements of nerve conduction rate were taken on the 15th, 24th, and 34th days. At the end of the study, assessment of oxidative biomarkers in rat tissues was performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Body Weight\u003c/h2\u003e\u003cp\u003eThe body weight of each animal was monitored at 0th, 4th, 15th, 24th, and 34th day using a digital weighing balance. The changes in body weight throughout the research experiment were carefully recorded. The initial body weight was measured before administering STZ, referred to as the 0th day body weight. After the diabetes confirmation, referred to as the 4th day, and during treatment, body weight was measured at different intervals, referred to as the 15th, 24th, and 34th day [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Estimation of Blood Glucose Level and Lipid Profile\u003c/h2\u003e\u003cp\u003eRats were fasted overnight, and their blood samples were withdrawn (under anesthesia) by retro-orbital puncture method at different intervals on 0th, 4th, 15th, 24th, and 34th day. The serum was separated by centrifugation at 4000 rpm for 5 min. The isolated serum was used to analyse blood glucose, triglycerides, and cholesterol, LDL, and HDL levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Behavioral Parameters\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.10.1. Assessment of Locomotor Activity\u003c/h2\u003e\u003cp\u003eAn actophotometer test was conducted to evaluate the spontaneous motor behaviour of the rats. Every animal was monitored for a period of ten minutes in a confined square activity enclosure. The equipment automatically kept track of how many times the light beams were interrupted by animals. The beam of light was cut off as the rat started to explore. The digital recorder in the instrument automatically picked up and tallied each interruption. The digital counter stopped recording as the animal became immobile. When the animal started to walk, the action was recorded again [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.11.1 Evaluation of Mechanical Hyperalgesia\u003c/h2\u003e\u003cp\u003eThe Randall\u0026ndash;Selitto test assesses allodynia or neuropathic pain in animals by measuring the paw withdrawal threshold. Allodynia, a heightened sensitivity to pain stimuli, is caused by nerve injury. Prior to the test, all animals were subjected to 5 min of handling to become used to being handled. The test entailed applying more and more mechanical pressure on the animal's experimental limb. Pressure was exerted on the medial region of the plantar surface of the left hind paw of each rat until a withdrawal reaction was elicited. The pressure or weight given to elicit the withdrawal reaction was recorded for each animal [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.11.2 Assessment of Thermal Hyperalgesia\u003c/h2\u003e\u003cp\u003eEddy\u0026rsquo;s hot plate was used for thermal hyperalgesia assessment. A heated plate maintained at a temperature of 53\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C was employed for animal placement. An electronic timer was utilized to record the latency of reaction, either by hind-paw licking or jumping. A time limit of 30 s was implemented to safeguard tissue against harm. Every rat had triple measurements at 10 min intervals [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Nerve Conduction Velocity Measurements\u003c/h2\u003e\u003cp\u003eThe nerve conduction velocity (NCV) was assessed in the sciatic nerve-injured rats. The NCV evaluation utilized an AD Instrument's 8-channel Power Lab, together with an animal nerve-stimulating electrode and needle electrodes. Animals were anaesthetized with Ketamine (90 mg/kg i.p.) and Xylazine (5 mg/kg i.p.) and secured to a board. Following the cleaning of the proximal and distal ends of the lower leg with alcohol, the electrodes were positioned. An action potential was created by attaching a stimulating electrode at the proximal end and recording from the distal end. The test was performed under the methods specified in references [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Histopathological Analysis\u003c/h2\u003e\u003cp\u003eAt the termination of the study, the animals were sacrificed by cervical dislocation for tissue isolation (nerve, pancreas, and brain). Flanks measuring 1.5 cm in length from the crushed location on the sciatic nerves were collected and preserved in 10% neutral-buffered formalin. Furthermore, all the sciatic nerve samples for histopathological examination were processed for H and E staining [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The pancreas was carefully removed, washed in ice-cold saline, and embedded in 10% formalin for histological examination [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The brain was harvested for oxidative stress biomarker assessment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Assessment of Oxidative Stress Biomarkers\u003c/h2\u003e\u003cp\u003eAnalysis for malondialdehyde (MDA), catalase, reduced glutathione (GSH), and superoxide dismutase (SOD) was performed using homogenized sciatic nerve, pancreas, and brain tissues. These assays were performed using the procedure indicated earlier [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The data of lipid profile, behavioral, and biochemical estimations were statistically analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test using GraphPad Instat version 3.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of Liposomes\u003c/h2\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1. Particle Size\u003c/h2\u003e\u003cp\u003eThe results of particle-size analysis of liposomes are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The observed average hydrodynamic diameter varied among the tested formulations as blank chitosan-coated liposomes (129.61 nm), capsaicin-loaded liposomes (183.50 nm), capsaicin-loaded chitosan-coated liposomes (252.90 nm), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (291.40 nm). The blank chitosan-coated liposomes showed the lowest particle size, which increased with capsaicin loading, subsequent chitosan coating, as well as co-loading.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2. Entrapment Efficiency\u003c/h2\u003e\u003cp\u003eEntrapment efficiencies also varied among tested formulations and were as follows: capsaicin-loaded liposomes (78.94%), capsaicin-loaded chitosan-coated liposomes (80.20%), capsaicin in the co-loaded chitosan-coated system (68.92%), gabapentin (55.0%), and metformin (47.0%).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Body Weight\u003c/h2\u003e\u003cp\u003eRats in the normal control group showed no significant difference in body weight over the study period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, after the administration of STZ, all diabetic rats showed a significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) drop in body weight compared to normal control rats by day 4. Positive control rats showed a continuous decrease in body weight (153.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 g to 144.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 g) from day 4 to day 34 compared to normal control rats. On day 15, among all drug-treated groups, rats subjected to gabapentin (Group 3) treatment (181.6\u0026thinsp;\u0026plusmn;\u0026thinsp;29.0 g), chitosan-coated liposomes co-loaded with capsaicin, gabapentin and metformin (Group 9) treatment (178.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 g) showed significant gain (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in percentage (20.3%) of body weight. However, rats (Group 7) received low dose of capsaicin-loaded chitosan-coated liposomes (175.8\u0026thinsp;\u0026plusmn;\u0026thinsp;18.6 g) began to gain weight slightly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) compared to the positive control group (148.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 g). However, a highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement in body weight was observed in all drug-treated groups compared to the positive control by day 24 and day 34, respectively. On day 34, treatment (Group 7) with low dose of capsaicin-loaded chitosan-coated liposomes (234.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2g) and chitosan-coated liposomes co-loaded with capsaicin, gabapentin and metformin (Group 9) (241.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 g) showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) increase in body weight, whereas (Group 6) capsaicin-loaded liposome (219.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 g) exhibited non-significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) compared to gabapentin (221.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75g) and metformin (216.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6g). Administration of high dose of capsaicin-loaded chitosan-coated liposomes (Group 8, 225.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 g) exhibited a highly significant increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) than metformin and showed a non-significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) compared to gabapentin (Group 3). However, no significant difference was noted in the body weight of rats treated with gabapentin and metformin in comparison with each other from day 15 to day 34. Overall, the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy in Group 9 demonstrated the highest improvement in body weight over the other drug treatments, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Blood Glucose Level and Lipid Profile Estimation\u003c/h2\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Blood Glucose Level\u003c/h2\u003e\u003cp\u003eBlood glucose level was increased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in all experimental groups except the normal control after the induction of STZ at day 4, and levels were drastically increased from 301.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 to 310.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 mg/dl by the 34th day, as shown in the positive control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Continuous administration of metformin and capsaicin-loaded liposome formulations for 30 days reduced the level of blood glucose significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) as compared to the positive control. Compared to metformin, the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group demonstrated the highest improvement in blood glucose level over the other treated drugs from 300.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 to 130.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mg/dl during the treatment phase. On the 34th day, a more significant drop (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in glucose level was observed from all drug-treated groups except gabapentin and the blank chitosan-coated liposomes group compared to the positive control. However, the blank chitosan-coated liposomes group (303.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg/dl) showed less significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) improvement, and the gabapentin treated group (306.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 mg/dl) exhibited no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) compared to the positive control. Treatment with capsaicin-loaded liposome (170.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (141.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 and 133.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (130.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mg/dl) showed more significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in glucose level than the gabapentin-treated group. Compared to metformin (130.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 mg/dl), a non-significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) was observed with high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin. Moreover, the metformin-treated group demonstrated a marked reduction in glucose level than the gabapentin-treated group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2.Effect of Capsaicin-Loaded Liposome Individually and in Combination on Triglyceride Levels\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the triglyceride levels in all experimental animals throughout the study. However, after the administration of STZ, all rats demonstrated an increasing level of triglycerides with a significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) as compared to normal control rats at day 4. These increasing levels are found to decrease on continuous treatment with drugs over the study period. On 15th day, metformin (189.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 mg/dl), capsaicin-loaded liposome (206.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (185.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 and 193.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group (182.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 mg/dl) showed more significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in triglyceride level compared to the positive control (225.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 mg/dl). However, compared to the positive control, a slight improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) was observed with the blank chitosan-coated liposomes group by day 24, which became significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) by day 34. Gabapentin showed no improvement (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) in triglyceride level over the study period. On day 34, treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes (135.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 and 143.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mg/dl) exhibited no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (123\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mg/dl) showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement in triglyceride level compared to metformin-treated group (139.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 mg/dl). Moreover, all capsaicin-loaded liposome administered groups showed marked reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in triglyceride level compared to the gabapentin-treated group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Total Cholesterol Levels\u003c/h2\u003e\u003cp\u003eIn normal control rats, cholesterol levels were at their basal range, but they were constantly increased in positive control rats over the study period. However, it was found that the total cholesterol level among all drug-treated groups had declined, whilst the positive control group showed an increment from day 4 to day 34 (192.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 to 203.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 mg/dl). Compared to the positive control, a highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) reduction was observed in the metformin-treated group, whereas high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin exhibited a significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) drop in cholesterol level on day 15, which became more significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) by day 24. On day 34, rats subjected to metformin (131.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0 mg/dl), capsaicin-loaded liposome (148.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (137.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.2 and 140.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (131.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 mg/dl) showed marked reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in cholesterol level, whereas gabapentin-treated group (191.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 mg/dl) showed no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) and blank chitosan-coated liposomes group (186.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mg/dl) exhibited less significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) decrease in cholesterol level compared to positive control. In comparison with the metformin-treated group, no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) was observed with low and high doses of capsaicin-loaded chitosan-coated liposomes, and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treated group. However, all drug-treated groups except blank chitosan-coated liposomes showed more significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) reduction in cholesterol level compared to the gabapentin-treated group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4. Effect of Capsaicin-Loaded Liposome Individually and in Combination on LDL Levels\u003c/h2\u003e\u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, LDL levels were increased drastically in positive control rats as compared to normal control rats throughout the study period. Continuous administration of all treated drugs except gabapentin and blank chitosan-coated liposomes showed a marked reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in LDL level compared to the positive control by day 24 and day 34. However, the gabapentin-treated group exhibited non-significant results (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) during the treatment phase, whereas the blank chitosan-coated liposomes group showed less significant \u003cem\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) improvement compared to the positive control on day 34. On 34th day, in contrast to metformin (106.2\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4 mg/dl), treatment with blank chitosan-coated liposomes (188.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 mg/dl), capsaicin-loaded liposome (147.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (133.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 and 146.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8 mg/dl) showed highly significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), although chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin group (129.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 mg/dl) exhibited less significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in reducing the LDL level. Moreover, all capsaicin-loaded liposome administered groups showed more significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) reduction in LDL level compared to the gabapentin treated group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003e3.3.5. Effect of capsaicin-loaded liposome individually and in combination on HDL levels\u003c/h2\u003e\u003cp\u003eHDL levels were drastically decreased in all experimental groups as compared to the normal control on day 4. However, positive control rats showed a continuous decrease in level from day 4 to day 34. On 15th day, metformin (26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg/dl), low and high doses of capsaicin-loaded chitosan-coated liposomes (23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 and 24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 mg/dl), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 mg/dl) showed highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), whereas capsaicin-loaded liposome (22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg/dl) exhibited less significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) increase in HDL level compared to positive control (16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mg/dl). Continuous administration of treated drugs improved the HDL level by day 24 and day 34, respectively, although treatment with gabapentin exhibited no significant improvement during the interval of time. On the 34th day, a highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement was observed in all drug-treated groups, except gabapentin, as compared to the positive control group (12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 mg/dl). Administration of capsaicin-loaded liposome (28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0), low and high doses of capsaicin-loaded chitosan-coated liposomes (36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 and 39.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (41.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0) showed a highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) increase in HDL levels as compared to the gabapentin-treated group (17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 mg/dl). However, in contrast to metformin (41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 mg/dl), low dose of capsaicin-loaded chitosan-coated liposomes showed less significant difference, while high dose of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin demonstrated no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Behavioral Analysis\u003c/h2\u003e\u003cdiv id=\"Sec36\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 Effect of Capsaicin-Loaded Liposome Individually and in Combination on Locomotor Activity Using Actophotometer\u003c/h2\u003e\u003cp\u003eLocomotor activity in all groups of experimental rats was drastically decreased compared to the normal control group rats after the administration of STZ on day 4. Administration of sciatic nerve injury on day 5 in the STZ-induced diabetes rats showed a reduction in locomotor behavior in all drug-treated rats with a highly significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) on day 15 compared to day 4. On day 15, groups treated with gabapentin (163.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1), low and high doses of capsaicin-loaded chitosan-coated liposomes (172.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4 and 162.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (185.6\u0026thinsp;\u0026plusmn;\u0026thinsp;18.2) showed a highly significant increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) as compared to positive control group (132.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6), whereas metformin (140.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5), blank chitosan-coated liposomes (133\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8) and capsaicin-loaded liposome (145.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2) exhibited non-significant results. However, maximum improvement was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin as compared to individual therapy of gabapentin and metformin. Furthermore, all drug-treated groups except blank chitosan-coated liposomes exhibited the highest increment (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in locomotor behavior compared to the positive control group on day 24 and day 34, respectively. On the 34th day, low dose of capsaicin-loaded chitosan-coated liposomes (267\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6) exhibited less significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treated group (287.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1) showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement in comparison with the gabapentin-treated group (252.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4). In comparison with the metformin-treated group (191.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8), a large improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) was observed (79.9%) with capsaicin-loaded liposome (226\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7), low and high doses of capsaicin-loaded chitosan-coated liposomes, and the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group (128.6%). Moreover, rats subjected to the gabapentin-treated group exhibited a more significant increment (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in locomotor activity compared to the metformin-treated group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Effect of Capsaicin-Loaded Liposome Individually and in Combination on Mechanical Hyperalgesia via Randall Selitto\u003c/h2\u003e\u003cp\u003eThe diabetic neuropathy affected the pain threshold of the rat and exhibited allodynia; therefore, a highly significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) was observed in paw withdrawal threshold when pressure was applied through Randall Selitto in all experimental groups as compared to the normal control on day 4. Rats subjected to the positive control group produced allodynia at a significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) compared to normal control rats from day 15 to day 34. Continuous administration of the standard drugs (gabapentin and metformin) and liposomal drug formulations, except the blank chitosan-coated liposomes group, enhanced the paw withdrawal threshold significantly during the treatment phase. On day 34, all drug-treated groups exhibited a highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), whereas the blank chitosan-coated liposomes group (3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 g) showed a non-significant difference compared to the positive control group (2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 g). However, chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 g) showed a significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) increment in paw withdrawal threshold than the gabapentin-treated group (18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 g). In comparison with the metformin-treated group (11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 g), treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes (19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 g and 17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 g), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin exhibited a highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/em\u003e increase in paw withdrawal threshold. Moreover, the gabapentin-treated group attenuated allodynia more significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) than the metformin-treated group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Thermal Hyperalgesia via Eddy\u0026rsquo;s Hot Plate\u003c/h2\u003e\u003cp\u003eThe duration was employed for each animal to exhibit a response (paw licking or jumping) to heat, or the latency period was recorded. The animals showed a prolonged latency period due to a disrupted sciatic nerve. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, on day 34, paw licking latency duration among all drug-treated groups except the blank chitosan-coated liposomes group was reduced over time when compared with the positive control group (280.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 s). Treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes (10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 s and 12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 s), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 s) showed no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) as compared to the gabapentin-treated group (8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 s). However, chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin and low-dose capsaicin-loaded chitosan-coated liposomes exhibited highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and at high dose of capsaicin-loaded chitosan-coated liposomes showed less significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) reduction in latency duration when compared to the metformin-treated group (21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 s). Additionally, in paw jumping latency time, positive control rats showed a higher latency period as compared to normal control rats throughout the experimental study. On 34th day, administration of gabapentin (9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 s), capsaicin-loaded liposome (15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 s), low and high doses of capsaicin-loaded chitosan-coated liposomes (13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 s, and 14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 s), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 s) showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), whereas metformin (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 s) exhibited slightly improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and blank chitosan-coated liposomes group (25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 s) showed no significant (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) improvement in latency duration as compared to positive control (29.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 s). Treatment with low and high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin demonstrated non-significant results (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) as compared to gabapentin, whilst in comparison with the metformin-treated group, these groups showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement in prolonged latency duration, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Notably, marked improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in both paw licking and paw jumping latency time was recorded in gabapentin-treated rats over the metformin-treated rats.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Nerve Conduction Velocity\u003c/h2\u003e\u003cp\u003eNerve conduction velocity in normal control rats was 89.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 m/s on day 15th, whilst 27.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 m/s in positive control rats, which was decreased to 23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 m/s over the study period. Administration of gabapentin (76.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 m/s), capsaicin-loaded liposome (58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 m/s), low and high doses of capsaicin-loaded chitosan-coated liposomes (67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 and 64.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4), and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin (79.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 m/s) showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement, whereas blank chitosan-coated liposomes group (30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 m/s) showed slightly improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in nerve conduction velocities as compared to the positive control group on 34th day. Treatment with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed maximum attenuation over the other drug-treated groups, and exhibited a non-significant (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) result when compared with the gabapentin-treated group. Moreover, no significant improvement (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) was observed in the metformin-treated group in comparison with the positive control throughout the experimental study, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Assessment of Oxidative Stress Biomarkers\u003c/h2\u003e\u003cp\u003e\u003cb\u003e3.6.1. Effect of Capsaicin-Loaded Liposome Individually and in Combination on MDA of Rat Organs (Sciatic nerve, Pancreas, and Brain)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMDA level in the sciatic nerve (6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nM/mg protein), pancreas (6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 nM/mg protein), and brain (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 nM/mg protein) of positive control rats increased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) compared to normal control rats. Continuous administration of treated drugs except gabapentin and blank chitosan-coated liposomes attenuated these increasing levels more significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in the sciatic nerve, pancreas, and brain compared to the positive control. On the other hand, blank chitosan-coated liposomes group showed highly significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in sciatic nerve (6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 nM/mg protein) and exhibited less significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in both pancreas (5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM/mg protein) and brain (7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nM/mg protein), whereas gabapentin treated group showed a slight reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in MDA level in rat organs (nerve, pancreas, and brain). In contrast to metformin, rats subjected to chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group showed less significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in rat organs MDA level, whereas low dose of capsaicin-loaded chitosan-coated liposomes exhibited no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) in pancreas and brain, but showed significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in sciatic nerve MDA level. Moreover, a highly significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in MDA level was observed in the metformin-treated group compared to the gabapentin-treated group in rat organs. Overall, the highest improvement was noted with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin group in the sciatic nerve, whereas in the pancreas and brain, low dose of capsaicin-loaded chitosan-coated liposomes showed more reduction in MDA level among all liposomal formulations, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6.2. Effect of Capsaicin-Loaded Liposome Individually and in Combination on Catalase Activity in Rat Organs (Sciatic nerve, Pancreas, and Brain)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, it was observed that the catalase activity in positive control rats was decreased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in the sciatic nerve (3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 U/mg protein), pancreas (11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 U/mg protein), and brain (30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 U/mg protein), compared to normal control rats. On continuous administration of treated drugs, catalase activity was restored in rat organs (nerve, pancreas, and brain). Compared to the positive control group, treatment with metformin, and all capsaicin-loaded liposome administered groups exhibited highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in sciatic nerve, pancreas, and brain catalase activity. However, the gabapentin-treated group showed a slight increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in rat organs (nerve, pancreas, and brain), whereas the blank chitosan-coated liposomes group exhibited a highly significant increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in pancreas, and showed less significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in sciatic nerve and brain catalase activity. Compared to the gabapentin-treated group, a more significant increase was observed with the metformin-treated group in rat organs (nerve, pancreas, and brain). In comparison to the metformin-treated group, a highly significant increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) was noted with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin in the sciatic nerve (17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 U/mg protein), and no significant difference was observed in pancreas (32.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 U/mg protein) catalase activity. Furthermore, in brain catalase activity, treatment with low dose of capsaicin-loaded chitosan-coated liposomes (51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 showed less significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) compared to the metformin-treated group (56.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 U/mg protein). Overall, in the sciatic nerve and pancreas, chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed maximum improvement, whereas in the brain, the highest improvement was observed with low dose of capsaicin-loaded chitosan-coated liposomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6.3\u003c/b\u003e. \u003cb\u003eEffect of Capsaicin-Loaded Liposome Individually and in Combination on GSH in Rat Organs (Sciatic nerve, Pancreas, and Brain)\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePositive control rats demonstrated a highly significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in GSH activity in rat organs (nerve, pancreas, and brain) as compared with normal control rats. All drug treatment groups showed a highly significant increment (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in GSH level, whereas the gabapentin-treated group showed a slight (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and the blank chitosan-coated liposomes group showed lesser significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) compared to the positive control rat organs (nerve, pancreas, and brain). Treatment with low doses of capsaicin-loaded chitosan-coated liposomes showed maximum improvement over other liposomal-treated groups with no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) compared to the metformin-treated group in brain and pancreas GSH activity, whereas in the sciatic nerve, the highest improvement was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group among other liposomal-treated groups and showed no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) compared to the metformin-treated group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6.4\u003c/b\u003e. \u003cb\u003eEffect of Capsaicin-Loaded Liposome Individually and in Combination on SOD in Rat Organs (Sciatic nerve and Pancreas)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRats in the positive control group showed a marked reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in SOD activity compared to the normal control group. It was found that all drug-treated groups demonstrated highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement in SOD activity, whereas the blank chitosan-coated liposomes group and gabapentin-treated group showed less improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) compared to the positive control in the sciatic nerve and pancreas. However, the metformin-treated group exhibited a more significant increment in SOD activity than the gabapentin-treated group in both organs. In contrast to the metformin-treated group, treatment with low dose of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) in nerve and pancreas SOD activity. Overall, maximum increment was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group over the other liposome-treated groups in both sciatic nerve and pancreas GSH activity, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Histopathological Evaluation of Sciatic Nerve and Pancreas\u003c/h2\u003e\u003cdiv id=\"Sec42\" class=\"Section3\"\u003e\u003ch2\u003e3.7.1. Histopathological Evaluation of Sciatic Nerve\u003c/h2\u003e\u003cp\u003eNo pathological changes were observed in the normal control group of the sciatic nerve (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The transverse section of the sciatic nerve demonstrated a collection of nerve fiber bundles, each surrounded by connective tissue, endoneurium, and encased with perineurium and epineurium. It contains axons of various sizes surrounded by myelin sheath and separated by an endoneurial connective tissue. However, in the diabetic control group, the sciatic nerve showed shrinkage, demyelination, necrosis, and degenerated nerve fibers surrounded by thin perineurium. Focal loss of endoneurium was also observed due to destructive reactions. Treatment with low dose of capsaicin-loaded chitosan-coated liposomes reduced the disturbance in morphological structure; only a few inflammatory cells were seen as compared to the high dose treated group. Blank chitosan-coated liposomes group of sciatic nerve showed axonal regrowth after treatment. The chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin formulation showed improvement in the regeneration of nerve fibers, where endoneurium was clearly seen. In addition, this formulation showed a better orderly arrangement in nerve fiber bundles. It also recovered the axons with the myelin sheet.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec43\" class=\"Section3\"\u003e\u003ch2\u003e3.7.2. Histopathological Evaluation of Pancreas\u003c/h2\u003e\u003cp\u003eIn the normal control group, the cells of the pancreas were all present in their normal proportions (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The duct was clearly seen. The islet cells are seen embedded within the acinar cells and surrounded by a fine capsule. In the positive control group, the islets were damaged, shrunken in size, and infiltration of lymphocytes was observed. The islet cells are seen to be normal in position. The size of the cell is to be back in its normal position after treatment with metformin and gabapentin. The islets of the blank chitosan-coated liposomes-treated group showed improvement after treatment. The islet cells are seen to be in normal position and more in numbers, which were also observed in the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group, as compared to their individual treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe incidence of diabetes is increasing more rapidly. A study reported that there are roughly 537\u0026nbsp;million individuals with diabetes globally and estimated that it will increase to 783\u0026nbsp;million by 2045 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During DPN, the peripheral nerve exhibits insulin resistance, and the insulin receptors on Schwann cells and axons become unresponsive to insulin. Insulin resistance impairs Schwann cell and axonal metabolism, which leads to neuronal demyelination and neuronal damage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Loss of myelin in myelinated nerves and unmyelinated nerve injury are the factors that affect nerve conduction velocity and abnormal sensory function. Hyperglycemia activates the polyol pathway, hexosamine pathway, protein kinase C pathway, and more metabolic pathways, causing detrimental effects on neurons, which result in inflammation and oxidative stress, ultimately leading to neuronal damage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The infiltrating macrophages within peripheral nerve cells stimulate the production of cytokines and chemokines, hence exacerbating inflammation and causing nerve fibre injury [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Various natural compounds have been targeted for the management of DPN, offering effective, safe, and beneficial therapeutic strategies with fewer side effects. Polyphenols, flavonoids, and other herbal extracts play a crucial role in addressing the underlying pathophysiological mechanisms of nerve damage [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Novel formulations, such as nanoparticle-based delivery systems, have been explored that accelerate progress in the field by enhancing the bioavailability and therapeutic approaches [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Capsaicin has great potential to treat neuropathic pain and the possible mechanisms of action are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNanotechnology-based formulations such as emulsions and liposomes are the drug-delivery systems that are being explored for topically administered capsaicin [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The limited bioavailability of capsaicin impedes future researchers in their formulations and significantly restricts clinical uses. Consequently, it is imperative to pursue an appropriate drug delivery strategy that may enhance both the solubility and bioavailability of capsaicin concurrently. Based on previous research, our current study looked at the protective effect of an oral capsaicin-loaded liposome in diabetes-induced peripheral neuropathy.\u003c/p\u003e\u003cp\u003eThe changes in particle size and entrapment efficiency are internally consistent and compatible with the biophysical behavior that is anticipated for polymer-coated vesicles and mixed lipophilic/hydrophilic payloads. The possible explanation for the increase in particle size in capsaicin-loaded liposomes is due to the drug\u0026rsquo;s lipophilic nature, which is expected to partition into the bilayer, increasing bilayer thickness and decreasing membrane curvature. Subsequent chitosan coating of the capsaicin-loaded liposomes further increases the size to 252.90 nm due to the formation of hydrated polymer shell around the vesicles. The largest mean diameter (291.40 nm) was observed for the co-loaded chitosan-coated liposomes, because the internal aqueous volume would have increased as a result of co-loading with hydrophilic small molecules (metformin and gabapentin), which may lead to fusion events that increase the population of bigger particles, changed curvature, or swelling of the vesicle lumen.\u003c/p\u003e\u003cp\u003eEntrapment efficiency results support the physicochemical partitioning theory. Capsaicin-loaded liposomes achieved the highest entrapment efficiency at 78.94%, indicating efficient incorporation of the highly lipophilic capsaicin into the phospholipid bilayer. The capsaicin-loaded, chitosan-coated liposomes exhibited a slightly higher entrapment efficiency (80.20%), suggesting that the positively charged chitosan coating may stabilize the lipid bilayer and reduce drug leakage during post-processing. This observation is consistent with previous findings reported [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, co-loading gabapentin and metformin with capsaicin led to a decrease in capsaicin entrapment (68.92%), while gabapentin and metformin showed moderate entrapment of 55% and 47%, respectively. This reduction in entrapment efficiency could be due to the presence of two additional hydrophilic drugs that likely competed for aqueous-core and bilayer interface spaces, disrupting optimal lipid packing and reducing the hydrophobic compartment available for capsaicin. In another investigation, the particle size and entrapment efficiency of capsaicin-loaded liposomes were found to be 52.2 nm and 81.9% [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDiabetes was induced by intraperitoneal injection of STZ. The dose of capsaicin administered (250 \u0026micro;g/kg or 500 \u0026micro;g/kg) was based on an earlier study based on lipid multi-particulate formulation in rats, wherein the dose was 0.2 to 1 mg/kg [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The current study showed a significant increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in body weight compared to the positive control. Low dose of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy found to increase maximum body weight and showed a highly significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) compared to gabapentin and metformin. In our study, all the capsaicin-loaded liposomal preparations showed highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) reduction in the increasing levels of glucose and triglyceride compared to the positive control from day 15 to day 34. In order to decrease glucose and triglyceride levels, the most effective outcomes were achieved with chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy, with the values of glucose (130.9 mg/dl) and triglyceride (123.0 mg/dl) levels. The glucose level from this therapy showed no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e), whereas the value of triglyceride showed 11.7% more reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) than the metformin-treated group (glucose: 130.8 mg/dl and triglyceride: 139.3 mg/dl) by the 34th day. On the other hand, a highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) was observed in total cholesterol, LDL, and HDL levels from all capsaicin-loaded liposome-treated groups compared to the positive control from day 24 to day 34. Treatment with chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin exhibited the highest improvement and showed a statistically significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in LDL level, and exhibited no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) in total cholesterol and HDL levels compared to the metformin-treated group. Blank chitosan-coated liposomes group showed less significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in glucose, triglyceride, total cholesterol, and LDL levels, whereas it exhibited highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in HDL levels compared to the positive control on day 34. Comparable findings were documented in earlier research involving capsaicin-encapsulated nano-emulsions targeting obesity and chitosan microparticles infused with antidiabetic medication formulations for the management of DM [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Research indicated that chitosan has a beneficial impact on DM. The efficacy of chitosan microparticles containing antidiabetic pharmaceuticals was enhanced compared to regular medications (metformin and glibenclamide) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Multiple research studies on DPN indicated that rats with sciatic nerve injury displayed various behavioural changes, including reduced locomotor activity, prolonged heat tolerance delay, development of allodynia, and diminished nerve conduction velocities. As the sciatic nerve experiences ongoing regeneration, these alterations will demonstrate a proportional response [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our current study, all capsaicin-loaded liposome administered groups demonstrated highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in locomotor activity and paw withdrawal threshold compared to the positive control from day 24 to day 34. The best results were obtained with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy, which showed more improvement than the gabapentin-treated group by 13.8% in locomotor activity and 24.4% in paw withdrawal threshold. The comparable outcomes were achieved with low and high doses of capsaicin-loaded chitosan-coated liposomes in paw withdrawal threshold, whereas in locomotor activity, the low dose of capsaicin-loaded chitosan-coated liposomes showed more increase than the gabapentin-treated group, with a significant difference (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) at day 34. In heat tolerance latency, treatment with all capsaicin liposomal formulations demonstrated a significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in paw licking and paw jumping latency compared to the positive control. Low and high doses of capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin therapy produced no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) compared to the gabapentin-treated group. However, maximum improvement was observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group by the 34th day. From day 15 to day 34, treatment with all capsaicin-loaded liposome formulations, as well as chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin, restored the decreased nerve conduction velocity drastically (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) compared to the positive control. Low and high doses of capsaicin-loaded chitosan-coated liposomes were found to improve nerve conduction velocity less significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) than the gabapentin-treated group, while the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treatment demonstrated no significant difference (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) by day 34. The protective role of the blank chitosan-coated liposomes in nerve regeneration was not proven in behavioral analysis, except for nerve conduction velocity. In nerve conduction velocity, the blank chitosan-coated liposomes group displayed a slight improvement over the positive control at the 34th day. No research has been conducted on the sciatic nerve regeneration potential of capsaicin. A study demonstrated the transdermal administration of capsaicin nanoemulgel for diabetic neuropathy, showing superior enhancement of its antinociceptive effects compared to traditional gel [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. On the other hand, chitosan has a beneficial effect on peripheral nerve regeneration revealed by a study that supports our current findings [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Evidence suggests that hyperglycemia has been shown to cause oxidative stress, which plays a key role in the evolution of DPN, by causing proteins and monosaccharides to oxidize on their own. The disparity between the production of ROS, such as free radicals, and the body\u0026rsquo;s own antioxidant enzymes leads to the development of oxidative stress, i.e., increasing MDA level and decreasing SOD, GSH, and CAT concentrations [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Our current study showed that all the capsaicin-loaded liposome administered groups effectively suppressed free radical activity. The study findings demonstrated a significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in malondialdehyde (MDA) levels with all capsaicin encapsulated liposomal formulations treatment in the sciatic nerve, pancreas, and brain compared to the positive control. Additionally, catalase activity and GSH levels showed significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) improvement in the sciatic nerve, pancreas, and brain. SOD concentrations were increased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) in the sciatic nerve, while in the pancreas, low dose of capsaicin-loaded chitosan-coated liposomes, as well as chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin, showed highly significant improvement (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), whereas capsaicin-loaded liposomes and high dose of capsaicin-loaded chitosan-coated liposomes exhibited less significant increment (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in SOD concentrations compared to the positive control. Maximum reduction in MDA and improvement in GSH level in the sciatic nerve were observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treatment. In pancreas and brain, low dose of capsaicin-loaded chitosan-coated liposomes exhibited the highest attenuation in increasing MDA and showed an increase in GSH level. In sciatic nerve and pancreas catalase activity, the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin treatment showed maximum improvement, whereas in the brain, low dose of capsaicin-loaded chitosan-coated liposomes showed maximum increment. Treatment with a chitosan-coated liposome co-loaded with capsaicin, gabapentin, and metformin was also found to be more effective in increasing SOD concentrations in both the sciatic nerve and pancreas. In previous studies, similar findings were reported with capsaicin-loaded nanoliposome against liver oxidative stress [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and with capsaicin chitosan nanoparticles against rat mammary carcinogenesis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The histological findings of the rat\u0026rsquo;s sciatic nerve and pancreas provided supporting evidence, revealing differences between the liposomal-treated and positive control groups. The chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed a better orderly arrangement in nerve fiber bundles and also recovered the axons with the myelin sheet. The islet cells are seen to be in normal position and more in numbers, which were also observed in the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin-treated group as compared to individual treatment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe study\u0026rsquo;s results offer strong evidence that oral nanoformulation of capsaicin-loaded liposome, capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin has the potential to alleviate diabetes-induced peripheral neuropathy. The chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin showed the best outcomes over the other capsaicin formulations evaluated, compared to metformin in case of blood glucose and lipid profile estimation, and with gabapentin in behavioral analysis. Current formulations are unexplored to address pharmacokinetic and toxicological studies. Furthermore, more research is needed to explore the more promising results.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAMPK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAMP-activated protein kinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiabetes mellitus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDPN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiabetic peripheral neuropathy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEntrapment efficiency\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReduced glutathione\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHDL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHigh-density lipoprotein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehigh-performance liquid chromatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1-Interleukin-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e6-Interleukin-6\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLDL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLow-density lipoprotein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMalondialdehyde\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNCV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNerve conduction velocity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eкB-Nuclear factor kappa-B\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReactive oxygen species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSuperoxide dismutase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTZ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStreptozotocin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eα-Tumor necrosis factor-alpha\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTRPV1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransient receptor potential vanilloid 1 receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interest\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial and non-financial interests to disclose\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthical Approval\u003c/h2\u003e\u003cp\u003eThe animal study was approved by the Ethics Committee of the Institutional Animal Ethical Committee (IAEC Reference No. CAF/MMDU/25/IAEC-3).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cp\u003eNot Applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003cp\u003eNot Applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, Simran Saini and Sumeet Gupta; Data Curation, Simran Saini, Rama Devi, Soumyajit Panda, and Anroop B Nair; Formal Analysis, Kashish Wilson, Nidhi Gupta, Sarita Sharma, Seema Bansal, Bimal K Agrawal, and Reena V Saini; Writing-Original Draft Preparation, Simran Saini, Rama Devi, Soumyajit Panda, Kashish Wilson, Nidhi Gupta, Sarita Sharma, Seema Bansal, Bimal K Agrawal, and Reena V Saini; Writing-Review and Editing, Anroop B Nair, and Sumeet Gupta. All authors have contributed significantly to the work, have read, and approved the final manuscript for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to acknowledge Maharishi Markandeshwar College of Pharmacy, M.M. (DU), Mullana, Ambala, India, for providing numerous resources, guidance, technical support, and various facilities for the completion of the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data and supportive information are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang L, Wang Q, Su H, Cheng J (2021) Exosomes from adipose derived mesenchymal stem cells alleviate diabetic osteoporosis in rats through suppressing NLRP3 inflammasome activation in osteoclasts. J Biosci Bioeng 131:671\u0026ndash;678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jbiosc.2021.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiosc.2021.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHossain MJ, Al-Mamun M, Islam MR (2024) Diabetes mellitus, the fastest growing global public health concern: Early detection should be focused. \u003cem\u003eHealth science reports 7\u003c/em\u003e, e2004. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/hsr2.2004\u003c/span\u003e\u003cspan address=\"10.1002/hsr2.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu J, Hu Z, Luo Y, Liu Y, Luo W, Du X, Luo Z, Hu J, Peng S (2023) Diabetic peripheral neuropathy: pathogenetic mechanisms and treatment. Front Endocrinol 14:1265372. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2023.1265372\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2023.1265372\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTao Y, Zhang HY, MacGilchrist C, Kirwan E, McIntosh C (2025) Prevalence and risk factors of painful diabetic neuropathy: A systematic review and meta-analysis. Diabetes Res Clin Pract 222:112099. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.diabres.2025.112099\u003c/span\u003e\u003cspan address=\"10.1016/j.diabres.2025.112099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrevitali SC (2021) Peripheral Nerve Development and the Pathogenesis of Peripheral Neuropathy: the Sorting Point. Neurotherapeutics: J Am Soc Experimental Neurother 18:2156\u0026ndash;2168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13311-021-01080-z\u003c/span\u003e\u003cspan address=\"10.1007/s13311-021-01080-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoss KR, Saxena S (2025) Schwann Cells in Neuromuscular Disorders: A Spotlight on Amyotrophic Lateral Sclerosis. Cells 14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cells14010047\u003c/span\u003e\u003cspan address=\"10.3390/cells14010047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBosch-Queralt M, Fledrich R, Stassart RM (2023) Schwann cell functions in peripheral nerve development and repair. Neurobiol Dis 176:105952. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nbd.2022.105952\u003c/span\u003e\u003cspan address=\"10.1016/j.nbd.2022.105952\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eM SL, O P (2021) Inflammatory biomarkers as a part of diagnosis in diabetic peripheral neuropathy. J Diabetes Metab Disord 20:869\u0026ndash;882. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s40200-021-00734-1\u003c/span\u003e\u003cspan address=\"10.1007/s40200-021-00734-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNashtahosseini Z, Eslami M, Paraandavaji E, Haraj A, Dowlat BF, Hosseinzadeh E, Oksenych V, Naderian R (2025) Cytokine Signaling in Diabetic Neuropathy: A Key Player in Peripheral Nerve Damage. Biomedicines 13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines13030589\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines13030589\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChakraborty T, Gupta S, Nair A, Chauhan S, Saini V (2021) Wound healing potential of insulin-loaded nanoemulsion with Aloe vera gel in diabetic rats. J Drug Deliv Sci Technol 64:102601\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta S, Sharma H, Morsy MA, Nair AB, Kaur K, Kumar S, Dhanawat M (2021) Protective effect of Plumeria Obtusa leaves extracts on dexamethasone-induced insulin-resistance diabetes mellitus in rats. Indian J Pharm Educ Res 55:S184\u0026ndash;S192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5530/ijper.55.1s.49\u003c/span\u003e\u003cspan address=\"10.5530/ijper.55.1s.49\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeng J, Li X, Tian H, Liu C, Guo Y, Zhang S, Chu Y, Li J, Wang Y, Zhang L (2020) Neuroprotective effect of diosgenin in a mouse model of diabetic peripheral neuropathy involves the Nrf2/HO-1 pathway. BMC Complement Med Ther 20:126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12906-020-02930-7\u003c/span\u003e\u003cspan address=\"10.1186/s12906-020-02930-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang W, Zhang Y, Fan J, Feng Z, Song X (2024) Pharmacological activity of capsaicin: Mechanisms and controversies (Review). Mol Med Rep 29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/mmr.2024.13162\u003c/span\u003e\u003cspan address=\"10.3892/mmr.2024.13162\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng Y, Chen J, Wu X, Zhang X, Hu C, Kang Y, Lin J, Li J, Huang Y, Zhang X et al (2022) Enhanced Anti-Inflammatory Effects of Silibinin and Capsaicin Combination in Lipopolysaccharide-Induced RAW264.7 Cells by Inhibiting NF-κB and MAPK Activation. Front Chem 10:934541. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fchem.2022.934541\u003c/span\u003e\u003cspan address=\"10.3389/fchem.2022.934541\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang W, Lan Y, Chen C, Song M, Xiao J, Huang Q, Cao Y, Ho CT, Lu M (2023) Modulating effects of capsaicin on glucose homeostasis and the underlying mechanism. Crit Rev Food Sci Nutr 63:3634\u0026ndash;3652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10408398.2021.1991883\u003c/span\u003e\u003cspan address=\"10.1080/10408398.2021.1991883\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHall OM, Broussard A, Range T, Carroll Turpin MA, Ellis S, Lim VM, Cornett EM, Kaye AD (2020) Novel Agents in Neuropathic Pain, the Role of Capsaicin: Pharmacology, Efficacy, Side Effects, Different Preparations. Curr Pain Headache Rep 24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11916-020-00886-4\u003c/span\u003e\u003cspan address=\"10.1007/s11916-020-00886-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDaeihamed M, Dadashzadeh S, Haeri A, Akhlaghi MF (2017) Potential of Liposomes for Enhancement of Oral Drug Absorption. Curr Drug Deliv 14:289\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1567201813666160115125756\u003c/span\u003e\u003cspan address=\"10.2174/1567201813666160115125756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohammed L, Nourddine H, Saad EF, Abdelali D, Hamid R (2021) Chitosan-covered liposomes as a promising drug transporter: nanoscale investigations. RSC Adv 11:1503\u0026ndash;1516. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d0ra08305d\u003c/span\u003e\u003cspan address=\"10.1039/d0ra08305d\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkrawi SH, Gorain B, Nair AB, Choudhury H, Pandey M, Shah JN, Venugopala KN (2020) Development and optimization of naringenin-loaded chitosan-coated nanoemulsion for topical therapy in wound healing. Pharmaceutics 12:1\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/pharmaceutics12090893\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics12090893\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumria R, Al-Dhubiab BE, Shah J, Nair AB (2018) Formulation and evaluation of chitosan-based buccal bioadhesive films of zolmitriptan. J Pharm Innov 13:133\u0026ndash;143\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu Y, Wang M, Zhang J, Peng W, Firempong CK, Deng W, Wang Q, Wang S, Shi F, Yu J et al (2015) Improved oral bioavailability of capsaicin via liposomal nanoformulation: preparation, in vitro drug release and pharmacokinetics in rats. Arch Pharm Res 38:512\u0026ndash;521. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12272-014-0481-7\u003c/span\u003e\u003cspan address=\"10.1007/s12272-014-0481-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorsy MA, Nair AB (2018) Prevention of rat liver fibrosis by selective targeting of hepatic stellate cells using hesperidin carriers. Int J Pharm 552:241\u0026ndash;250\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJahanfar S, Gahavami M, Khosravi-Darani K, Jahadi M, Mozafari MR (2021) Entrapment of rosemary extract by liposomes formulated by Mozafari method: physicochemical characterization and optimization. Heliyon 7:e08632. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2021.e08632\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2021.e08632\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlMulhim FM, Nair AB, Aldhubiab B, Shah H, Shah J, Mewada V, Sreeharsha N, Jacob S, Design (2023) Development, Evaluation, and In Vivo Performance of Buccal Films Embedded with Paliperidone-Loaded Nanostructured Lipid Carriers. Pharmaceutics 15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/pharmaceutics15112530\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics15112530\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhosh S, Haldar S, Gupta S, Bisht A, Chauhan S, Kumar V, Roy P, Lahiri D (2020) Anisotropically Conductive Biodegradable Scaffold with Coaxially Aligned Carbon Nanotubes for Directional Regeneration of Peripheral Nerves. ACS Appl Bio Mater 3:5796\u0026ndash;5812. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acsabm.0c00534\u003c/span\u003e\u003cspan address=\"10.1021/acsabm.0c00534\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSachan N, Saraswat N, Chandra P, Khalid M, Kabra A (2022) Isolation of Thymol from Trachyspermum ammi Fruits for Treatment of Diabetes and Diabetic Neuropathy in STZ-Induced Rats. \u003cem\u003eBioMed research international 2022\u003c/em\u003e, 8263999. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2022/8263999\u003c/span\u003e\u003cspan address=\"10.1155/2022/8263999\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGosavi DD, Kamdi AS, Kalambe SM, Bohra PN (2020) The spontaneous motor activity of aqueous extract of Withania coagulans fruits in Swiss albino mice by actophotometer. Int J Basic Clin Pharmacol 9:1440\u0026ndash;1443. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18203/2319-2003.ijbcp20203633\u003c/span\u003e\u003cspan address=\"10.18203/2319-2003.ijbcp20203633\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoshi A, Choudhury S, Baghel VS, Ghosh S, Gupta S, Lahiri D, Ananthasuresh GK, Chatterjee K (2023) 4D Printed Programmable Shape-Morphing Hydrogels as Intraoperative Self-Folding Nerve Conduits for Sutureless Neurorrhaphy. Adv Healthc Mater 12:e2300701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/adhm.202300701\u003c/span\u003e\u003cspan address=\"10.1002/adhm.202300701\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Huang F, Xu Y, Xiang W, Xie C (2021) TRPV1 is involved in the antinociceptive effects of resveratrol in paclitaxel-induced neuropathic pain. All Life 14:66\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/26895293.2020.1861111\u003c/span\u003e\u003cspan address=\"10.1080/26895293.2020.1861111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi X, Chen Y, Nadeem L, Xu G (2013) Beneficial effect of TNF-α inhibition on diabetic peripheral neuropathy. J Neuroinflamm 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1742-2094-10-69\u003c/span\u003e\u003cspan address=\"10.1186/1742-2094-10-69\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNurdiana S, Goh YM, Ahmad H, Dom SM, Syimal'ain Azmi N, Noor M, Zin NS, Ebrahimi M (2017) Changes in pancreatic histology, insulin secretion and oxidative status in diabetic rats following treatment with Ficus deltoidea and vitexin. BMC Complement Altern Med 17:290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12906-017-1762-8\u003c/span\u003e\u003cspan address=\"10.1186/s12906-017-1762-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNjan AA, Adenuga FO, Ajayi AM, Sotunde O, Ologe MO, Olaoye SO, Erdogan ON, Iwalewa OE (2020) Neuroprotective and memory-enhancing effects of methanolic leaf extract of Peristrophe bicalyculata in rat model of type 2 diabetes mellitus. Heliyon 6:e04011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.heliyon.2020.e04011\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2020.e04011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSandireddy R, Yerra VG, Komirishetti P, Areti A, Kumar A (2016) Fisetin Imparts Neuroprotection in Experimental Diabetic Neuropathy by Modulating Nrf2 and NF-κB Pathways. Cell Mol Neurobiol 36:883\u0026ndash;892. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10571-015-0272-9\u003c/span\u003e\u003cspan address=\"10.1007/s10571-015-0272-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEid SA, Rumora AE, Beirowski B, Bennett DL, Hur J, Savelieff MG, Feldman EL (2023) New perspectives in diabetic neuropathy. Neuron 111:2623\u0026ndash;2641. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neuron.2023.05.003\u003c/span\u003e\u003cspan address=\"10.1016/j.neuron.2023.05.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMerghany RM, Saleh RA, Hamed AA, El-Dessouki AM, El-Shiekh RA, Zaki ES, Mahfoz AM (2025) Natural therapy proposed for the management of diabetic peripheral neuropathy (DPN). Inflammopharmacology. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10787-025-01790-2\u003c/span\u003e\u003cspan address=\"10.1007/s10787-025-01790-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKabir MT, Tabassum N, Uddin MS, Aziz F, Behl T, Mathew B, Rahman MH, Akter R, Rauf A, Aleya L (2021) Therapeutic Potential of Polyphenols in the Management of Diabetic Neuropathy. \u003cem\u003eEvidence-based complementary and alternative medicine: eCAM 2021\u003c/em\u003e, 9940169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2021/9940169\u003c/span\u003e\u003cspan address=\"10.1155/2021/9940169\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePottathil S, Nain P, Morsy MA, Kaur J, Al-Dhubiab BE, Jaiswal S, Nair AB (2020) Mechanisms of antidiabetic activity of methanolic extract of Punica granatum leaves in nicotinamide/streptozotocin-induced type 2 diabetes in rats. Plants 9:1609\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhursheed R, Singh SK, Wadhwa S, Gulati M, Kapoor B, Awasthi A, Kr A, Kumar R, Pottoo FH, Kumar V et al (2021) Opening eyes to therapeutic perspectives of bioactive polyphenols and their nanoformulations against diabetic neuropathy and related complications. Expert Opin Drug Deliv 18:427\u0026ndash;448. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/17425247.2021.1846517\u003c/span\u003e\u003cspan address=\"10.1080/17425247.2021.1846517\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia T, Rao J, Zou L, Zhao S, Yi Z, Wu B, Li L, Yuan H, Shi L, Zhang C et al (2017) Nanoparticle-Encapsulated Curcumin Inhibits Diabetic Neuropathic Pain Involving the P2Y12 Receptor in the Dorsal Root Ganglia. Front NeuroSci 11:755. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnins.2017.00755\u003c/span\u003e\u003cspan address=\"10.3389/fnins.2017.00755\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSebaaly C, Trifan A, Sieniawska E, Greige-Gerges H (2021) Chitosan-coating effect on the characteristics of liposomes: A focus on bioactive compounds and essential oils: A review. Processes 9:445\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSahin K, Kucuk O, Orhan C, Sahin E, Fowler K, White T, Durkee S, Bellamine A (2021) Bioavailability of a Capsaicin Lipid Multi-particulate Formulation in Rats. Eur J Drug Metab Pharmacokinet 46:645\u0026ndash;650. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13318-021-00697-x\u003c/span\u003e\u003cspan address=\"10.1007/s13318-021-00697-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu M, Cao Y, Ho CT, Huang Q (2017) The enhanced anti-obesity effect and reduced gastric mucosa irritation of capsaicin-loaded nanoemulsions. Food Funct 8:1803\u0026ndash;1809. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c7fo00173h\u003c/span\u003e\u003cspan address=\"10.1039/c7fo00173h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAvram I, Confederat L, Constantin SM, Stan CI, Profire L Biological evaluation of chitosan-antidiabetic drug formulations for the treatment of diabetes mellitus\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePawar SH, Gite AA, Patil RA (2025) Amelioration of Paclitaxel Induced Neuropathy by Gymnema sylvestre in Wistar Rats. Asian J Biol Sci 18:602\u0026ndash;614\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaab M, Raafat K, El-Maradny H (2022) Transdermal Delivery of Capsaicin Nanoemulgel: Optimization, Skin Permeation and In Vivo Activity against Diabetic Neuropathy. Adv Pharm Bull 12:780\u0026ndash;790. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.34172/apb.2022.080\u003c/span\u003e\u003cspan address=\"10.34172/apb.2022.080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePop NL, Mitrea D-R, Urdă-C\u0026icirc;mpean AE, Glossa-Athanasoula A, Toma VA, Moldovan R, Nan A, Decea N, Filip A, Clichici S (2021) Chitosan oral administration stimulates regeneration after experimentally induced peripheral nerve injury. \u003cem\u003eHealth, Sports \u0026amp; Rehabilitation Medicine 22\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlharthy KM, Balaha MF, Devi S, Altharawi A, Yusufoglu HS, Aldossari RM, di Alam A (2023) Giacomo, V. Ameliorative Effects of Isoeugenol and Eugenol against Impaired Nerve Function and Inflammatory and Oxidative Mediators in Diabetic Neuropathic Rats. Biomedicines 11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines11041203\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines11041203\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGiri TK, Mukherjee P, Barman TK, Maity S (2016) Nano-encapsulation of capsaicin on lipid vesicle and evaluation of their hepatocellular protective effect. Int J Biol Macromol 88:236\u0026ndash;243. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijbiomac.2016.03.056\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2016.03.056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDhamodharan K, Vengaimaran M, Sankaran M (2021) Capsaicin Encapsulated Chitosan Nanoparticles Augments Anticarcinogenic and Antiproliferative Competency Against 7,12 Dimethylbenz(a)anthracene Induced Experimental Rat Mammary Carcinogenesis. J Pharm Res Int 33:126\u0026ndash;144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.9734/jpri/2021/v33i41A32311\u003c/span\u003e\u003cspan address=\"10.9734/jpri/2021/v33i41A32311\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Capsaicin-loaded liposomes, diabetic neuropathy, behavioural analysis, nerve conduction velocity, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-8011851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8011851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiabetic peripheral neuropathy is a major complication arising from diabetes mellitus. It is accelerated by oxidative stress, inflammation, ER stress, and mitochondrial dysfunction. Capsaicin possesses antioxidant, anti-inflammatory, antidiabetic, and neuroprotective properties but exhibits limited bioavailability. The present study aimed to develop an oral capsaicin liposomal formulation and investigate its potential against the diabetic peripheral neuropathy rat model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree formulations, namely capsaicin-loaded liposomes, capsaicin-loaded chitosan-coated liposomes, and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin, were formulated and characterized. Rats were divided into nine groups in order to determine the liposomal formulation's capability by conducting several experiments. These included blood glucose level, lipid profile estimation, behavioural parameters, nerve conduction velocity, oxidative stress biomarkers, and histological examinations of tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe particle size of capsaicin-loaded liposomes, capsaicin-loaded chitosan-coated liposomes and chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin was 183.45 nm, 252.90 nm and 291.4 nm, respectively. The entrapment efficiency of capsaicin in formulations varied between 69–80%. Liposomal formulations of capsaicin reduced the levels of blood glucose and lipid profile. Additionally, significant improvement was observed in nerve conduction velocity, behavioural parameters, and oxidative stress biomarkers. Histological findings demonstrated that capsaicin liposomal formulations alleviated the sciatic nerve degeneration and showed improvement in the islet cells of the pancreas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormulated capsaicin exhibits sciatic nerve regeneration, antidiabetic, and antioxidant properties as evidenced by increased nerve conduction velocity and effective regulation of metabolic parameters. The best outcomes were observed with the chitosan-coated liposomes co-loaded with capsaicin, gabapentin, and metformin. These findings support that an oral formulation of capsaicin-loaded liposomes has great potential against diabetic peripheral neuropathy.\u003c/p\u003e","manuscriptTitle":"Assessment of Capsaicin Liposomes in a Rat Model of Diabetes-induced Peripheral Neuropathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 11:04:05","doi":"10.21203/rs.3.rs-8011851/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0af88582-6169-48f3-a8c4-c3adcd3cde65","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-25T10:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 11:04:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8011851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8011851","identity":"rs-8011851","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}