Polymeric nanomicelles for piperine delivery: Preparation, characterization and evaluation of antioxidant and in-vivo anti-inflammatory properties

Polymeric nanomicelles for piperine delivery: Preparation, characterization and evaluation of antioxidant and in-vivo anti-inflammatory properties | 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 Polymeric nanomicelles for piperine delivery: Preparation, characterization and evaluation of antioxidant and in-vivo anti-inflammatory properties Mahboobeh Zare, Hakime Gavzan, Malihe Noruzi Sarkati, Mohammad Ali Mobaraki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5945336/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 Purpose Piperine, one of the bioactive alkaloids, exhibits restricted therapeutic effectiveness owing to its inadequate solubility and bioavailability. Nanomicelles hold great potential as drug delivery systems for drugs with low water solubility. This study aimed to create micelles that contain tragacanth gum, vitamin D, and folic acid to encapsulate piperine. Methods Piperine was isolated from black pepper and subsequently loaded on synthesized polymeric micelles. The antioxidant activity of piperine-loaded micelles was tested by DPPH⋅ radical method. Moreover, the peripheral anti-inflammatory activity of nanomicelles was assessed using the formalin-induced paw edema method. Results According to the results obtained from this investigation, the utilization of nanomicelles demonstrated an enhancement in the radical scavenging activity of piperine, consequently leading to a notable reduction in the required dosage of piperine for its anti-inflammatory effects. Conclusion The findings indicate that the utilization of synthesized micelle as an innovative targeted delivery system could potentially augment the therapeutic characteristics of piperine. Micelle Tragacanth gum Piperine Anti-inflammatory Antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Phytochemicals, primarily sourced from plants, possess biological activities and can be utilized for treating diverse illnesses [ 1 ]. Numerous natural herbal extracts with validated anti-inflammatory properties have been discovered through research utilizing in vitro and in vivo inflammation models [ 2 ]. Black pepper ( Piper nigrum L. ) has been employed as a culinary condiment and possesses medicinal properties [ 3 ]. Black pepper serves multiple functions beyond culinary use, including medicinal applications, preservation, and incorporation in perfumes. Numerous physiological impacts of black pepper, its derivatives, and its primary bioactive compound, PIP (piperine), have been documented in scientific literature over the past few years [ 4 ]. PIP has demonstrated a diverse array of pharmacological properties in various in vitro and in vivo experimental studies. These encompass antiproliferative, antitumor, antiangiogenic, antioxidant, antidiabetic, anti-obesity, cardioprotective, antimicrobial, antiaging, and immunomodulatory effects. Furthermore, PIP has been extensively studied for its hepatoprotective, anti-allergic, anti-inflammatory, and neuroprotective properties [ 5 – 7 ]. PIP effectively suppressed the release of proinflammatory and inflammatory agents [ 8 – 10 ] and mitigated tissue edema in the experimental models of inflammation [ 9 , 11 ]. The assessment of the influence of PIP on S. aureus endometritis in a murine model revealed that PIP exhibits the ability to mitigate inflammatory lesions in S. aureus endometritis to a considerable extent, employing various mechanisms [ 12 ]. However, the application of PIP for therapeutic intentions is constrained by its insufficient solubility in aqueous solutions [ 13 ]. A potential solution to address this limitation can be found within the realm of nanotechnology, a field that focuses on manipulating matter at the atomic level to create innovative structures, materials, and devices. The core principle of nanotechnology involves synthesizing artificial nanoparticles with unique attributes such as small size, high surface-to-mass ratio, shape, crystallinity, surface charge, reactive surface groups, dissolution rate, agglomeration state, and dispersal, which grant them properties that differ significantly from bulk particles of the same composition [ 13 ]. Nanocarriers are utilized for the transportation of active substances, particularly drugs or biologics, in therapeutic applications. The effectiveness of drug delivery has been extensively investigated using various nanocarriers, including micelles, polymers, carbon-based materials, lipid-based substances, and others [ 14 ]. Nanomicelles are self-assembled nanoscale colloidal particles with a hydrophobic/hydrophilic core-shell structure, which are suitable as carriers for poorly water-soluble drugs and are regarded as one of the most promising drug delivery systems. Amphiphilic copolymers are constructed by grafting hydrophobic molecules onto hydrophilic polymers and can readily form self-assembled micelles in an aqueous solution. The use of natural polysaccharides as hydrophilic micelle shells is widely recommended due to their high bioactivity, abundant availability, and easy modification which makes them suitable for sustained and controlled drug delivery, targeting ability, and long-circulation [ 15 – 17 ]. TG (Tragacanth gum), a neutral polymer, has garnered considerable interest as a hydrophilic backbone in this delivery system. TG is a natural plant gum that is used for various applications in industries and biomedicine. Its cost-effectiveness and ready availability, coupled with its desirable biocompatibility and biodegradability, make it an advantageous polysaccharide [ 18 ]. On the other hand, VD (Vitamin D), known as cholecalciferol, is classified as a fat-soluble secosteroid [ 19 ]. FA (Folic acid) is applicable to inflammation-targeting drug delivery systems because recent studies have shown that the folate receptor beta, which has a high affinity for FA, is specifically expressed by activated macrophages [ 20 ]. This study outlines a simple method for the synthesis of a novel nanocopolymeric micelle composed of TG as the hydrophilic component, VD as the hydrophobic segment and FA as the targeting ligand for targeted drug delivery systems (Scheme 1 ). The morphology and size of synthesized nano micelle was investigated and the antioxidant activity and anti-inflammatory profile of this drug were analyzed in detail. 2. Methodology 2.1 Equipments and materials Thionyl chloride, FA and organic solvents were procured from Merck. The FT-IR spectra were recorded using a spectrometer (AVATAR, Thermo, USA). The Proton nuclear magnetic resonance ( 1 H NMR) spectra were obtained using a 400 MHz Bruker instrument. Atomic force microscopy (DME company. Denmark) was used for the study of morphology. Particle size and zeta potential were monitored using dynamic light scattering (DLS) instrument (HORIBA, SZ-100, Kyoto, Japan). 2.2 Extraction of PIP PIP was extracted and purified from black pepper utilizing a previously documented technique [ 21 ]. In summary, a quantity of 100 g of black pepper seed powder was subjected to soxhlet extraction using 500 mL of petroleum ether, followed by filtration and subsequent extraction with 300 mL of ethanol for a duration of 2 h. The resulting solution was concentrated to a volume of 30 mL using a rotary evaporator, and subsequently combined with 2 mL of alcoholic KOH (10%). After a period of 30 minutes, the mixture was filtered and stored in a refrigerator overnight, yielding yellowish-brown needles of PIP. These needles were further purified through recrystallization in ethanol, resulting in the production of pure PIP needles. The melting point of the obtained PIP ranged from 127–130°C. 2.3 Synthesis of PIP-loaded micelles Firstly, to synthesize folic acid chloride (FA-Cl), 0.500 g of FA was combined with 15 ml of thionyl chloride and stirred for a duration of 2 h. Subsequently, the resulting mixture was evaporated and subjected to acetone washing, yielding FA-Cl [ 22 ]. Synthesis of vitamin D chloride (VD-Cl) was done by combining VD with 15 ml of thionyl chloride and stirring the mixture for a duration of 2 h. Subsequently, the excess thionyl chloride was evaporated, and the resulting reaction mixture was subjected to acetone washing, resulting in the formation of VD-Cl. Polymeric micelles (FA-TG-VD) were obtained from the reaction of TG with FA-Cl and VD-Cl. For this reaction, solution of VD-Cl (0.32 g in DMSO) and FA-Cl (0.28 g in DMSO) were added separately to a solution of TG (0.5 g in H 2 O). The reaction mixture was stirred for 36 h, resulting in the formation of polymeric micelles through esterification and etherification reactions between the OH groups of TG and the COCl groups of FA-Cl and C-Cl groups of VD-Cl. The product was purified by dialysis against distilled water, followed by lyophilization, yielding a yellow powder of FA-TG-VD. Finally, the incorporation of PIP into FA-TG-VD micelles was done by dropwise addition of PIP solution (0.5 g in 25 mL of acetone) in to FA-TG-VD mixture (0.5 g in 150 mL of H 2 O), followed by stirring for 24 hours. The acetone was then removed using a rotary evaporator, and the resulting mixture was subjected to centrifugation. The supernatant obtained was subsequently subjected to lyophilization, resulting in the production of PIP-loaded FA-TG-VD micelles [ 23 ]. 2.4 Evaluation of drug loading (DL) The PIP loading was quantified by employing a UV-Vis spectrophotometer set at a wavelength of 342 nm. A solution of 1 mg of PIP-loaded synthesized micelles in 10 mL of DMSO was prepared and subjected to 24 hours of agitation. Subsequently, the resulting supernatant was collected and the PIP concentration was determined using a previously established methodology [ 24 ]. 2.5 Evaluation of antioxidant activity The DPPH radical scavenging method was employed to monitor the radical scavenging capacity of PIP-loaded micelles [ 25 ]. Five varying concentrations of PIP-loaded micelles were dispersed in 2.5 mL of distilled ethanol, followed by the addition of 2.5 mL of DPPH solution (200µM). The reaction mixtures were incubated for 60 minutes, and subsequently, the absorbance was determined at 517 nm. Additionally, parallel experiments were conducted using pure PIP and FA-TG-VD to compare their interactions with DPPH. 2.6 Evaluation of anti-inflammatory activities The peripheral anti-inflammatory activity of PIP micelles was assessed by evaluating the formalin-induced paw edema using the prescribed methodology. Adult male NMRI mice (20–30gr, Pasteur Institute of Iran, Amol) were housed under standard environmental conditions, including controlled temperature (22 ± 2°C), humidity of 40–50%, a 12-hour light and dark cycle, and provided with unlimited access to food and water. All animal housing and experiments were conducted in strict accordance with the institutional Guidelines for Care and Use of Laboratory Animals at Amol University of Special Modern Technologies. The present study's experiments were granted approval by the Institutional Animals Ethics Committee of Amol University of Special Modern Technologies, with the authorization code IR.ASMT.REC.1403.001. The primary objective was to reduce the number of animals used and mitigate their associated suffering. PIP was solubilized in a suspension containing Tween-80 and normal saline (0.9% NaCl) at a 1:25 ratio, serving as the vehicle [ 8 ]. Formalin, diclofenac, and PIP micelle were dissolved in a solution of normal saline. To elicit peripheral inflammation and pain, mice were subcutaneously injected with 20 µl of 2.5% formalin solution in the subplantar region of the right hind paw. The animals were allocated randomly into five groups, with eight animals in each group. They received either vehicle, PIP (15 mg/kg), PIP micelle (10 and 15 mg/kg), or diclofenac (20 mg/kg). Formalin injection was administered 15 minutes prior to the administration of vehicle, PIP and PIP micelle, and 30 minutes prior to diclofenac [ 26 ]. The degree of paw edema was assessed by measuring paw thickness with a digital caliper at various time intervals (pre-injection, 1, 2, 3, 4 and 24 h post-injection). The extent of edema suppression was calculated using a designated equation. ∆v = v2 – v1 v1: the initial thickness of the paw prior to the administration of formalin, v2: the subsequent thickness of the paw post formalin injection. 3. Results and discussion As shown in Fig. 1 , the FTIR spectra of PIP exhibit a prominent peak at 1634 cm − 1 , which corresponds to the amide group. Additionally, the range between 2802–2939 cm − 1 displays peaks indicative of aliphatic C-H bonds, while the range between 3000–3300 cm − 1 is associated with aromatic C-H bonds. In the 1 HNMR spectrum of PIP illustrated in Fig. 2 , the chemical shifts for cycloaliphatic, aliphatic (methylenedioxy), alkenic, and aromatic hydrogen were observed at 1.59–1.67 and 3.53–3.63, 5.98, 6.43–6.79 and 6.88–7.43 ppm respectively. The characterization of PIP-loaded synthesized micelles was conducted using FT-IR as illustrated in Fig. 3 , DLS as shown in Fig. 4 , and AFM that is shown in Fig. 5 . FTIR measurement was employed to analyze synthesized micelles and PIP-loaded micelles. The spectra revealed a broad peak at 3000–3500 cm − 1 , which was attributed to the stretching vibrations of the -OH group of gum and FA. Additionally, bands at approximately 1098, 1387, and 2925 cm − 1 were observed, which were related to stretching vibrations of C-O and C-H groups of TG. The FT-IR spectrum of the FA-TG-VD showed peaks at 1690 cm − 1 , 1600 cm − 1 , and 1300 − 1100 cm − 1 , confirming that VD and FA were grafted to TG. Encapsulation of PIP in the micelle was confirmed by a peak at 1000–1300 cm − 1 , assigned to the stretching vibrations of C-O in the ether group of PIP. The dimensions and morphology of self-assembled micelle nanoparticles containing PIP were analyzed using light-scattering measurements and AFM. The PIP-loaded micelles displayed a mean diameter of 181 nm, with a narrow size distribution in the DLS experiment that is shown in Fig. 4 . As shown in Fig. 5 , the AFM revealed that the micelles were spherical in shape, and the size observed was consistent with that measured by DLS [ 27 ]. Nanoparticles exhibiting a high zeta potential demonstrate favorable physical stability due to the electrostatic repulsion between individual particles [ 28 , 29 ]. As shown in Fig. 6 , Zeta potential value of PIP-loaded micelles was − 24.5 mV. The results suggest that the prepared nanoparticle systems were in a relatively stable condition. Based on a previously investigation concerning PIP-encapsulated nano-sized liposomes, it was observed that liposomes incorporating PIP demonstrated a notable level of stability [ 30 ]. The drug loading capacity of FA-TG-VD micelles was determined to be approximately 50% in the current study, indicating effective loading of PIP into the nanocarrier. Various studies demonstrated that the incorporation of a self-emulsifying drug delivery system in a formulation significantly improved the oral bioavailability of PIP [ 30 , 31 ]. The DPPH assay was applied to investigate the antioxidant properties of prepared micelles. As shown in Fig. 7 , the IC50 values of PIP-loaded micelles and PIP were 2.4 and 4.2 mg/mL respectively. The scavenging activity of PIP-loaded FA-TG-VD micelles was improved by increasing its concentrations. The carrier as a blank had a less antioxidant activity thus the free radical scavenging activity of PIP-loaded FA-TG-VD micelles are mainly due to the presence of PIP. The incorporation of PIP into the micelle nanoparticles enhanced antioxidant effects of PIP. This observation highlights the significant positive impact of these nanoparticles on augmenting the antioxidant properties of PIP. Various studies showed that PIP has antioxidant actions [ 6 , 32 ]. Anti-inflammatory activity PIP and PIP micelle showed significant anti-inflammatory effects up to 24 hours. The results revealed that the inflammation was significantly inhibited by 15 mg/kg dose of PIP and 15 mg/kg dose of PIP micelle as well as 20 mg/kg dose of standard drug (diclofenac) as compared to the empty micelle in 1, 2, 3, and 24 h (p < 0.001) after injection of formalin. PIP has been known as a potential anti-inflammatory substance, and its molecular mechanism may involve regulating the key factors of the NF-B and MAPK signalling pathways. This alkaloid also inhibits the expression of IL6 and MMP13 and reduces the production of PGE2 in a dose-dependent manner [ 9 , 33 ]. The present study has also demonstrated the anti-inflammatory efficacy of PIP. Furthermore, we observed the impact of the nano drug delivery system on the anti-inflammatory characteristics of PIP. In a comparable investigation, the paw edema and leukocyte migration activity were notably diminished in rats that received intraperitoneal injections of nanoformulations as opposed to free PIP [ 34 ]. As illustrated in Fig. 8 , PIP micelle siginificantly reduced the formalin- induced paw edema as well as PIP and standard drug (P < 0.001) starting from 1hr of formalin injection and continued to 24 h compared to vehicle group. It is noteworthy that there was no significant difference among PIP, PIP micelle and diclofenac in 1, 2, and 3hrs after formalin injection. Given that the drug loading capacity within this drug delivery system is approximated to be 50%, it can be inferred that the anti-inflammatory impact of PIP-loaded nanomicelle is not markedly distinct from the anti-inflammatory effect of PIP administered at twice the dosage. Essentially, employing this drug delivery system allows for a substantial reduction in the PIP dosage without compromising its anti-inflammatory efficacy. The anti-inflammatory properties of PIP were demonstrated in the in vitro and in vivo studies and our results are in line with the previous works. PIP attenuated LPS-induced inflammatory cytokine production in BV2 microglia [ 35 ]. In addition, PIP was found to inhibit cerebral ischemia- reperfusion-induced inflammation [ 36 ]. Also, PIP exhibited a notable reduction in the production of TNF-α and IL-1β induced by LPS. Furthermore, it was demonstrated that PIP significantly impeded the infiltration of neutrophils induced by LPS and the production of inflammatory cytokines [ 37 ]. In addition, PIP caused a reduction in the expression of proinflammatory cytokines in paw tissue and spinal cord samples in mice that were subjected to the formalin test [ 10 ]. This finding suggests that this substance has the capacity to alleviate both peripheral and central inflammation. Based on the aforementioned findings, it is possible to optimize the utilization of bioactive substances, specifically those with anti-inflammatory properties, through the development of nano-drug delivery systems. The utilization of this drug delivery system is deemed more advantageous than the conventional employment of PIP, as it not only enhances the nanoscale characteristics and augments the antioxidant impact, but it has also been meticulously engineered to elicit a distinct influence on inflammation. 4. Conclusion Extensive research has been conducted on the medicinal properties of PIP, a naturally occurring substance derived from plants. However, the clinical application of PIP has faced challenges due to its low bioavailability, poor water solubility, and complex release kinetics. To overcome these obstacles, a proposed solution involves the use of a nanoparticle delivery system for PIP. This study suggests that nano micelles hold promise as a nanocarrier for PIP, as they have the potential to enhance its antioxidant and anti-inflammatory. Moreover, the nano micelle formulation, which incorporates FA and encapsulates PIP, exhibits unique characteristics that improve its bioavailability and water solubility. These distinctive properties enable targeted interactions with inflammatory cells, making it a specialized design for modulating inflammatory processes. Looking ahead, the development of nanoparticles with specific designs will facilitate efficient and targeted management of pathogenic processes, thereby improving both the duration and quality of life for patients. Declarations CRediT author statement Mahboobeh Zare: Conceptualization, Methodology, Validation, Investigation,Resources, Writing - Review and Editing,Supervision Hakime Gavzan: Conceptualization, Methodology, Validation,Formal analysis, Investigation, Resources, Writing - Review & Editing Malihe Noruzi Sarkati: Investigation, Resources Mohammad Ali Mobaraki : Writing - Original Draft Acknowledgment The authors acknowledge the financial support received from the Research Council of Amol University of Special Modern Technologies. Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: Supported by the Research Council of Amol University of Special Modern Technologies. Availability of data and materials The data supporting the results of this study can be obtained from the corresponding authors upon reasonable request. References N. Mendoza and E. M. E. Silva, "Introduction to phytochemicals: secondary metabolites from plants with active principles for pharmacological importance," Phytochemicals: Source of antioxidants and role in disease prevention, vol. 25, pp. 1-5, 2018. F. Maione, R. Russo, H. Khan, and N. Mascolo, "Medicinal plants with anti-inflammatory activities," Natural product research, vol. 30, no. 12, pp. 1343-1352, 2016. Z. Zarai, E. Boujelbene, N. B. Salem, Y. Gargouri, and A. Sayari, "Antioxidant and antimicrobial activities of various solvent extracts, piperine and piperic acid from Piper nigrum," Lwt-Food science and technology, vol. 50, no. 2, pp. 634-641, 2013. K. Srinivasan, "Black pepper and its pungent principle-piperine: a review of diverse physiological effects," Critical reviews in food science and nutrition, vol. 47, no. 8, pp. 735-748, 2007. I. U. Haq, M. Imran, M. Nadeem, T. Tufail, T. A. Gondal, and M. S. Mubarak, "Piperine: A review of its biological effects," Phytotherapy research, vol. 35, no. 2, pp. 680-700, 2021. R. Mittal and R. Gupta, "In vitro antioxidant activity of piperine," Methods and findings in experimental and clinical pharmacology, vol. 22, no. 5, pp. 271-274, 2000. R. Nasrnezhad, S. Halalkhor, F. Sadeghi, and F. Pourabdolhossein, "Piperine improves experimental autoimmune encephalomyelitis (EAE) in lewis rats through its neuroprotective, anti-inflammatory, and antioxidant effects," Molecular neurobiology, vol. 58, no. 11, pp. 5473-5493, 2021. R. A. Gupta, M. N. Motiwala, N. G. Dumore, K. R. Danao, and A. B. Ganjare, "Effect of piperine on inhibition of FFA induced TLR4 mediated inflammation and amelioration of acetic acid induced ulcerative colitis in mice," Journal of ethnopharmacology, vol. 164, pp. 239-246, 2015. J. S. Bang et al. , "Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1β-stimulated fibroblast-like synoviocytes and in rat arthritis models," Arthritis research & therapy, vol. 11, pp. 1-9, 2009. P. Boonrueng, P. W. D. Wasana, n. Hasriadi, O. Vajragupta, P. Rojsitthisak, and P. Towiwat, "Combination of curcumin and piperine synergistically improves pain-like behaviors in mouse models of pain with no potential CNS side effects," Chinese Medicine, vol. 17, no. 1, p. 119, 2022. F. Tasleem, I. Azhar, S. N. Ali, S. Perveen, and Z. A. Mahmood, "Analgesic and anti-inflammatory activities of Piper nigrum L," Asian Pacific journal of tropical medicine, vol. 7, pp. S461-S468, 2014. W.-j. Zhai et al. , "Piperine plays an anti‐inflammatory role in Staphylococcus aureus endometritis by inhibiting activation of NF‐κB and MAPK pathways in mice," Evidence‐Based Complementary and Alternative Medicine, vol. 2016, no. 1, p. 8597208, 2016. T. Ren et al. , "Piperine-loaded nanoparticles with enhanced dissolution and oral bioavailability for epilepsy control," European Journal of Pharmaceutical Sciences, vol. 137, p. 104988, 2019. V. Girdhar, S. Patil, S. Banerjee, and G. Singhvi, "Nanocarriers for drug delivery: mini review," Current Nanomedicine (Formerly: Recent Patents on Nanomedicine), vol. 8, no. 2, pp. 88-99, 2018. Y. Ding et al. , "Development and evaluation of a novel drug delivery: Soluplus®/TPGS mixed micelles loaded with piperine in vitro and in vivo," Drug development and industrial pharmacy, vol. 44, no. 9, pp. 1409-1416, 2018. U. Kedar, P. Phutane, S. Shidhaye, and V. Kadam, "Advances in polymeric micelles for drug delivery and tumor targeting," Nanomedicine: Nanotechnology, Biology and Medicine, vol. 6, no. 6, pp. 714-729, 2010. N. Zhang, P. R. Wardwell, and R. A. Bader, "Polysaccharide-based micelles for drug delivery," Pharmaceutics, vol. 5, no. 2, pp. 329-352, 2013. Z. Nazemi, M. Sahraro, M. Janmohammadi, M. S. Nourbakhsh, and H. Savoji, "A review on tragacanth gum: A promising natural polysaccharide in drug delivery and cell therapy," International Journal of Biological Macromolecules, vol. 241, p. 124343, 2023. R. Liang, Z. Bao, B. Su, H. Xing, and Q. Ren, "Solubility of vitamin D3 in six organic solvents at temperatures from (248.2 to 273.2) K," Journal of Chemical & Engineering Data, vol. 57, no. 8, pp. 2328-2331, 2012. A. Rollett et al. , "Folic acid-functionalized human serum albumin nanocapsules for targeted drug delivery to chronically activated macrophages," International journal of pharmaceutics, vol. 427, no. 2, pp. 460-466, 2012. F. F. Alyaseen, B. A. Hassan, and H. S. Abdulhussein, "Extraction, isolation and chemical identification of piperine alkaloid from black pepper seeds and its antibacterial activity," 2018. N. S. Russel, P. K. Paul, M. Karim, and B. Song, "Synthesis of Folate-Appended β-Cyclodextrin Using Phenanthroline as Linker for Cancer Targeting Drug Delivery," International Journal of Organic Chemistry, vol. 9, no. 1, pp. 47-66, 2019. M. Gou et al. , "Curcumin-loaded biodegradable polymeric micelles for colon cancer therapy in vitro and in vivo," Nanoscale, vol. 3, no. 4, pp. 1558-1567, 2011. M. Akrami et al. , "Evaluation of multilayer coated magnetic nanoparticles as biocompatible curcumin delivery platforms for breast cancer treatment," RSC Advances, vol. 5, no. 107, pp. 88096-88107, 2015. M. Zare and M. N. Sarkati, "Chitosan‐functionalized Fe3O4 nanoparticles as an excellent biocompatible nanocarrier for silymarin delivery," Polymers for Advanced Technologies, vol. 32, no. 10, pp. 4094-4100, 2021. R. R. Simões et al. , "Oral treatment with essential oil of Hyptis spicigera Lam.(Lamiaceae) reduces acute pain and inflammation in mice: Potential interactions with transient receptor potential (TRP) ion channels," Journal of ethnopharmacology, vol. 200, pp. 8-15, 2017. A. Sahu, U. Bora, N. Kasoju, and P. Goswami, "Synthesis of novel biodegradable and self-assembling methoxy poly (ethylene glycol)–palmitate nanocarrier for curcumin delivery to cancer cells," Acta biomaterialia, vol. 4, no. 6, pp. 1752-1761, 2008. E. Joseph and G. Singhvi, "Multifunctional nanocrystals for cancer therapy: a potential nanocarrier," Nanomaterials for drug delivery and therapy, pp. 91-116, 2019. J. D. Clogston and A. K. Patri, "Zeta potential measurement," Characterization of nanoparticles intended for drug delivery, pp. 63-70, 2011. D. Pentak, "In vitro spectroscopic study of piperine-encapsulated nanosize liposomes," European biophysics journal, vol. 45, pp. 175-186, 2016. B. Shao et al. , "Enhanced oral bioavailability of piperine by self-emulsifying drug delivery systems: in vitro, in vivo and in situ intestinal permeability studies," Drug delivery, vol. 22, no. 6, pp. 740-747, 2015. B. Chopra, A. K. Dhingra, R. P. Kapoor, and D. N. Prasad, "Piperine and its various physicochemical and biological aspects: A review," Open Chemistry Journal, vol. 3, no. 1, 2016. Z. Duan, H. Xie, S. Yu, S. Wang, and H. Yang, "Piperine derived from Piper nigrum L. inhibits LPS-induced inflammatory through the MAPK and NF-κB signalling pathways in RAW264. 7 Cells," Foods, vol. 11, no. 19, p. 2990, 2022. K. AbouAitah et al. , "Anti-inflammatory and antioxidant effects of nanoformulations composed of metal-organic frameworks delivering rutin and/or piperine natural agents," Drug Delivery, vol. 28, no. 1, pp. 1478-1495, 2021. X. Ying et al. , "Piperine inhibits LPS induced expression of inflammatory mediators in RAW 264.7 cells," Cellular immunology, vol. 285, no. 1-2, pp. 49-54, 2013. K. Vaibhav et al. , "Piperine suppresses cerebral ischemia–reperfusion-induced inflammation through the repression of COX-2, NOS-2, and NF-κB in middle cerebral artery occlusion rat model," Molecular and cellular biochemistry, vol. 367, pp. 73-84, 2012. S. Yu, X. Liu, D. Yu, E. Changyong, and J. Yang, "Piperine protects LPS-induced mastitis by inhibiting inflammatory response," International immunopharmacology, vol. 87, p. 106804, 2020. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Graphical abstract Scheme.png Scheme 1 A schematic illustration of the formation of PIP loaded synthesized micelles. 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-5945336","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":411029240,"identity":"d017320f-7f0f-411e-8b88-3316af5cc7b7","order_by":0,"name":"Mahboobeh Zare","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYPACGwSTjUgtaSCCsYEULYcRWggC3Qb2h58rKs7LG5w/nf6AocaOgU/6AH4tZgcYkiXPnLltuOFG7sYGhmPJDGx8CQS1HJBsbLvNuOEGL1AL2wEGNh4CDjM7wNj8s/HfOfsN588CtfwjSgszm2Rjw4HEDQeADmNsI0bLYTY2y4ZjyckzgX6ZkdiXzENYy/H2xzcbauxs+86f3fDhwzc7OfkeAloYmJE5CQwMhOwYBaNgFIyCUUAMAADtUEFihgNFVwAAAABJRU5ErkJggg==","orcid":"","institution":"Amol University of Special Modern Technologies","correspondingAuthor":true,"prefix":"","firstName":"Mahboobeh","middleName":"","lastName":"Zare","suffix":""},{"id":411029241,"identity":"5443ad2e-f511-43d4-8de5-eb4a7ab8bc50","order_by":1,"name":"Hakime Gavzan","email":"","orcid":"","institution":"Amol University of Special Modern Technologies","correspondingAuthor":false,"prefix":"","firstName":"Hakime","middleName":"","lastName":"Gavzan","suffix":""},{"id":411029242,"identity":"7d0bb707-c8fa-4561-b820-bf53b1125072","order_by":2,"name":"Malihe Noruzi Sarkati","email":"","orcid":"","institution":"Amol University of Special Modern Technologies","correspondingAuthor":false,"prefix":"","firstName":"Malihe","middleName":"Noruzi","lastName":"Sarkati","suffix":""},{"id":411029243,"identity":"789cdaa1-c3a8-4d2c-bee8-aa680c4efbac","order_by":3,"name":"Mohammad Ali Mobaraki","email":"","orcid":"","institution":"Amol University of Special Modern Technologies","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Mobaraki","suffix":""}],"badges":[],"createdAt":"2025-02-02 10:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5945336/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5945336/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75499455,"identity":"566c1ccf-5524-43c3-9f58-1643be48a3ae","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62606,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR\u003csup\u003e \u003c/sup\u003espectra of PIP\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/99280619c8681e6f83915b00.png"},{"id":75499457,"identity":"0c9983cc-41c0-4eab-8ff7-1914973544ae","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-NMR spectra of PIP\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/d24a3a853682046b0a3680e7.png"},{"id":75499464,"identity":"d02b45e8-31d4-4552-838b-f0c6db2061e8","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63301,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of TG, FA-TG-VD, and PIP-loaded micelle\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/bf6132179e449f66a5598d7c.png"},{"id":75500808,"identity":"a4fbe603-ea8b-4813-8442-e4d529c14cfe","added_by":"auto","created_at":"2025-02-05 08:56:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26109,"visible":true,"origin":"","legend":"\u003cp\u003eSize distributions for the PIP-loaded FA-TG-VD micelles by DLS\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/673649feb8a3e98e14d4b421.png"},{"id":75500797,"identity":"d0f6a250-dcee-4661-954a-c8806594ff29","added_by":"auto","created_at":"2025-02-05 08:56:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151172,"visible":true,"origin":"","legend":"\u003cp\u003eAFM image of PIP-loaded micelles\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/7b19fb9a208b0741eacd7292.png"},{"id":75500806,"identity":"e5d59dc2-aac4-4c66-a152-b60d768f5b16","added_by":"auto","created_at":"2025-02-05 08:56:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36160,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential distribution graphic of PIP-loaded micelles\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/52a7fa937a1c28ded7de8421.png"},{"id":75499472,"identity":"e8945161-5b81-45f0-a2c1-f80336033ad5","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":23183,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of PIP,micelles andPIP-loaded micelles against DPPH free radicals\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/cfa39e44d2158f0e1abd7a92.png"},{"id":75499477,"identity":"09bf9694-bf9a-4a25-8d41-5022860bc38a","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":55590,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PIP, PIP-loaded micelle and diclofenac sodium on formalin-induced paw edema\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/1a6db4566977809fd8f301dc.png"},{"id":76563034,"identity":"f60919d2-b0a4-4277-be33-13eec7a3d0bb","added_by":"auto","created_at":"2025-02-18 12:17:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1063721,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/615699bc-6863-415e-9d00-2390b1785f13.pdf"},{"id":75499460,"identity":"963b3eb2-a3d2-4e4f-ba2b-1fabddf362aa","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":169142,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/b0af73bf2a7b4858beeb0dc3.png"},{"id":75499458,"identity":"e73e22ca-2e3e-44e6-95fd-f1a09228fcdf","added_by":"auto","created_at":"2025-02-05 08:48:41","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":155521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 \u003c/strong\u003eA schematic illustration of the formation of PIP loaded synthesized micelles.\u003c/p\u003e","description":"","filename":"Scheme.png","url":"https://assets-eu.researchsquare.com/files/rs-5945336/v1/9d0c7b113235162929d86ea9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polymeric nanomicelles for piperine delivery: Preparation, characterization and evaluation of antioxidant and in-vivo anti-inflammatory properties","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePhytochemicals, primarily sourced from plants, possess biological activities and can be utilized for treating diverse illnesses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Numerous natural herbal extracts with validated anti-inflammatory properties have been discovered through research utilizing in vitro and in vivo inflammation models [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Black pepper (\u003cem\u003ePiper nigrum L.\u003c/em\u003e) has been employed as a culinary condiment and possesses medicinal properties [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Black pepper serves multiple functions beyond culinary use, including medicinal applications, preservation, and incorporation in perfumes. Numerous physiological impacts of black pepper, its derivatives, and its primary bioactive compound, PIP (piperine), have been documented in scientific literature over the past few years [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PIP has demonstrated a diverse array of pharmacological properties in various in vitro and in vivo experimental studies. These encompass antiproliferative, antitumor, antiangiogenic, antioxidant, antidiabetic, anti-obesity, cardioprotective, antimicrobial, antiaging, and immunomodulatory effects. Furthermore, PIP has been extensively studied for its hepatoprotective, anti-allergic, anti-inflammatory, and neuroprotective properties [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. PIP effectively suppressed the release of proinflammatory and inflammatory agents [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and mitigated tissue edema in the experimental models of inflammation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The assessment of the influence of PIP on S. aureus endometritis in a murine model revealed that PIP exhibits the ability to mitigate inflammatory lesions in S. aureus endometritis to a considerable extent, employing various mechanisms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the application of PIP for therapeutic intentions is constrained by its insufficient solubility in aqueous solutions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A potential solution to address this limitation can be found within the realm of nanotechnology, a field that focuses on manipulating matter at the atomic level to create innovative structures, materials, and devices. The core principle of nanotechnology involves synthesizing artificial nanoparticles with unique attributes such as small size, high surface-to-mass ratio, shape, crystallinity, surface charge, reactive surface groups, dissolution rate, agglomeration state, and dispersal, which grant them properties that differ significantly from bulk particles of the same composition [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nanocarriers are utilized for the transportation of active substances, particularly drugs or biologics, in therapeutic applications. The effectiveness of drug delivery has been extensively investigated using various nanocarriers, including micelles, polymers, carbon-based materials, lipid-based substances, and others [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nanomicelles are self-assembled nanoscale colloidal particles with a hydrophobic/hydrophilic core-shell structure, which are suitable as carriers for poorly water-soluble drugs and are regarded as one of the most promising drug delivery systems. Amphiphilic copolymers are constructed by grafting hydrophobic molecules onto hydrophilic polymers and can readily form self-assembled micelles in an aqueous solution. The use of natural polysaccharides as hydrophilic micelle shells is widely recommended due to their high bioactivity, abundant availability, and easy modification which makes them suitable for sustained and controlled drug delivery, targeting ability, and long-circulation [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. TG (Tragacanth gum), a neutral polymer, has garnered considerable interest as a hydrophilic backbone in this delivery system. TG is a natural plant gum that is used for various applications in industries and biomedicine. Its cost-effectiveness and ready availability, coupled with its desirable biocompatibility and biodegradability, make it an advantageous polysaccharide [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. On the other hand, VD (Vitamin D), known as cholecalciferol, is classified as a fat-soluble secosteroid [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. FA (Folic acid) is applicable to inflammation-targeting drug delivery systems because recent studies have shown that the folate receptor beta, which has a high affinity for FA, is specifically expressed by activated macrophages [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This study outlines a simple method for the synthesis of a novel nanocopolymeric micelle composed of TG as the hydrophilic component, VD as the hydrophobic segment and FA as the targeting ligand for targeted drug delivery systems (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The morphology and size of synthesized nano micelle was investigated and the antioxidant activity and anti-inflammatory profile of this drug were analyzed in detail.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Equipments and materials\u003c/h2\u003e \u003cp\u003eThionyl chloride, FA and organic solvents were procured from Merck. The FT-IR spectra were recorded using a spectrometer (AVATAR, Thermo, USA). The Proton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR) spectra were obtained using a 400 MHz Bruker instrument. Atomic force microscopy (DME company. Denmark) was used for the study of morphology. Particle size and zeta potential were monitored using dynamic light scattering (DLS) instrument (HORIBA, SZ-100, Kyoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Extraction of PIP\u003c/h2\u003e \u003cp\u003ePIP was extracted and purified from black pepper utilizing a previously documented technique [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In summary, a quantity of 100 g of black pepper seed powder was subjected to soxhlet extraction using 500 mL of petroleum ether, followed by filtration and subsequent extraction with 300 mL of ethanol for a duration of 2 h. The resulting solution was concentrated to a volume of 30 mL using a rotary evaporator, and subsequently combined with 2 mL of alcoholic KOH (10%). After a period of 30 minutes, the mixture was filtered and stored in a refrigerator overnight, yielding yellowish-brown needles of PIP. These needles were further purified through recrystallization in ethanol, resulting in the production of pure PIP needles. The melting point of the obtained PIP ranged from 127\u0026ndash;130\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Synthesis of PIP-loaded micelles\u003c/h2\u003e \u003cp\u003eFirstly, to synthesize folic acid chloride (FA-Cl), 0.500 g of FA was combined with 15 ml of thionyl chloride and stirred for a duration of 2 h. Subsequently, the resulting mixture was evaporated and subjected to acetone washing, yielding FA-Cl [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSynthesis of vitamin D chloride (VD-Cl) was done by combining VD with 15 ml of thionyl chloride and stirring the mixture for a duration of 2 h. Subsequently, the excess thionyl chloride was evaporated, and the resulting reaction mixture was subjected to acetone washing, resulting in the formation of VD-Cl.\u003c/p\u003e \u003cp\u003ePolymeric micelles (FA-TG-VD) were obtained from the reaction of TG with FA-Cl and VD-Cl. For this reaction, solution of VD-Cl (0.32 g in DMSO) and FA-Cl (0.28 g in DMSO) were added separately to a solution of TG (0.5 g in H\u003csub\u003e2\u003c/sub\u003eO). The reaction mixture was stirred for 36 h, resulting in the formation of polymeric micelles through esterification and etherification reactions between the OH groups of TG and the COCl groups of FA-Cl and C-Cl groups of VD-Cl. The product was purified by dialysis against distilled water, followed by lyophilization, yielding a yellow powder of FA-TG-VD.\u003c/p\u003e \u003cp\u003eFinally, the incorporation of PIP into FA-TG-VD micelles was done by dropwise addition of PIP solution (0.5 g in 25 mL of acetone) in to FA-TG-VD mixture (0.5 g in 150 mL of H\u003csub\u003e2\u003c/sub\u003eO), followed by stirring for 24 hours. The acetone was then removed using a rotary evaporator, and the resulting mixture was subjected to centrifugation. The supernatant obtained was subsequently subjected to lyophilization, resulting in the production of PIP-loaded FA-TG-VD micelles [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Evaluation of drug loading (DL)\u003c/h2\u003e \u003cp\u003eThe PIP loading was quantified by employing a UV-Vis spectrophotometer set at a wavelength of 342 nm. A solution of 1 mg of PIP-loaded synthesized micelles in 10 mL of DMSO was prepared and subjected to 24 hours of agitation. Subsequently, the resulting supernatant was collected and the PIP concentration was determined using a previously established methodology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Evaluation of antioxidant activity\u003c/h2\u003e \u003cp\u003eThe DPPH radical scavenging method was employed to monitor the radical scavenging capacity of PIP-loaded micelles [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Five varying concentrations of PIP-loaded micelles were dispersed in 2.5 mL of distilled ethanol, followed by the addition of 2.5 mL of DPPH solution (200\u0026micro;M). The reaction mixtures were incubated for 60 minutes, and subsequently, the absorbance was determined at 517 nm. Additionally, parallel experiments were conducted using pure PIP and FA-TG-VD to compare their interactions with DPPH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Evaluation of anti-inflammatory activities\u003c/h2\u003e \u003cp\u003eThe peripheral anti-inflammatory activity of PIP micelles was assessed by evaluating the formalin-induced paw edema using the prescribed methodology. Adult male NMRI mice (20\u0026ndash;30gr, Pasteur Institute of Iran, Amol) were housed under standard environmental conditions, including controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), humidity of 40\u0026ndash;50%, a 12-hour light and dark cycle, and provided with unlimited access to food and water. All animal housing and experiments were conducted in strict accordance with the institutional Guidelines for Care and Use of Laboratory Animals at Amol University of Special Modern Technologies. The present study's experiments were granted approval by the Institutional Animals Ethics Committee of Amol University of Special Modern Technologies, with the authorization code IR.ASMT.REC.1403.001. The primary objective was to reduce the number of animals used and mitigate their associated suffering. PIP was solubilized in a suspension containing Tween-80 and normal saline (0.9% NaCl) at a 1:25 ratio, serving as the vehicle [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Formalin, diclofenac, and PIP micelle were dissolved in a solution of normal saline. To elicit peripheral inflammation and pain, mice were subcutaneously injected with 20 \u0026micro;l of 2.5% formalin solution in the subplantar region of the right hind paw. The animals were allocated randomly into five groups, with eight animals in each group. They received either vehicle, PIP (15 mg/kg), PIP micelle (10 and 15 mg/kg), or diclofenac (20 mg/kg). Formalin injection was administered 15 minutes prior to the administration of vehicle, PIP and PIP micelle, and 30 minutes prior to diclofenac [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The degree of paw edema was assessed by measuring paw thickness with a digital caliper at various time intervals (pre-injection, 1, 2, 3, 4 and 24 h post-injection). The extent of edema suppression was calculated using a designated equation.\u003c/p\u003e \u003cp\u003e \u003cem\u003e∆v\u0026thinsp;=\u0026thinsp;v2 \u0026ndash; v1\u003c/em\u003e \u003c/p\u003e \u003cp\u003ev1: the initial thickness of the paw prior to the administration of formalin, v2: the subsequent thickness of the paw post formalin injection.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the FTIR spectra of PIP exhibit a prominent peak at 1634 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which corresponds to the amide group. Additionally, the range between 2802\u0026ndash;2939 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e displays peaks indicative of aliphatic C-H bonds, while the range between 3000\u0026ndash;3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with aromatic C-H bonds. In the \u003csup\u003e1\u003c/sup\u003eHNMR spectrum of PIP illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the chemical shifts for cycloaliphatic, aliphatic (methylenedioxy), alkenic, and aromatic hydrogen were observed at 1.59\u0026ndash;1.67 and 3.53\u0026ndash;3.63, 5.98, 6.43\u0026ndash;6.79 and 6.88\u0026ndash;7.43 ppm respectively.\u003c/p\u003e \u003cp\u003eThe characterization of PIP-loaded synthesized micelles was conducted using FT-IR as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, DLS as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and AFM that is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFTIR measurement was employed to analyze synthesized micelles and PIP-loaded micelles. The spectra revealed a broad peak at 3000\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was attributed to the stretching vibrations of the -OH group of gum and FA. Additionally, bands at approximately 1098, 1387, and 2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed, which were related to stretching vibrations of C-O and C-H groups of TG. The FT-IR spectrum of the FA-TG-VD showed peaks at 1690 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1300\u0026thinsp;\u0026minus;\u0026thinsp;1100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, confirming that VD and FA were grafted to TG. Encapsulation of PIP in the micelle was confirmed by a peak at 1000\u0026ndash;1300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, assigned to the stretching vibrations of C-O in the ether group of PIP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe dimensions and morphology of self-assembled micelle nanoparticles containing PIP were analyzed using light-scattering measurements and AFM. The PIP-loaded micelles displayed a mean diameter of 181 nm, with a narrow size distribution in the DLS experiment that is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the AFM revealed that the micelles were spherical in shape, and the size observed was consistent with that measured by DLS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNanoparticles exhibiting a high zeta potential demonstrate favorable physical stability due to the electrostatic repulsion between individual particles [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Zeta potential value of PIP-loaded micelles was \u0026minus;\u0026thinsp;24.5 mV. The results suggest that the prepared nanoparticle systems were in a relatively stable condition. Based on a previously investigation concerning PIP-encapsulated nano-sized liposomes, it was observed that liposomes incorporating PIP demonstrated a notable level of stability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe drug loading capacity of FA-TG-VD micelles was determined to be approximately 50% in the current study, indicating effective loading of PIP into the nanocarrier. Various studies demonstrated that the incorporation of a self-emulsifying drug delivery system in a formulation significantly improved the oral bioavailability of PIP [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe DPPH assay was applied to investigate the antioxidant properties of prepared micelles. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the IC50 values of PIP-loaded micelles and PIP were 2.4 and 4.2 mg/mL respectively. The scavenging activity of PIP-loaded FA-TG-VD micelles was improved by increasing its concentrations. The carrier as a blank had a less antioxidant activity thus the free radical scavenging activity of PIP-loaded FA-TG-VD micelles are mainly due to the presence of PIP. The incorporation of PIP into the micelle nanoparticles enhanced antioxidant effects of PIP. This observation highlights the significant positive impact of these nanoparticles on augmenting the antioxidant properties of PIP. Various studies showed that PIP has antioxidant actions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-inflammatory activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePIP and PIP micelle showed significant anti-inflammatory effects up to 24 hours. The results revealed that the inflammation was significantly inhibited by 15 mg/kg dose of PIP and 15 mg/kg dose of PIP micelle as well as 20 mg/kg dose of standard drug (diclofenac) as compared to the empty micelle in 1, 2, 3, and 24 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) after injection of formalin.\u003c/p\u003e \u003cp\u003ePIP has been known as a potential anti-inflammatory substance, and its molecular mechanism may involve regulating the key factors of the NF-B and MAPK signalling pathways. This alkaloid also inhibits the expression of IL6 and MMP13 and reduces the production of PGE2 in a dose-dependent manner [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The present study has also demonstrated the anti-inflammatory efficacy of PIP. Furthermore, we observed the impact of the nano drug delivery system on the anti-inflammatory characteristics of PIP. In a comparable investigation, the paw edema and leukocyte migration activity were notably diminished in rats that received intraperitoneal injections of nanoformulations as opposed to free PIP [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, PIP micelle siginificantly reduced the formalin- induced paw edema as well as PIP and standard drug (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) starting from 1hr of formalin injection and continued to 24 h compared to vehicle group. It is noteworthy that there was no significant difference among PIP, PIP micelle and diclofenac in 1, 2, and 3hrs after formalin injection. Given that the drug loading capacity within this drug delivery system is approximated to be 50%, it can be inferred that the anti-inflammatory impact of PIP-loaded nanomicelle is not markedly distinct from the anti-inflammatory effect of PIP administered at twice the dosage. Essentially, employing this drug delivery system allows for a substantial reduction in the PIP dosage without compromising its anti-inflammatory efficacy.\u003c/p\u003e \u003cp\u003eThe anti-inflammatory properties of PIP were demonstrated in the in vitro and in vivo studies and our results are in line with the previous works. PIP attenuated LPS-induced inflammatory cytokine production in BV2 microglia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, PIP was found to inhibit cerebral ischemia- reperfusion-induced inflammation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Also, PIP exhibited a notable reduction in the production of TNF-α and IL-1β induced by LPS. Furthermore, it was demonstrated that PIP significantly impeded the infiltration of neutrophils induced by LPS and the production of inflammatory cytokines [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, PIP caused a reduction in the expression of proinflammatory cytokines in paw tissue and spinal cord samples in mice that were subjected to the formalin test [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This finding suggests that this substance has the capacity to alleviate both peripheral and central inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the aforementioned findings, it is possible to optimize the utilization of bioactive substances, specifically those with anti-inflammatory properties, through the development of nano-drug delivery systems. The utilization of this drug delivery system is deemed more advantageous than the conventional employment of PIP, as it not only enhances the nanoscale characteristics and augments the antioxidant impact, but it has also been meticulously engineered to elicit a distinct influence on inflammation.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eExtensive research has been conducted on the medicinal properties of PIP, a naturally occurring substance derived from plants. However, the clinical application of PIP has faced challenges due to its low bioavailability, poor water solubility, and complex release kinetics. To overcome these obstacles, a proposed solution involves the use of a nanoparticle delivery system for PIP. This study suggests that nano micelles hold promise as a nanocarrier for PIP, as they have the potential to enhance its antioxidant and anti-inflammatory. Moreover, the nano micelle formulation, which incorporates FA and encapsulates PIP, exhibits unique characteristics that improve its bioavailability and water solubility. These distinctive properties enable targeted interactions with inflammatory cells, making it a specialized design for modulating inflammatory processes. Looking ahead, the development of nanoparticles with specific designs will facilitate efficient and targeted management of pathogenic processes, thereby improving both the duration and quality of life for patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMahboobeh Zare: \u003c/strong\u003eConceptualization, Methodology, Validation, Investigation,Resources, Writing - Review and Editing,Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHakime Gavzan:\u003c/strong\u003e Conceptualization, Methodology, Validation,Formal analysis, Investigation, Resources, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Malihe Noruzi Sarkati: \u003c/strong\u003eInvestigation, Resources\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMohammad Ali Mobaraki\u003cem\u003e:\u003c/em\u003e \u003c/strong\u003e Writing - Original Draft\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Acknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the financial support received from the Research Council of Amol University of Special Modern Technologies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Declaration of conflicting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Funding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSupported by \u003c/em\u003ethe Research Council of Amol University of Special Modern Technologies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe data supporting the results of this study can be obtained from the corresponding authors upon reasonable request.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eN. Mendoza and E. M. E. Silva, \u0026quot;Introduction to phytochemicals: secondary metabolites from plants with active principles for pharmacological importance,\u0026quot; \u003cem\u003ePhytochemicals: Source of antioxidants and role in disease prevention, \u003c/em\u003evol. 25, pp. 1-5, 2018.\u003c/li\u003e\n\u003cli\u003eF. Maione, R. Russo, H. Khan, and N. Mascolo, \u0026quot;Medicinal plants with anti-inflammatory activities,\u0026quot; \u003cem\u003eNatural product research, \u003c/em\u003evol. 30, no. 12, pp. 1343-1352, 2016.\u003c/li\u003e\n\u003cli\u003eZ. Zarai, E. Boujelbene, N. B. Salem, Y. Gargouri, and A. Sayari, \u0026quot;Antioxidant and antimicrobial activities of various solvent extracts, piperine and piperic acid from Piper nigrum,\u0026quot; \u003cem\u003eLwt-Food science and technology, \u003c/em\u003evol. 50, no. 2, pp. 634-641, 2013.\u003c/li\u003e\n\u003cli\u003eK. Srinivasan, \u0026quot;Black pepper and its pungent principle-piperine: a review of diverse physiological effects,\u0026quot; \u003cem\u003eCritical reviews in food science and nutrition, \u003c/em\u003evol. 47, no. 8, pp. 735-748, 2007.\u003c/li\u003e\n\u003cli\u003eI. U. Haq, M. Imran, M. Nadeem, T. Tufail, T. A. Gondal, and M. S. Mubarak, \u0026quot;Piperine: A review of its biological effects,\u0026quot; \u003cem\u003ePhytotherapy research, \u003c/em\u003evol. 35, no. 2, pp. 680-700, 2021.\u003c/li\u003e\n\u003cli\u003eR. Mittal and R. Gupta, \u0026quot;In vitro antioxidant activity of piperine,\u0026quot; \u003cem\u003eMethods and findings in experimental and clinical pharmacology, \u003c/em\u003evol. 22, no. 5, pp. 271-274, 2000.\u003c/li\u003e\n\u003cli\u003eR. Nasrnezhad, S. Halalkhor, F. Sadeghi, and F. Pourabdolhossein, \u0026quot;Piperine improves experimental autoimmune encephalomyelitis (EAE) in lewis rats through its neuroprotective, anti-inflammatory, and antioxidant effects,\u0026quot; \u003cem\u003eMolecular neurobiology, \u003c/em\u003evol. 58, no. 11, pp. 5473-5493, 2021.\u003c/li\u003e\n\u003cli\u003eR. A. Gupta, M. N. Motiwala, N. G. Dumore, K. R. Danao, and A. B. Ganjare, \u0026quot;Effect of piperine on inhibition of FFA induced TLR4 mediated inflammation and amelioration of acetic acid induced ulcerative colitis in mice,\u0026quot; \u003cem\u003eJournal of ethnopharmacology, \u003c/em\u003evol. 164, pp. 239-246, 2015.\u003c/li\u003e\n\u003cli\u003eJ. S. Bang\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1\u0026beta;-stimulated fibroblast-like synoviocytes and in rat arthritis models,\u0026quot; \u003cem\u003eArthritis research \u0026amp; therapy, \u003c/em\u003evol. 11, pp. 1-9, 2009.\u003c/li\u003e\n\u003cli\u003eP. Boonrueng, P. W. D. Wasana, n. Hasriadi, O. Vajragupta, P. Rojsitthisak, and P. Towiwat, \u0026quot;Combination of curcumin and piperine synergistically improves pain-like behaviors in mouse models of pain with no potential CNS side effects,\u0026quot; \u003cem\u003eChinese Medicine, \u003c/em\u003evol. 17, no. 1, p. 119, 2022.\u003c/li\u003e\n\u003cli\u003eF. Tasleem, I. Azhar, S. N. Ali, S. Perveen, and Z. A. Mahmood, \u0026quot;Analgesic and anti-inflammatory activities of Piper nigrum L,\u0026quot; \u003cem\u003eAsian Pacific journal of tropical medicine, \u003c/em\u003evol. 7, pp. S461-S468, 2014.\u003c/li\u003e\n\u003cli\u003eW.-j. Zhai\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Piperine plays an anti‐inflammatory role in Staphylococcus aureus endometritis by inhibiting activation of NF‐\u0026kappa;B and MAPK pathways in mice,\u0026quot; \u003cem\u003eEvidence‐Based Complementary and Alternative Medicine, \u003c/em\u003evol. 2016, no. 1, p. 8597208, 2016.\u003c/li\u003e\n\u003cli\u003eT. Ren\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Piperine-loaded nanoparticles with enhanced dissolution and oral bioavailability for epilepsy control,\u0026quot; \u003cem\u003eEuropean Journal of Pharmaceutical Sciences, \u003c/em\u003evol. 137, p. 104988, 2019.\u003c/li\u003e\n\u003cli\u003eV. Girdhar, S. Patil, S. Banerjee, and G. Singhvi, \u0026quot;Nanocarriers for drug delivery: mini review,\u0026quot; \u003cem\u003eCurrent Nanomedicine (Formerly: Recent Patents on Nanomedicine), \u003c/em\u003evol. 8, no. 2, pp. 88-99, 2018.\u003c/li\u003e\n\u003cli\u003eY. Ding\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Development and evaluation of a novel drug delivery: Soluplus\u0026reg;/TPGS mixed micelles loaded with piperine in vitro and in vivo,\u0026quot; \u003cem\u003eDrug development and industrial pharmacy, \u003c/em\u003evol. 44, no. 9, pp. 1409-1416, 2018.\u003c/li\u003e\n\u003cli\u003eU. Kedar, P. Phutane, S. Shidhaye, and V. Kadam, \u0026quot;Advances in polymeric micelles for drug delivery and tumor targeting,\u0026quot; \u003cem\u003eNanomedicine: Nanotechnology, Biology and Medicine, \u003c/em\u003evol. 6, no. 6, pp. 714-729, 2010.\u003c/li\u003e\n\u003cli\u003eN. Zhang, P. R. Wardwell, and R. A. Bader, \u0026quot;Polysaccharide-based micelles for drug delivery,\u0026quot; \u003cem\u003ePharmaceutics, \u003c/em\u003evol. 5, no. 2, pp. 329-352, 2013.\u003c/li\u003e\n\u003cli\u003eZ. Nazemi, M. Sahraro, M. Janmohammadi, M. S. Nourbakhsh, and H. Savoji, \u0026quot;A review on tragacanth gum: A promising natural polysaccharide in drug delivery and cell therapy,\u0026quot; \u003cem\u003eInternational Journal of Biological Macromolecules, \u003c/em\u003evol. 241, p. 124343, 2023.\u003c/li\u003e\n\u003cli\u003eR. Liang, Z. Bao, B. Su, H. Xing, and Q. Ren, \u0026quot;Solubility of vitamin D3 in six organic solvents at temperatures from (248.2 to 273.2) K,\u0026quot; \u003cem\u003eJournal of Chemical \u0026amp; Engineering Data, \u003c/em\u003evol. 57, no. 8, pp. 2328-2331, 2012.\u003c/li\u003e\n\u003cli\u003eA. Rollett\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Folic acid-functionalized human serum albumin nanocapsules for targeted drug delivery to chronically activated macrophages,\u0026quot; \u003cem\u003eInternational journal of pharmaceutics, \u003c/em\u003evol. 427, no. 2, pp. 460-466, 2012.\u003c/li\u003e\n\u003cli\u003eF. F. Alyaseen, B. A. Hassan, and H. S. Abdulhussein, \u0026quot;Extraction, isolation and chemical identification of piperine alkaloid from black pepper seeds and its antibacterial activity,\u0026quot; 2018.\u003c/li\u003e\n\u003cli\u003eN. S. Russel, P. K. Paul, M. Karim, and B. Song, \u0026quot;Synthesis of Folate-Appended \u0026beta;-Cyclodextrin Using Phenanthroline as Linker for Cancer Targeting Drug Delivery,\u0026quot; \u003cem\u003eInternational Journal of Organic Chemistry, \u003c/em\u003evol. 9, no. 1, pp. 47-66, 2019.\u003c/li\u003e\n\u003cli\u003eM. Gou\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Curcumin-loaded biodegradable polymeric micelles for colon cancer therapy in vitro and in vivo,\u0026quot; \u003cem\u003eNanoscale, \u003c/em\u003evol. 3, no. 4, pp. 1558-1567, 2011.\u003c/li\u003e\n\u003cli\u003eM. Akrami\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Evaluation of multilayer coated magnetic nanoparticles as biocompatible curcumin delivery platforms for breast cancer treatment,\u0026quot; \u003cem\u003eRSC Advances, \u003c/em\u003evol. 5, no. 107, pp. 88096-88107, 2015.\u003c/li\u003e\n\u003cli\u003eM. Zare and M. N. Sarkati, \u0026quot;Chitosan‐functionalized Fe3O4 nanoparticles as an excellent biocompatible nanocarrier for silymarin delivery,\u0026quot; \u003cem\u003ePolymers for Advanced Technologies, \u003c/em\u003evol. 32, no. 10, pp. 4094-4100, 2021.\u003c/li\u003e\n\u003cli\u003eR. R. Sim\u0026otilde;es\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Oral treatment with essential oil of Hyptis spicigera Lam.(Lamiaceae) reduces acute pain and inflammation in mice: Potential interactions with transient receptor potential (TRP) ion channels,\u0026quot; \u003cem\u003eJournal of ethnopharmacology, \u003c/em\u003evol. 200, pp. 8-15, 2017.\u003c/li\u003e\n\u003cli\u003eA. Sahu, U. Bora, N. Kasoju, and P. Goswami, \u0026quot;Synthesis of novel biodegradable and self-assembling methoxy poly (ethylene glycol)\u0026ndash;palmitate nanocarrier for curcumin delivery to cancer cells,\u0026quot; \u003cem\u003eActa biomaterialia, \u003c/em\u003evol. 4, no. 6, pp. 1752-1761, 2008.\u003c/li\u003e\n\u003cli\u003eE. Joseph and G. Singhvi, \u0026quot;Multifunctional nanocrystals for cancer therapy: a potential nanocarrier,\u0026quot; \u003cem\u003eNanomaterials for drug delivery and therapy, \u003c/em\u003epp. 91-116, 2019.\u003c/li\u003e\n\u003cli\u003eJ. D. Clogston and A. K. Patri, \u0026quot;Zeta potential measurement,\u0026quot; \u003cem\u003eCharacterization of nanoparticles intended for drug delivery, \u003c/em\u003epp. 63-70, 2011.\u003c/li\u003e\n\u003cli\u003eD. Pentak, \u0026quot;In vitro spectroscopic study of piperine-encapsulated nanosize liposomes,\u0026quot; \u003cem\u003eEuropean biophysics journal, \u003c/em\u003evol. 45, pp. 175-186, 2016.\u003c/li\u003e\n\u003cli\u003eB. Shao\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Enhanced oral bioavailability of piperine by self-emulsifying drug delivery systems: in vitro, in vivo and in situ intestinal permeability studies,\u0026quot; \u003cem\u003eDrug delivery, \u003c/em\u003evol. 22, no. 6, pp. 740-747, 2015.\u003c/li\u003e\n\u003cli\u003eB. Chopra, A. K. Dhingra, R. P. Kapoor, and D. N. Prasad, \u0026quot;Piperine and its various physicochemical and biological aspects: A review,\u0026quot; \u003cem\u003eOpen Chemistry Journal, \u003c/em\u003evol. 3, no. 1, 2016.\u003c/li\u003e\n\u003cli\u003eZ. Duan, H. Xie, S. Yu, S. Wang, and H. Yang, \u0026quot;Piperine derived from Piper nigrum L. inhibits LPS-induced inflammatory through the MAPK and NF-\u0026kappa;B signalling pathways in RAW264. 7 Cells,\u0026quot; \u003cem\u003eFoods, \u003c/em\u003evol. 11, no. 19, p. 2990, 2022.\u003c/li\u003e\n\u003cli\u003eK. AbouAitah\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Anti-inflammatory and antioxidant effects of nanoformulations composed of metal-organic frameworks delivering rutin and/or piperine natural agents,\u0026quot; \u003cem\u003eDrug Delivery, \u003c/em\u003evol. 28, no. 1, pp. 1478-1495, 2021.\u003c/li\u003e\n\u003cli\u003eX. Ying\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Piperine inhibits LPS induced expression of inflammatory mediators in RAW 264.7 cells,\u0026quot; \u003cem\u003eCellular immunology, \u003c/em\u003evol. 285, no. 1-2, pp. 49-54, 2013.\u003c/li\u003e\n\u003cli\u003eK. Vaibhav\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Piperine suppresses cerebral ischemia\u0026ndash;reperfusion-induced inflammation through the repression of COX-2, NOS-2, and NF-\u0026kappa;B in middle cerebral artery occlusion rat model,\u0026quot; \u003cem\u003eMolecular and cellular biochemistry, \u003c/em\u003evol. 367, pp. 73-84, 2012.\u003c/li\u003e\n\u003cli\u003eS. Yu, X. Liu, D. Yu, E. Changyong, and J. Yang, \u0026quot;Piperine protects LPS-induced mastitis by inhibiting inflammatory response,\u0026quot; \u003cem\u003eInternational immunopharmacology, \u003c/em\u003evol. 87, p. 106804, 2020.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"Micelle, Tragacanth gum, Piperine, Anti-inflammatory, Antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-5945336/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5945336/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePiperine, one of the bioactive alkaloids, exhibits restricted therapeutic effectiveness owing to its inadequate solubility and bioavailability. Nanomicelles hold great potential as drug delivery systems for drugs with low water solubility. This study aimed to create micelles that contain tragacanth gum, vitamin D, and folic acid to encapsulate piperine.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePiperine was isolated from black pepper and subsequently loaded on synthesized polymeric micelles. The antioxidant activity of piperine-loaded micelles was tested by DPPH\u0026sdot; radical method. Moreover, the peripheral anti-inflammatory activity of nanomicelles was assessed using the formalin-induced paw edema method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to the results obtained from this investigation, the utilization of nanomicelles demonstrated an enhancement in the radical scavenging activity of piperine, consequently leading to a notable reduction in the required dosage of piperine for its anti-inflammatory effects.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe findings indicate that the utilization of synthesized micelle as an innovative targeted delivery system could potentially augment the therapeutic characteristics of piperine.\u003c/p\u003e","manuscriptTitle":"Polymeric nanomicelles for piperine delivery: Preparation, characterization and evaluation of antioxidant and in-vivo anti-inflammatory properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 08:48:36","doi":"10.21203/rs.3.rs-5945336/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":"35011ed6-1a6e-4356-a515-542b3380466a","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-18T12:08:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-05 08:48:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5945336","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5945336","identity":"rs-5945336","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}