Matrix stiffness regulates lipid nanoparticle-mRNA delivery in cell-laden hydrogels.

mRNA therapeutics have increased in popularity, largely due to the transient and fast nature of protein expression and the low risk of off-target effects. This has increased drastically with the remarkable success of mRNA-based vaccines for COVID-19. Despite advances in lipid nanoparticle (LNP)-based delivery, the mechanisms that regulate efficient endocytic trafficking and translation of mRNA remain poorly understood. Although it is widely acknowledged that the extracellular matrix (ECM) regulates uptake and expression of exogenous nano-complexed genetic material, its specific effects on mRNA delivery and expression have not yet been examined. Here, we demonstrate a critical role for matrix stiffness in modulating both mRNA transfection and expression and uncover distinct mechano-regulatory mechanisms for endocytosis of mRNA through RhoA mediated mTOR signaling and cytoskeletal dynamics. Our findings have implications for effective delivery of therapeutic mRNA to targeted tissues that may be differentially affected by tissue and matrix stiffness.

Redox-Active Profile Characterization of Remirea maritima Extracts and Its Cytotoxic Effect in Mouse Fibroblasts (L929) and Melanoma (B16F10) Cells.

Remirea maritima is a tropical plant with a reticulated root system belonging to the family Cyperaceae, also known to have biologically active secondary metabolites. However, very few data on R. maritima's biological actions are available and there are no reports regarding the redox-active profile of this plant. In this study, we examined the total phenolic content of Remirea maritima hydroalcoholic (RMHA) extracts, redox properties against different reactive species generated in vitro and their cytotoxic effect against fibroblasts (L929) and melanoma (B16F10) cells. Total reactive antioxidant potential index (TRAP) and total antioxidant reactivity (TAR) results revealed that RMHA at all concentrations tested showed significant antioxidant capacity. RMHA was also effective against hydroxyl radical formation, reduction of Fe3+ to Fe2+ and in scavenging nitric oxide (NO) radicals. In vitro, the level of lipid peroxidation was reduced by RMHA extract and the data showed significant oxidative damage protection. The RMHA cytotoxicity was evaluated by a neutral red assay in fibroblast (L929) and melanome (B16F10) cells. The obtained results showed that the RMHA (40 and 80 µg/mL, respectively) reduced 70% of the viable cells. In conclusion, this study represents the first report regarding the antioxidant and anti-proliferative potential of R. maritima against B16F10 melanoma cells.

Cyclodextrin-complexed Ocimum basilicum leaves essential oil increases Fos protein expression in the central nervous system and produce an antihyperalgesic effect in animal models for fibromyalgia.

O. basilicum leaves produce essential oils (LEO) rich in monoterpenes. The short half-life and water insolubility are limitations for LEO medical uses. ß-Cyclodextrin (ß-CD) has been employed to improve the pharmacological properties of LEO. We assessed the antihyperalgesic profile of LEO, isolated or complexed in ß-CD (LEO/ß-CD), on an animal model for fibromyalgia. Behavioral tests: mice were treated every day with either LEO/ß-CD (25, 50 or 100 mg/kg, p.o.), LEO (25 mg/kg, p.o.), tramadol (TRM 4 mg/kg, i.p.) or vehicle (saline), and 60 min after treatment behavioral parameters were assessed. Therefore, mice were evaluated for mechanical hyperalgesia (von Frey), motor coordination (Rota-rod) and muscle strength (Grip Strength Metter) in a mice fibromyalgia model. After 27 days, we evaluated the central nervous system (CNS) pathways involved in the effect induced by experimental drugs through immunofluorescence protocol to Fos protein. The differential scanning analysis (DSC), thermogravimetry/derivate thermogravimetry (TG/DTG) and infrared absorption spectroscopy (FTIR) curves indicated that the products prepared were able to incorporate the LEO efficiently. Oral treatment with LEO or LEO-ßCD, at all doses tested, produced a significant reduction of mechanical hyperalgesia and we were able to significantly increase Fos protein expression. Together, our results provide evidence that LEO, isolated or complexed with ß-CD, produces analgesic effects on chronic non-inflammatory pain as fibromyalgia.

Involvement of nuclear factor κB and descending pain pathways in the anti-hyperalgesic effect of ß-citronellol, a food ingredient, complexed in ß-cyclodextrin in a model of complex regional pain syndrome - Type 1.

Complex regional pain syndrome type 1 (CRPS-1) is a painful syndrome without effective treatment. In order to explore possible new treatments, we used an animal model of CRPS-1 to examine the effects of ß-Citronellol (ßCT), a monoterpene found in a variety of plants that has been shown to have analgesic effects. We aimed to assess its effects alone, and complexed with ß-cyclodextrin (ßCD), which has been previously used to enhance the effects of a number of medicines. The ßCT-ßCD was characterized physiochemically using high performance liquid chromatography (HPLC) and differential scanning calorimetry (DSC) and shown to have 80% efficiency. In the animal model, Swiss mice were treated with ßCT, ßCT-ßCD, vehicle, pregabalin or sham and evaluated for hyperalgesia and motor coordination. Inflammatory mediators were measured by Western blot or ELISA and the descending pain pathway by immunofluorescence. ßCT was shown to have an anti-hyperalgesic effect (without affecting motor coordination) that reduced inflammatory mediators and activated the descending pain pathway, and these effects were increased with complexation in ßCD. Our results showed ßCT-ßCD to be a promising treatment for CRPS-1.

Micropatterned hydrogels and cell alignment enhance the odontogenic potential of stem cells from apical papilla in-vitro.

INTRODUCTION: An understanding of the extracellular matrix characteristics which stimulate and guide stem cell differentiation in the dental pulp is fundamental for the development of enhanced dental regenerative therapies. Our objectives, in this study, were to determine whether stem cells from the apical papilla (SCAP) responded to substrate stiffness, whether hydrogels providing micropatterned topographical cues stimulate SCAP self-alignment, and whether the resulting alignment could influence their differentiation towards an odontogenic lineage in-vitro. METHODS: Experiments utilized gelatin methacryloyl (GelMA) hydrogels of increasing concentrations (5, 10 and 15%). We determined their compressive modulus via unconfined compression and analyzed cell spreading via F-actin/DAPI immunostaining. GelMA hydrogels were micropatterned using photolithography, in order to generate microgrooves and ridges of 60 and 120µm, onto which SCAP were seeded and analyzed for self-alignment via fluorescence microscopy. Lastly, we analyzed the odontogenic differentiation of SCAP using alkaline phosphatase protein expression (ANOVA/Tukey α=0.05). RESULTS: SCAP appeared to proliferate better on stiffer hydrogels. Both 60 and 120µm micropatterned hydrogels guided the self-alignment of SCAP with no significant difference between them. Similarly, both 60 and 120µm micropattern aligned cells promoted higher odontogenic differentiation than non-patterned controls. SIGNIFICANCE: In summary, both substrate mechanics and geometry have a statistically significant influence on SCAP response, and may assist in the odontogenic differentiation of dental stem cells. These results may point toward the fabrication of cell-guiding scaffolds for regenerative endodontics, and may provide cues regarding the development of the pulp-dentin interface during tooth formation.

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

Carvacrol/ß-cyclodextrin inclusion complex inhibits cell proliferation and migration of prostate cancer cells.

Carvacrol, a phenolic monoterpene derived from thyme oil has gained wide interest recently because of its anticancer activities. To improve the solubility of carvacrol, the formation of inclusion complexes with ß-cyclodextrin was performed by ultrasound and freeze-drying methods and characterized using thermal analysis, FTIR, XRD, SEM, NMR and HPLC analysis. From these results, carvacrol was successfully complexed within ß-cyclodextrin cavity. Moreover, HPLC analysis demonstrated a higher entrapment efficiency for freeze-drying method (81.20 ±â€¯0.52%) in contrast to ultrasound method (34.02 ±â€¯0.67%). Hence, freeze-drying inclusion complex was evaluated for its antiproliferative effect and cytotoxicity against prostate cancer cell line (PC3) in vitro. Further, freeze-drying complex led to a dose-dependent inhibition in tumor cell growth in 2D and 3D cell culture systems. Altogether, the inclusion of carvacrol in ß-cyclodextrin led to the formation of stable complexes with potent antiproliferative effects against PC3 cells, in vitro. Such an improved cytotoxic effect can be attributed to the enhanced the aqueous solubility and bioavailability of carvacrol by effective complexation in ß-cyclodextrin.

Molecular mechanism underlying orofacial antinociceptive activity of Vanillosmopsis arborea Baker (Asteraceae) essential oil complexed with ß-cyclodextrin.

BACKGROUND: Vanillosmopsis arborea Baker has recognized economic value owing to the high content of (-)-α-bisabolol (BISA) in the essential oil of its stem (EOVA). The antinociceptive effect of EVOA has already been demonstrated, and ß-cyclodextrin (ßCD) is known to improve the analgesic effect of various substances. PURPOSE: Thus, we aimed to evaluate the orofacial antinociceptive effect of a complex containing EOVA-ßCD in rodents. METHODS: EOVA was obtained by simple hydrodistillation, and the essential oil was complexed with ßCD. The animals (n = 6/group) were treated orally with EOVA-ßCD (10 or 50 mg/kg), or vehicle (control), and subjected to cutaneous orofacial nociception (formalin, capsaicin, acidic saline or glutamate), corneal (hypertonic saline) or temporomandibular (formalin) tests. The expression of FOS protein was analyzed in the spinal cord. Molecular docking was performed using the 5-HT3 and M2 receptors and BISA. RESULTS: The oral administration of EOVA-ßCD reduced nociceptive behaviour. Moreover, EOVA-ßCD decreased FOS expression. The molecular docking study indicates that BISA interacts with 5-HT3 and M2 receptors, indicating the potential mechanism of action of the tested compound. CONCLUSIONS: Our results indicate that EOVA-ßCD possesses orofacial antinociceptive effect, indicating that this complex can be used in analgesic drug development.

Effect of Pulsed Therapeutic Ultrasound and Diosmin on Skeletal Muscle Oxidative Parameters.

Cyclodextrins (CDs) have been widely used as a promising alternative in the formation of inclusion complexes with poorly soluble molecules. From this perspective, the present study aimed to study the inclusion complexes of diosmin in ß-cyclodextrin, chemically quantify the diosmin-in-gel preparation and analyze the stability of the gels. Furthermore, we evaluated the effect of therapeutic pulsed ultrasound (TPU) in association with the gel-diosmin complex on the parameters of muscle damage and oxidative stress in rats. Serum creatine kinase (CK) levels were used as an indicator of skeletal muscle injury. Lipid peroxidation (thiobarbituric acid-reactive substances [TBARS]) and superoxide dismutase and catalase activities were used as indicators of oxidative stress. The results obtained indicated that the inclusion complex obtained by co-evaporation had the highest complexation efficiency and stability; there was no change in the features of diosmin on incorporation into the Carbopol gel. Additionally, a significant (p <0.05) decrease was observed in CK levels (TPU plus gel-diosmin: 178.4 ± 85.3 U/L) relative to the untreated group (527.8 ± 46.1 U/L). Levels of TBARS were lower in the TPU plus gel-diosmin group (0.008 ± 0.0004 nmol malondialdehyde/mg protein, p <0.05) compared with the untreated group (0.081 ± 0.011 nmol malondialdehyde/mg protein, p <0.05, n = 6). Catalase activity did not statistically significantly differ between the treatment groups, and superoxide dismutase activity was lower in the diosmin-treated group (0.320 ± 0.11 U/mg protein) compared with the untreated group (0.983 ± 0.40 U/mg protein). These results suggest that TPU in association with the diosmin-gel complex is effective in reducing muscle damage and oxidative stress after mechanical trauma.

The inclusion complex of carvacrol and ß-cyclodextrin reduces acute skeletal muscle inflammation and nociception in rats.

BACKGROUND: Skeletal muscle inflammation is strongly associated with pain and may impair regeneration and functional recovery after injury. Since anti-inflammatory and antinociceptive effects have been described for the inclusion complex of carvacrol and ß-cyclodextrin (ßCD-carvacrol), this study investigated the effects of ßCD-carvacrol in a model of acute skeletal muscle inflammation. METHODS: Muscle injury was induced in male Wistar rats by injection of 3% carrageenan in the gastrocnemius muscle. Rats were orally pretreated with saline (vehicle) or ßCD-carvacrol (20, 40, 80 and 180 mg/kg) one hour before administration of carrageenan. RESULTS: The injection of carrageenan in the gastrocnemius muscle increased tissue myeloperoxidase (MPO) activity (p < 0.001), edema (p < 0.001) and the levels of tumoral necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, macrophage inflammatory protein (MIP-2), but not IL-10 levels. Also, it increased mechanical hyperalgesia and decreased the grip force of animals. Pretreatment with ßCD-carvacrol (80 or 160 mg/kg) significantly decreased muscle MPO activity and edema 24 h after injury in comparison to vehicle-pretreated group. Animals pretreated with ßCD-carvacrol (160 mg/kg) presented significantly lower levels of IL-1ß, IL-6 and MIP-2 and higher levels of IL-10 six hours after induction and lower levels of TNF-α and MIP-2 after 24 h when compared to the vehicle group. Pretreatment with ßCD-carvacrol also reduced mechanical hyperalgesia and limited the decrease of grip force (80 or 160 mg/kg; p < 0.001) 6 and 24 h after injury. CONCLUSION: These results show that ßCD-carvacrol reduces inflammation and nociception in a model of acute injury to skeletal muscles.

Anti-hyperalgesic effect of Lippia grata leaf essential oil complexed with ß-cyclodextrin in a chronic musculoskeletal pain animal model: Complemented with a molecular docking and antioxidant screening.

BACKGROUND: Due to its unclear pathophysiology, the pharmacological treatment of fibromyalgia is a challenge for researchers. Studies using medicinal plants, such as those from the genus Lippia, complexed with cyclodextrins (CDs) have shown innovative results. OBJECTIVE: The present research intended to evaluate the effect of an inclusion complex containing ß-cyclodextrin (ßCD) inclusion complex with Lippia grata (LG) essential oil in a chronic musculoskeletal pain model, its central activity and its possible interaction with neurotransmitters involved in pain. METHODS: After acid saline-induced chronic muscle pain, male mice were evaluated for primary and secondary hyperalgesia and muscle strength. Moreover, an antagonist assay was performed to assess the possible involvement of the opioidergic, serotonergic and noradrenergic pathways. In addition, Fos protein in the spinal cord was assessed, and a docking study and antioxidant assays were performed. RESULTS: The treatment with LG-ßCD, especially in the dose of 24mg/kg, was able to significantly decrease (p<0.05) the paw withdrawal and muscle threshold. Furthermore, LG-ßCD was shown to affect the opioidergic and serotonergic pathways. There were no significant changes in muscle strength. Fos protein immunofluorescence showed a significant decrease in expression in the dorsal horn of the spinal cord. The main compounds of LG showed through the docking study interaction energies with the alpha-adrenergic and µOpioid receptors. In all antioxidant assays, LG exhibited stronger antioxidant activities than LG-ßCD. CONCLUSION: This study suggested that LG-ßCD could be considered as a valuable source for designing new drugs in the treatment of chronic pain, especially musculoskeletal pain.

Physico-chemical characterization and antibacterial activity of inclusion complexes of Hyptis martiusii Benth essential oil in ß-cyclodextrin.

Cyclodextrins (CDs) have been used as important pharmaceutical excipients for improve the physicochemical properties of the drugs of low solubility as the essential oil of Hyptis martiusii. This oil is important therapeutically, but the low solubility and bioavailability compromises your use. Therein, the aim of this study was to obtain and to characterize physico-chemically the samples obtained by physical mixture (PM), paste complexation (PC) and slurry complexation (SC) of the essential oil Hyptis martiusii (EOHM) in ß-CD, and to compare the antibacterial and modulatory-antibiotic activity of products obtained and oil free. The physicochemical characterization was performed by differential scanning calorimetry (DSC), thermogravimetry/derivative thermogravimetry (TG/DTG), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Karl Fischer titration. Additionally, the antibacterial tests were performed by microdilution technique. Thus, it was observed that the PM method showed low complexing capacity, unlike PC and SC in which it was observed the formation of inclusion complexes. In addition, the second stage of the TG/DTG curves showed that SC was the best method inclusion with mass loss of 6.9% over the PC that was 6.0%. The XRD results corroborate with the results above suggesting the formation of new solid phase and the SEM photomicrographs showed the porous surface of the samples PC and SC. The essential oil alone demonstrated an antibacterial and modulatory effect against the S. aureus and the Gram negative strain, respectively. However, the ß-CD and the inclusion complex did not demonstrate any biological activity in the performed antibacterial assays.

Inclusion complex between ß-cyclodextrin and hecogenin acetate produces superior analgesic effect in animal models for orofacial pain.

Hecogenin acetate (HA) is a steroidal sapogenin-acetylated with pharmacological properties which have already been described in the literature such as, anti-inflammatory, anti-hyperalgesic and antinociceptive, but it has low solubility in aqueous media. Therefore, in an attempt to overcome this, we set out to create inclusion complexes between HA and b-cyclodextrin (b-CD) and evaluate the antinociceptive effects in the orofacial nociception in mice. The complexes were prepared using different methods in the molar ratios 1:1 and 1:2 and characterized physicochemically. The results of the physicochemical characterization elucidated inclusion complexes formation between b-CD and HA by freeze drying method in the molar ratio 1:2, which obtained a complexation efficiency of 92% and produced superior analgesic effect in animal models for orofacial pain at a lower dose when compared to HA alone.

D-limonene exhibits superior antihyperalgesic effects in a ß-cyclodextrin-complexed form in chronic musculoskeletal pain reducing Fos protein expression on spinal cord in mice.

Chronic musculoskeletal pain is one of the main symptoms found in Fibromyalgia with unclear etiology and limited pharmacological treatment. The aim of this study was to complex LIM in ß-cyclodextrin (LIM-ßCD) and then evaluate its antihyperalgesic effect in an animal model of chronic musculoskeletal pain. Differential scanning calorimetry and scanning electron microscopy was used for the characterization of the inclusion complex. Male Swiss mice were used for experimental procedures where mechanical hyperalgesia, thermal hyperalgesia, muscular strength, Fos immunofluorescence was studied after induction of hyperalgesia. Mechanism of action was also investigated through tail flick test and capsaicin-induced nociception. Endothermic events and morphological changes showed that the slurry complex method was the best method for the complexation. After induction of hyperalgesia, the oral administration of LIM-ßCD (50mg/kg) significantly increased the paw withdrawal threshold compared to uncomplexed limonene. Fos immunofluorescence showed that both compounds significantly decreased the number of Fos-positive cells in the dorsal horn. In nociceptive tests, FLU was able to reverse the antinociceptive effect of LIM-ßCD. After intraplantar administration of capsaicin, LIM was able to significantly decrease time to lick. LIM-ßCD has antihyperalgesic action superior to its uncomplexed form, with possible action in the dorsal horn of the spinal cord. These results suggest the possible applicability of LIM, uncomplexed or complexed with ßCD, in conditions such as FM and neuropathic pain, for which there are currently only limited pharmacological options.

Inclusion of terpenes in cyclodextrins: Preparation, characterization and pharmacological approaches.

Terpenes constitute the largest class of natural products and are important resources for the pharmaceutical, food and cosmetics industries. However, due to their low water solubility and poor bioavailability there has been a search for compounds that could improve their physicochemical properties. Cyclodextrins (natural and derived) have been proposed for this role and have been complexed with different types of terpenes. This complexation has been demonstrated by using analytical techniques for characterizing complexes such as DSC, NMR, XRD, FTIR, and TGA. The formation of inclusion complexes has been able to improve drug characteristics such as bioavailability, solubility and stability; and to enhance biological activity and efficacy. This review shows strong experimental evidence that cyclodextrins improve the pharmacological properties of terpenes, and therefore need to be recognized as being possible targets for clinical use.

Docking, characterization and investigation of ß-cyclodextrin complexed with citronellal, a monoterpene present in the essential oil of Cymbopogon species, as an anti-hyperalgesic agent in chronic muscle pain model.

BACKGROUND: Citronellal (CT) is a monoterpene with antinociceptive acute effect. ß-Cyclodextrin (ßCD) has enhanced the analgesic effect of various substances. HYPOTHESIS/PURPOSE: To evaluate the effect of CT both complexed in ß-cyclodextrin (CT-ßCD) and non-complexed, in a chronic muscle pain model (CMP) in mice. STUDY DESIGN: The complex containing CT in ßCD was obtained and characterized in the laboratory. The anti-hyperalgesic effect of CT and CT-ßCD was evaluated in a pre-clinical in vivo study in a murine CMP. METHODS: The complex was characterized through differential scanning calorimetry, derivative thermogravimetry, moisture determination, infrared spectroscopy and scanning electron microscopy. Male Swiss mice were pre-treated with CT (50mg/kg, po), CT-ßCD (50mg/kg, po), vehicle (isotonic saline, po) or standard drug (tramadol4 mg/kg, ip). 60 min after the treatment and then each 1h, the mechanic hyperalgesia was evaluated to obtain the time effect. In addition, the muscle strength using grip strength meter and hyperalgesia were also performed daily, for 7 days. We assessed by immunofluorescence for Fos protein on brains and spinal cords of mice. The involvement of the CT with the glutamatergic system was studied with molecular docking. RESULTS: All characterization methods showed the CT-ßCD complexation. CT-induced anti-hyperalgesic effect lasted until 6h (p <0.001) while CT-ßCD lasted until 8h (p <0.001vs vehicle and p <0.001vs CT from the 6th h). CT-ßCD reduced mechanical hyperalgesia on all days of treatment (p <0.05), without changing muscle strength. Periaqueductal gray (p <0.01) and rostroventromedular area (p <0.05) showed significant increase in the Fos protein expression while in the spinal cord, there was a reduction (p <0.001). CT showed favorable energy binding (-5.6 and -6.1) to GluR2-S1S2J protein based in the docking score function. CONCLUSION: We can suggest that ßCD improved the anti-hyperalgesic effect of CT, and that effect seems to involve the descending pain-inhibitory mechanisms, with a possible interaction of the glutamate receptors, which are considered as promising molecules for the management of chronic pain such as CMP.

ß-caryophyllene, a dietary cannabinoid, complexed with ß-cyclodextrin produced anti-hyperalgesic effect involving the inhibition of Fos expression in superficial dorsal horn.

AIMS: Evaluate the anti-hyperalgesic effect of the complex containing ß-caryophyllene (ßCP) and ß-cyclodextrin (ßCD) in a non-inflammatory chronic muscle pain mice model and investigated its action on superficial dorsal horn of the lumbar spinal cord. MAIN METHODS: The ßCP-ßCD complex were prepared and characterized through the DSC, TG/DTG, FTIR, XRD and SEM. The model of chronic muscle pain was induced by two injections of pH4.0 saline (20µL) into left gastrocnemius 5days apart. After confirming hyperalgesia, male mice were treated with ßCP-ßCD (10 or 20mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 9days. 1h after, the mechanical hyperalgesia, muscle withdrawal thresholds and motor performance were evaluated. To evaluate the ßCP-ßCD action on spinal cord, animals induced with chronic muscle pain were treated with ßCP-ßCD (20mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) and 90min. after, were perfused, the lumbar spinal cord collected, crioprotected, cut and submitted in an immunofluorescence protocol for Fos protein. KEY FINDINGS: The characterization tests indicated that ßCP were efficiently incorporated into ßCD. The oral treatment with ßCP-ßCD, at all doses tested, produced a significant (p<0.05) reduction on mechanical hyperalgesia and a significant (p<0.05) increase in muscle withdrawal thresholds, without produce any alteration in force. In addition, ßCP-ßCD was able to significantly (p<0.05) decrease Fos expression in the superficial dorsal horn. SIGNIFICANCE: Thus, ßCP-ßCD attenuates the non-inflammatory chronic muscle pain in mice and inhibits the Fos expression in the lumbar spinal cord.

Characterization and Antihypertensive Effect of the Complex of (-)-ß- pinene in ß-cyclodextrin.

This work aimed to characterize and evaluate the antihypertensive effect of the (-)-ß-pinene/ß-cyclodextrin (ßP/ß-CD) complex. The complex was prepared through physical mixture and slurry complexation methods and was analyzed through differential scanning calorimetry, thermogravimetry/derivative thermogravimetry, fourier transform infrared spectroscopy, diffraction X-ray, docking and scanning electron microscopy. Normotensive or L-NAME-induced hypertensive rats were used in pharmacological experiments. Mean arterial pressure (MAP) was determined with direct blood pressure measurements from the abdominal aorta. The drugs were orally administrated and their effects were recorded during 48 hours. Vascular effects of ßP were evaluated in isolated ring of mesenteric artery. The physicochemical characterization showed ßP/ß-CD complex formation. In hypertensive rats (MAP = 156±16 mmHg), the complex, but not ßP alone, promoted hypotension at 36 and 48 hours after administration (MAP = 124±3 and 110±5 mmHg, respectively). In arterial rings, ßP vasorelaxed rings precontracted with phenylephrine (Emax = 105±6%), which was not changed after the removal of the vascular endothelium (Emax = 108±4%), after the pre-contraction with KCl 80 mM (Emax = 107±8%) or S(-)-BayK8644 (Emax = 107±5%), or after incubation with TEA (Emax = 113±4%). Finally, ßP inhibited CaCl2- and sodium-orthovanadate-induced contractions. In conclusion, the slurry complexation method was the best among them. Pharmacological results demonstrated that the complex promoted antihypertensive effect. Furthermore, ßP induced endothelium- independent vasorelaxation possibly caused by the inhibition of the Ca2+ influx through L-type Ca2+ channel associated to a decrease in calcium sensitivity.

In vivo antitumor effect, induction of apoptosis and safety of Remirea maritima Aubl. (Cyperaceae) extracts.

BACKGROUND: Remirea maritima has been widely used in the treatment of diarrhea, kidney disease, and high fever and for therapeutic purposes, such as an analgesic and anti-inflammatory. However, few scientific research studies on its medicinal properties have been reported. PURPOSE: The present study aimed to investigate the anticancer potential of aqueous extract (AE), 40% hydroalcoholic extracts (40HA) and 70% (70HA) from R. maritima in experimental models and to identify its phytochemical compounds. METHODS: The chemical composition of AE, 40HA and 70HA was assessed by HPLC-DAD and ESI-IT-MS/MS. In vitro activity was determined on cultured tumor cell, NCI-H385N (Broncho-alveolar carcinoma), OVCAR-8 (Ovarian carcinoma) and PC-3M (prostate carcinoma) by the MTT assay, and the in vivo antitumor activity was assessed in Sarcoma 180-bearing mice. Toxicological parameters were also evaluated as well as the humoral immune response. RESULTS: Among the aqueous and hydroalcoholic extracts of R. maritima, only 40HA showed in vitro biological effect potential, presenting IC50 values of 27.08, 46.62 and >50µg/ml for OVCAR-8, NCI-H385M and PC-3M cells lines, respectively. Regarding chemical composition, a mixture of isovitexin-2''-O-ß-D-glucopyranoside, vitexin-2''-O-ß-D-glucopyranoside, luteolin-7-O-glucuronide and 1-O-(E)-caffeoyl-ß-D-glucose were identified as the major phytochemical compounds of the extracts. In the in vivo study, the tumor inhibition rates were 57.16-62.57% at doses of 25mg/kg and 50mg/kg, respectively, and the tumor morphology presented increasing numbers of apoptotic cells. Additionally, 40HA also demonstrated significantly increased of OVA-specific total Ig. CONCLUSIONS: 40HA exhibited in vitro and in vivo anticancer properties without substantial toxicity that could be associated with its immunostimulating properties.

α-Terpineol, a monoterpene alcohol, complexed with ß-cyclodextrin exerts antihyperalgesic effect in animal model for fibromyalgia aided with docking study.

The anti-hyperalgesic effect of the complex containing α-terpineol (αTPN) and ß-cyclodextrin (ßCD) was analyzed in a non-inflammatory chronic muscle pain model, as well as its mechanism of action through docking study for a possible interaction with receptors. The αTPN-ßCD complex was prepared and characterized through the thermogravimetry/derivate thermogravimetry (TG/DTG), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The model of chronic muscle pain was induced by two injections of pH 4.0 saline (20 µl) into the left gastrocnemius 5 days apart. After confirming hyperalgesia, male mice were treated with αTPN-ßCD (25, 50 or 100 mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 10 days. 1 h after the mechanical hyperalgesia, motor performance was evaluated. In addition, the systemic administration of naloxone and ondansetron tested the analgesic effect on the active opioid and serotonin receptors, respectively. The characterization tests indicated that αTPN was efficiently incorporated into ßCD. The oral treatment with αTPN-ßCD, at all doses tested, produced a significant (p < 0.001) decrease in the mechanical hyperalgesia, without causing any alteration in the force and in motor performance. This analgesic effect was reversed by the systemic administration of naloxone or ondansetron. These findings are corroborated by the docking study described in the present study, which verified a possible interaction of αTPN-ßCD with opioid (MU, Kappa, Delta) and 5-HT receptors. Thus, it can be concluded that αTPN-ßCD reduced the hyperalgesia followed by the chronic muscle pain model, probably evoked by the descending inhibitory pain system, specifically by opioid and serotoninergic receptors.