Milk miRNA expression in buffaloes as a potential biomarker for mastitis.

BACKGROUND: Buffaloes have the highest potential for production due to a promising gene pool that is being enhanced and upgraded. Mastitis is a significant health impediment that greatly diminishes milk yield and quality, affecting rural farmers' livelihoods. The traditional gold standard used for diagnosing mastitis or subclinical mastitis is CMT, but it has the drawback of false positive or negative results. Subclinical mastitis, if not treated promptly, can lead to mammary tumors. To address the gap in early diagnosis of subclinical mastitis in CMT-negative milk of buffaloes, we performed a retrospective analysis and evaluated the milk miRNA expression profiles as potential biomarkers. RESULTS: Thirty buffalo milk samples based on clinical signs and CMT were divided into normal, subclinical, and clinical mastitis. SCC evaluation showed significant differences between the groups. The data analysis demonstrated that the elevation of miR-146a and miR-383 differed substantially between normal, subclinical, and clinical mastitis milk of buffaloes with 100% sensitivity and specificity. The relationship of SCC with miR-146a and miR-383 in normal/healthy and subclinical mastitis was positively correlated. CONCLUSION: The overexpression of miR-146a and miR-383 is associated with inflammation. It can be a valuable prognostic and most sensitive biomarker for early mastitis detection in buffaloes with SCC below 2 lakhs and CMT-ve, enhancing the accuracy of subclinical mastitis diagnosis.

CEA, CA 15-3, and miRNA expression as potential biomarkers in canine mammary tumors.

The most often detected tumor in intact bitches is mammary tumors and represents a significant clinical problem throughout the world. Mammary neoplasms in canine have heterogeneous morphology, so the choice of the most appropriate biomarker is the biggest challenge in CMT detection. We performed a retrospective analysis and evaluated the canine cancer antigens and miRNA expression profiles as potential biomarkers. Sixty dogs based on histological examination divided into three groups, viz., dogs with a benign mammary tumor, malignant mammary tumor, and control/healthy. The CA 15-3 was found more sensitive than CEA but detection of both will increase sensitivity. miR-21 expression differed significantly in all three groups. miR-29b expression differed significantly between the control and benign group and control and malignant group. The miR-21 overexpression and miR-29b downregulation with CMT are associated with clinical stage and can be used as non-invasive diagnostic and prognostic biomarkers. Hence, evaluation of CA 15-3 along with CEA would be a non-invasive technique for detecting canine mammary tumors. Evaluation of deregulated circulating miR-21 could be a valuable prognostic marker for early detection of mammary tumors in canines while miR-29b can add sensitivity in the detection of the canine mammary tumors if evaluated with miR-21.

Microemulsion-based drug delivery system for transnasal delivery of Carbamazepine: preliminary brain-targeting study.

This study reports the development and evaluation of Carbamazepine (CMP)-loaded microemulsions (CMPME) for intranasal delivery in the treatment of epilepsy. The CMPME was prepared by the spontaneous emulsification method and characterized for physicochemical parameters. All formulations were radiolabeled with (99m)Tc (technetium) and biodistribution of CMP in the brain was investigated using Swiss albino rats. Brain scintigraphy imaging in rats was also performed to determine the uptake of the CMP into the brain. CMPME were found crystal clear and stable with average globule size of 34.11 ± 1.41 nm. (99m)Tc-labeled CMP solution (CMPS)/CMPME/CMP mucoadhesive microemulsion (CMPMME) were found to be stable and suitable for in vivo studies. Brain/blood ratio at all sampling points up to 8 h following intranasal administration of CMPMME compared to intravenous CMPME was found to be 2- to 3-fold higher signifying larger extent of distribution of the CMP in brain. Drug targeting efficiency and direct drug transport were found to be highest for CMPMME post-intranasal administration compared to intravenous CMP. Rat brain scintigraphy also demonstrated higher intranasal uptake of the CMP into the brain. This investigation demonstrates a prompt and larger extent of transport of CMP into the brain through intranasal CMPMME, which may prove beneficial for treatment of epilepsy.

Evaluation of brain targeting efficiency of intranasal microemulsion containing olanzapine: pharmacodynamic and pharmacokinetic consideration.

The objective of this study was to develop and evaluate olanzapine (OZP) -loaded microemulsions (OZPME) for intranasal delivery in the treatment of schizophrenia. The OZPME was formulated by the spontaneous microemulsification method and characterized for physicochemical parameters. Pharmacodynamic assessments (apomorphine - induced compulsive behavior and spontaneous locomotor activity) were performed using mice. All formulations were radiolabeled with technetium-99 ((99m)Tc), and biodistribution of drug in the brain was investigated using Swiss albino rats. Brain scintigraphy imaging in rabbits was performed to determine the uptake of the OZP into the brain. OZPME were found clear and stable with average globule size of 23.87 ± 1.07 nm. In pharmacodynamic assessments, significant (p < 0.05) difference in parameters estimated were found between the treated and control groups. (99m)Tc-labeled OZP solution (OZPS)/OZPME/OZP mucoadhesive microemulsion (OZPMME) were found to be stable and suitable for in vivo studies. Brain/blood ratio at all sampling points up to 8 h following intranasal administration of OZPMME compared to intravenous OZPME was found to be five to six times higher signifying larger extent of distribution of the OZP in brain. Drug targeting efficiency and direct drug transport were found to be highest for intranasal OZPMME, compared to intravenous OZPME. Furthermore, rabbit brain scintigraphy also demonstrated higher intranasal uptake of the OZP into the brain. This investigation demonstrates a prompt and larger extent of transport of OZP into the brain through intranasal OZPMME, which may prove beneficial for treatment of schizophrenia.

Paliperidone microemulsion for nose-to-brain targeted drug delivery system: pharmacodynamic and pharmacokinetic evaluation.

OBJECTIVE: The objective of present study was to develop and evaluate paliperidone (PALI) loaded microemulsion (PALI-ME) for intranasal delivery in the treatment of schizophrenia. MATERIAL AND METHODS: The PALI-ME was formulated by the spontaneous microemulsification method and characterized for physicochemical parameters. Pharmacodynamic assessments (apomorphine-induced compulsive behavior and spontaneous motor activity) were performed using mice. All formulations were tagged with (99m)Tc (technetium). Pharmacokinetic evaluation of PALI in the brain was investigated using Swiss albino rats. Brain scintigraphy imaging was performed in rabbits. RESULTS AND DISCUSSION: PALI-ME was found stable with average droplet size of 20.01 ± 1.28 nm. In pharmacodynamic studies, significant (p < 0.05) deference in parameters estimated, were found between the treated and control groups. (99m)Tc-tagged PALI solution (PALI-SOL)/PALI-ME/PALI muco-adhesive ME (PALI-MME) was found to be stable and suitable for in vivo studies. Brain-to-blood ratio at all sampling points up to 8 h following intranasal administration of PALI-MME compared to intravenous PALI-ME was found to be 6-8 times higher signifying greater extent of distribution of the PALI in brain. Rabbit brain scintigraphy demonstrated higher intranasal uptake of the PALI into the brain. CONCLUSION: This investigation demonstrates a prompt and larger extent of transport of PALI into the brain through intranasal PALI-MME, which may prove beneficial for treatment of schizophrenia.

Preparation and Evaluation of Surface Modified Lactose Particles for Improved Performance of Fluticasone Propionate Dry Powder Inhaler.

BACKGROUND: Dry powder inhalers (DPI) are generally formulated by mixing micronized drug particles with coarse lactose carrier particles to assist powder handling during the manufacturing and powder aerosol delivery during patient use. METHODS: In the present study, surface modified lactose (SML) particles were produced using force control agents, and their in vitro performance on dry powder inhaler (DPI) formulation of Fluticasone propionate was studied. With a view to reduce surface passivation of high surface free energy sites on the most commonly used DPI carrier, α- lactose monohydrate, effects of various force control agents such as Pluronic F-68, Cremophor RH 40, glyceryl monostearate, polyethylene glycol 6000, magnesium stearate, and soya lecithin were studied. RESULTS: DPI formulations prepared with SML showed improved flow properties, and atomic force microscopy (AFM) studies revealed decrease in surface roughness. The DSC and X-ray diffraction patterns of SML showed no change in the crystal structure and thermal behavior under the experimental conditions. The fine particle fraction (FPF) values of lactose modified with Pluronic F-68, Cremophor RH 40, glyceryl monostearate were improved, with increase in concentration up to 0.5%. Soya lecithin and PEG 6000 modified lactose showed decrease in FPF value with increase in concentration. Increase in FPF value was observed with increasing concentration of magnesium stearate. Two different DPI devices, Rotahaler(®) and Diskhaler(®), were compared to evaluate the performance of SML formulations. FPF value of all SML formulations were higher using both devices as compared to the same formulations prepared using untreated lactose. One month stability of SML formulations at 40°C/75% RH, in permeable polystyrene tubes did not reveal any significant changes in FPF values. CONCLUSION: SML particles can help in reducing product development hindrances and improve inhalational properties of DPI.

Comparative evaluation of polymeric nanoparticles of rifampicin comprising Gantrez and poly(ethylene sebacate) on pharmacokinetics, biodistribution and lung uptake following oral administration.

The present study reports the comparative pharmacokinetic evaluation and biodistribution of rifampicin (RIF) following oral administration of nanoparticles of a bioadhesive polymer, Gantrez and a hydrophobic polymer poly(ethylene sebacate) (PES). A specific objective of the study was to evaluate lung uptake of the nanoparticles following oral administration. Nanoparticles were obtained in the size range 350-450 nm with rifampicin loading of 12-14% w/w. Zeta potential confirmed colloidal stability. PES nanoparticles revealed high macrophage uptake compared to Gantrez nanoparticles, and direct correlation was observed between hydrophobicity (contact angle) and macrophage uptake (r2 -0.940). Enhanced RIF uptake with folic acid anchoring suggested folate receptor mediated uptake. RIF nanoparticles exhibited significantly higher Cmax and AUC, delayed Tmax and sustained release compared to plain RIF. More importantly the plasma concentration of RIF with the nanoparticles was significantly greater than the MIC of RIF (0.25 microng/mL) over 24 h. While gamma scintigraphy revealed higher lung accumulation of nanoparticles, the concentration with Gantrez nanoparticles was significantly higher. HPLC evaluation of lung concentration correlated with scintigraphy data. The significantly higher bioavailability and lung accumulation with Gantrez nanoparticle over PES nanoparticles was attributed mucoadhesion and high affinity of Gantrez to the Peyer's patches. Our study suggests Gantrez nanoparticles as a promising carrier for enhancing lung accumulation of drugs.

Buparvaquone loaded solid lipid nanoparticles for targeted delivery in theleriosis.

BACKGROUND: Buparvaquone (BPQ), a hydroxynaphthoquinone derivative, has been investigated for the treatment of many infections and is recommended as the gold standard for the treatment of theileriosis. Theileriosis, an intramacrophage infection is localized mainly in reticuloendotheileial system (RES) organs. The present study investigates development of solid lipid nanoparticles (SLN) of BPQ for targeted delivery to the RES. MATERIALS AND METHODS: BPQ SLN was prepared using melt method by adding a molten mixture into aqueous Lutrol F68 solution (80°C). Larger batches were prepared up to 6 g of BPQ with GMS: BPQ, 2:1. SLN of designed size were obtained using ultraturrax and high pressure homogenizer. A freeze and thaw study was used to optimize type and concentration of cryoprotectant with Sf: Mean particle size, Si: Initial particle size <1.3. Differential scanning calorimetry (DSC), powder X-ray diffraction (XRD) and scanning electron microscope (SEM) study was performed on optimized formulation. Formulation was investigated for in vitro serum stability, hemolysis and cell uptake study. Pharmacokinetic and biodistribution study was performed in Holtzman rat. RESULTS: Based on solubility in lipid; glyceryl monostearate (GMS) was selected for preparation of BPQ SLN. Batches of BPQ SLN were optimized for average particle size and entrapment efficiency at <100 mg solid content. A combination of Solutol HS-15 and Lutrol F68 at 2% w/v and greater enabled the desired Sf/Si < 1.3. Differential scanning calorimetry and powder X-ray diffraction revealed decrease in crystallinity of BPQ in BPQ SLN while, scanning electron microscope revealed spherical morphology. BPQ SLN revealed good stability at 4°C and 25°C. Low hemolytic potential (<8%) and in vitro serum stability up to 5 h was observed. Cytotoxicity of SLN to the U937 cell was low. The macrophage cell line revealed high (52%) uptake of BPQ SLN in 1 h suggesting the potential to RES uptake. SLN revealed longer circulation and biodistrbution study confirmed high RES uptake (75%) in RES organs like liver lung spleen etc. CONCLUSION: The high RES uptake suggests BPQ SLN as a promising approach for targeted and improved delivery in theileriosis.

Genotoxicity evaluation of asymmetric lipid polymer hybrid nanoparticles of doxycycline hydrochloride following intravenous administration.

Nanoparticles, being small (<1,000 nm) in size, provide high surface area-to-volume ratio as compared with the bulk materials which increase the concern about their potential toxicities. The present investigation was undertaken to evaluate the genotoxic potential of asymmetric lipid polymer hybrid nanoparticles of doxycycline hydrochloride (DH lipomer) following intravenous route. DH lipomer was prepared by modified nano-precipitation method as reported earlier. Doxycyline loading was found to be 20 ± 2.5 %. Average particle size of DH lipomer and blank lipomer was 512 ± 8 and 520 ± 6 nm, respectively. Micronucleus (MN) assay was performed in adult healthy Swiss mice whereas chromosomal aberration (CA) test and comet assay were performed in healthy Holtzman rats following intravenous administration. Animals were divided into two sets, male and female, each set comprising of six groups (n = 5/group), viz., three test groups, blank lipomer (BL), vehicle control (VC), and positive control. Groups treated with 1.5 mg/kg BW DH lipomer did not show micronuclei formation in bone marrow cell, DNA damage, and CA, respectively, as compared with VC, suggesting no genotoxicity. On the other hand 3 and 6 mg/kg BW revealed significant (P > 0.001) increase in micronuclei formation, DNA damage, and chromosomal aberrations. Furthermore, BL (6 mg/kg BW) did not reveal genotoxic response in any of the tests, suggesting lipomer components as non-genotoxic. No sex-dependent variation in genotoxicity was observed. This study therefore suggests the potential safety of the proposed dose of DH lipomer at 1 mg/kg BW. An interesting highlight of the study is safety of lipomer matrix which could be exploited for other biomedical application.

Self nanoprecipitating preconcentrate of tamoxifen citrate for enhanced bioavailability.

We disclose a self nanoprecipitating preconcentrate (SNP) of tamoxifen citrate (TMX), which forms TMX loaded polymeric nanoparticles, on dilution with aqueous media. SNP comprised TMX, polymer (Kollidon SR) and surfactant/s dissolved in a pharmaceutically acceptable vehicle. Binary surfactant mixtures of Aerosol OT (AOT) with Tween 80 revealed synergistic reduction in surface tension to enable both high entrapment efficiency (EE) and low particle size (PS). Synergism of the surfactants was confirmed by molecular interaction parameter(ß(σ)). Combination of AOT and Tween 80 resulted in EE (∼85%) and PS (<250nm). Formation of TMX-KSR nanoparticles in situ was reproducible under most experimental conditions and exhibited pH independent behavior. Dilution volume (>80mL) influenced both PS and EE while dilution temperature influenced only PS. Marginal increase in size was evident at the end of 1h nevertheless was not of concern as TMX SNP exhibited near complete release in 1h. DSC and XRD studies revealed amorphous nature of TMX in nanoparticles. FTIR imaging confirmed uniform distribution of TMX in nanoparticles. ESEM and TEM revealed spherical nanoparticles. Biodistribution studies of (99m)Tc labeled TMX SNP in rats revealed no significant absorption however oral pharmacokinetics revealed enhanced oral bioavailability of TMX (165%) compared to TMX suspension. SNP presents a new in situ approach, for design of drug loaded polymeric nanoparticles.

Polyethylene sebacate-doxorubicin nanoparticles for hepatic targeting.

The present study discusses polyethylene sebacate (PES)-doxorubicin (DOX) nanoparticles (PES-DOX NP) using pullulan as asialoglycoprotein receptor (ASGPR) ligand for hepatic targeting. Pullulan, a hydrophilic polymer served as ligand and as stealth agent. PES-DOX NP were prepared by modified nanoprecipitation using PES and Gantrez AN 119 (Gantrez), as complexing agent in the organic phase, while DOX was dissolved in the aqueous phase. Pullulan was adsorbed on the formed nanoparticles (PES-DOX-PUL). Intimate association of PES and Gantrez, and ionic complexation of DOX with Gantrez (confirmed by FTIR), coupled with rapidity of nanoprecipitation resulted in nanoparticles with high entrapment efficiency and high drug loading. Nanoparticles were successfully freeze dried. Drug release from PES NP followed zero order kinetics. PES-DOX NP and PES-DOX-PUL exhibited low hemolytic potential and good serum stability. Comparative biodistribution study in rats using (99m)Tc labeled formulations revealed higher blood concentration and lower liver concentration of PES-DOX-PUL, confirming the long circulating nature of PES-DOX-PUL, and thereby the possibility of improved targeting to hepatocytes. Nanoparticles revealed lower DOX concentration in the heart suggestive of low cardiotoxicity. Our study presents a radically different yet simple approach for the design of PES-DOX nanoparticles with high drug loading for improved therapy in hepatic cancer.

Particle shape: a new design parameter for passive targeting in splenotropic drug delivery.

The role of particle size and surface modification on biodistribution of nanocarriers is widely reported. We report for the first time the role of nanoparticle shape on biodistribution. Our study demonstrates that irregular shaped polymer lipid nanoparticles (LIPOMER) evade kupffer cells and localize in the spleen. We also demonstrate the macrophage-evading characteristic of the irregular-shaped LIPOMER. Our results suggest particle shape as an important tool for passive targeting of nanocarriers in splenotropic drug delivery.