Novel lactoferrin-conjugated gallium complex to treat Pseudomonas aeruginosa wound infection.

Pseudomonas aeruginosa is one of the leading causes of opportunistic infections such as chronic wound infection that could lead to multiple organ failure and death. Gallium (Ga3+) ions are known to inhibit P. aeruginosa growth and biofilm formation but require carrier for localized controlled delivery. Lactoferrin (LTf), a two-lobed protein, can deliver Ga3+ at sites of infection. This study aimed to develop a Ga-LTf complex for the treatment of wound infection. The characterisation of the Ga-LTf complex was conducted using differential scanning calorimetry (DSC), Infra-Red (FTIR) and Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES). The antibacterial activity was assessed by agar disc diffusion, liquid broth and biofilm inhibition assays using the colony forming units (CFUs). The healing capacity and biocompatibility were evaluated using a P.aeruginosa infected wound in a rat model. DSC analyses showed thermal transition consistent with apo-lactoferrin; FTIR confirmed the complexation of gallium to lactoferrin. ICP-OES confirmed the controlled local delivery of Ga3+. Ga-LTf showed a 0.57 log10 CFUs reduction at 24 h compared with untreated control in planktonic liquid broth assay. Ga-LTf showed the highest antibiofilm activity with a 2.24 log10 CFUs reduction at 24 h. Furthermore, Ga-LTf complex is biocompatible without any adverse effect on brain, kidney, liver and spleen of rats tested in this study. Ga-LTf can be potentially promising novel therapeutic agent to treat pathogenic bacterial infections.

Gallium-Curcumin Nanoparticle Conjugates as an Antibacterial Agent against Pseudomonas aeruginosa: Synthesis and Characterization.

Combating antibiotic resistance has found great interest in the current clinical scenario. Pseudomonas aeruginosa, an opportunistic multidrug-resistant pathogen, is well known for its deadly role in hospital-acquired infections. Infections by P. aeruginosa are among the toughest to treat because of its intrinsic and acquired resistance to antibiotics. In this study, we project gallium-curcumin nanoparticle (GaCurNP) conjugates as a prospective candidate to fight against P. aeruginosa. The synthesized GaCurNPs were spherical with an average size ranging from 25-35 nm. Analysis by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy deduced the nature of interaction between gallium and curcumin. Conjugate formation with gallium was found to improve the stability of curcumin at the physiological pH. When tested after 24 h of contact, at the physiological pH and 37 °C, the degradation of curcumin bound in the GaCurNPs was 26%, while that of native curcumin was 95%. The minimum inhibitory concentration (MIC) of GaCurNPs was found to be 82.75 µg/mL for P. aeruginosa (ATCC 27853). GaCurNPs also showed excellent biofilm inhibition at 4MIC concentration. Raman spectroscopic analysis showed that GaCurNPs are capable of disrupting the cells of P. aeruginosa within 3 h of contact. Live/dead imaging also confirmed the compromised membrane integrity in cells treated with GaCurNPs. Scanning electron microscopy analysis showed membrane lysis and cell structure damage. The AlamarBlue assay showed that when L929 cell lines were treated with GaCurNPs with concentrations as high as 350 µg/mL, the cell viability elicited by the nanoparticles was 70.89%, indicating its noncytotoxic nature. In short, GaCurNPs appear to be a promising antibacterial agent capable of fighting a clinically significant pathogen, P. aeruginosa.

Vanadium pentoxide nanoparticle mediated perturbations in cellular redox balance and the paradigm of autophagy to apoptosis.

The redox-active transition metals such as copper, iron, chromium, vanadium, and silica are known for its ROS generation via mechanisms such as Haber-Weiss and Fenton-type reactions. Nanoparticles of these metals induce oxidative stress due to acellular factors owing to their small size and more reactive surface area, leading to various cellular responses. The intrinsic enzyme-like activity of nano vanadium has fascinated the scientific community. However, information concerning their cellular uptake and time-dependent induced effects on their cellular organelles and biological activity is lacking. This comprehensive study focuses on understanding the precise molecular interactions of vanadium pentoxide nanoparticles (VnNp) and evaluate their specific "nano" induced effects on MDA-MB-231 cancer cells. Understanding the mechanism behind NP-induced ROS generation could help design a model for selective NP induced toxicity, useful for cancer management. The study demonstrated the intracellular persistence of VnNp and insights into its molecular interactions with various organelles and its overall effects at the cellular level. Where triple-negative breast cancer MDA-MB-231 cells resulted in 59.6% cell death towards 48 h of treatment and the normal fibroblast cells showed only 15.4% cell death, indicating an inherent anticancer property of VnNp. It acts as an initial reactive oxygen species quencher, by serving itself as an antioxidant, while; it was also found to alter the cellular antioxidant system with prolonged incubation. The VnNp accumulated explicitly in the lysosomes and mitochondria and modulated various cellular processes including impaired lysosomal function, mitochondrial damage, and autophagy. At more extended time points, VnNp influenced cell cycle arrest, inhibited cell migration, and potentiated the onset of apoptosis. Results are indicative of the fact that VnNp selectively induced breast cancer cell death and hence could be developed as a future drug molecule for breast cancer management. This could override the most crucial challenge of chemo-resistance that still remain as the main hurdle to cancer therapy.

Organ distribution and biological compatibility of surface-functionalized reduced graphene oxide.

Graphene is an sp2 hybridized allotrope of carbon with a honeycomb lattice structure that has many applications in biomedicine owing to its unique physico-chemical properties. Graphene has attracted much interest from scientists for its biomedical potential, including in drug/gene delivery, fluorescent labeling of target analytes, tissue engineering, regenerative medicine and MRI contrast enhancement. However, there are very limited data available concerning the toxicity of graphene, and efforts have been made to study the bio-nano interactions of Pluronic functionalized reduced graphene oxide (rGO-P) in animal models. The present study aimed to evaluate the systemic toxicity of rGO-P and its ability to cross the blood-brain barrier in Swiss Albino mice subject to acute exposure to 10 mg kg-1 body weight of rGO-P. Prolonged exposure was evaluated in female Wistar rats by analyzing feto-placental transmission and any associated developmental neurotoxicity after intravenous administration of 5 mg kg-1 and 10 mg kg-1 body weight of rGO-P. Biodistribution analysis using confocal Raman mapping indicated that tiny amounts of rGO-P accumulated in major organs of both dams and pups, with no evident toxic response. The accumulation of rGO-P in various tissues of rat pups born to treated dams is ample evidence of feto-placental transmission. The present study clearly suggests that rGO-P is not toxic under any of the experimental conditions. These findings can therefore be carried forward for application of rGO-P in drug/gene delivery, early diagnosis and treatment of various diseases in neonates and adults. The results of the study show that rGO-P is an auspicious and promising material for future healthcare applications.

Raman spectroscopy for the detection of organ distribution and clearance of PEGylated reduced graphene oxide and biological consequences.

Graphene, a 2D carbon material has found vast application in biomedical field because of its exciting physico-chemical properties. The large planar sheet like structure helps graphene to act as an effective carrier of drug or biomolecules in enormous amount. However, limited data available on the biocompatibility of graphene upon interaction with the biological system prompts us to evaluate their toxicity in animal model. In this study organ distribution, clearance and toxicity of PEGylated reduced nanographene (PrGO) on Swiss Albino mice was investigated after intraperitoneal and intravenous administration. Biodistribution and blood clearance was monitored using confocal Raman mapping and indicated that PrGO was distributed on major organs such as brain, liver, kidney, spleen and bone marrow. Presence of PrGO in brain tissue suggests that it has the potential to cross blood brain barrier. Small amount of injected PrGO was found to excrete via urine. Repeated administration of PrGO induced acute liver injury, congestion in kidney and increased splenocytes proliferation in days following exposure. Hence the result of the study recommended that PrGO should undergo intensive safety assessment before clinical application or validated to be safe for medical use.

Europium Doped Calcium Deficient Hydroxyapatite as Theranostic Nanoplatforms: Effect of Structure and Aspect Ratio.

We present the development of theranostic nanoplatforms (NPs) based on a europium (Eu3+) doped calcium deficient hydroxyapatite (CDHA) core functionalized with cyclodextrin (ß-CD) and cucurbitural (CB[7]). The composition, crystalline structure, aspect ratio, surface area, morphology, and luminescence property of the NPs were investigated by means of X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray analysis (EDAX), the Brunauer-Emmett-Teller (BET) method, transmission electron microscopy (TEM), and fluorescence spectroscopy. The perceivable effects of Eu3+ doping appear in the minor peak shift to larger angles attributed to lower crystallite size and smaller aspect ratios coupled with greater structural strain in the rod shaped theranostic NPs and a shift in their zeta potential toward less negative values. Cell parameter calculations suggest that the doping of Eu3+ would cause the a-axis parameter to decrease slightly as the ionic radius of Eu3+ is smaller than that of Ca2+. Moreover drug release profiles employing 5-fluorouracil (5FU) suggest that these luminescent NPs depict controlled and sustained release profiles. Further the emissive intensities of the NPs in the carrier systems increase with cumulative released amounts of 5FU, suggesting that release of the drug can be monitored by changes in luminescent intensity. In addition, native NPs manifest commendable cytocompatibility as demonstrated by MTT and live/dead protocols, whereas the 5FU loaded NPs demonstrated over 80% HeLa cell death, signifying their therapeutic potential. We envision that these NPs can serve as effective and practical multifunctional probes for theranostic applications.

Neodymium doped hydroxyapatite theranostic nanoplatforms for colon specific drug delivery applications.

Theranostic nanoplatforms integrate therapeutic payloads with diagnostic agents, and help monitor therapeutic response. In this regard, stimuli responsive nanoplatforms further favour combinatorial therapeutic approach that can considerably improve efficacy and specificity of treatment. Herein, we present the engineering of a smart theranostic nanoplatform based on neodymium doped hydroxyapatite (HAN). The presence of neodymium endows the HAN nanoplatforms with near-infrared fluorescence capability. These HAN nanoparticles were then subsequently modified with alginic acid (HANA) to confer pH responsiveness to the synthesized nanoplatforms delivering them to the colon after oral administration. These nanoplatforms possessing optimum size, needle shaped morphology and negative zeta potential, are conducive to cellular internalization. On excitation at 410nm they exhibit near infrared emission at 670nm unraveling their theranostic capabilities. Cytotoxic effects systematically assessed using MTT and live dead assays reveal excellent viability. Raman microscopic imaging technique used to visualize uptake in HeLa cells demonstrate increased uptake from 4 to 16h, with growing cluster size and localization in the cytoplasm. Moreover the concomitant presence of alginic acid manifested advantages of augmented loading and pH dependent release profiles of the model drug, 4 acetyl salicylic acid (4ASA). We could thus establish a theranostic system for early tumour detection, targeted tumour therapy and monitoring of colon cancer that can be administered via the oral route.

Aggregation induced Raman scattering of squaraine dye: Implementation in diagnosis of cervical cancer dysplasia by SERS imaging.

The extent of squaraine dye aggregation that reflects on surface enhanced Raman signal scattering (SERS) intensity upon adsorption on nano-roughened gold surface has been investigated. Here we have synthesized a serious of six squaraine dyes consisting of two different electron donor moiety i.e. 1,1,2-trimethyl-1H-benzo[e]indole and 2-methylbenzo[d]thiazole which modulates the chemisorptions and hydrophobicity being designated as SQ1, SQ2, SQ3, SQ4, SQ5 and SQ6. Interestingly, SQ2 (mono lipoic acid appended), SQ5 and SQ6 (conjugated with hexyl and dodecyl side chain) squaraine derivatives having more tendency of aggregation in DMSO-water mixed solvent showed significant increase of Raman scattering in the fingerprint region when chemisorbed on spherical gold nanoparticles. Two sets of SERS nanotags were prepared with colloidal gold nanoparticle (Au-NPs size: 40 nm) by incorporating Raman reporters SQ2 and SQ5 followed by thiolated PEG encapsulation (SH-PEG, SH-PEG-COOH) denoted as AuNPs-SQ2-PEG and AuNPs-SQ5-PEG. Further conjugation of these nanotag with monoclonal antibodies specific to over expressed receptors, EGFR and p16/Ki-67 in cervical cancer cell, HeLa showed prominent SERS mapping intensity and selectivity towards cell surface and nucleus. The fast and accurate recognition obtained by antibody triggered SERS-nanotag has been compared with conventional time consuming immunocytochemistry technique which prompted us to extend further investigation using real patient cervical smear sample for a non-invasive, ultrafast and accurate diagnosis.

Chitosan scaffold co-cultured with keratinocyte and fibroblast heals full thickness skin wounds in rabbit.

This study evaluated the modulatory effect of chitosan sponge co-cultured with keratinocyte and fibroblast on wound healing. Dermal fibroblasts and keratinocyte isolated from rabbit skin were co-cultured on chitosan sponge, to fabricate cell-loaded chitosan tissue engineered construct. Full thickness excision wounds created on the rabbit dorsum were treated with three types of graft materials ­ a noncellular chitosan graft, homologous keratinocyte fibroblast loaded chitosan, and a commercial product. Postgraft skin-wound samples were examined histomorphologically at 7th, 14th, and 28th day after staining with hematoxylin and eosin, picrosirius red and/or immunohistochemistry. Wound healing parameters considered were the extent of re-epithelialization, collagen deposition, and neoangiogenesis. The number of proliferating cells, vimentin positive cells, and alpha smooth muscle actin cells were also quantified. The histology results suggested that the grafts aided wound healing but, the cell-loaded graft induced a differential pattern of healing and had lower scarring tendency. The cell-loaded tissue construct may be useful as a therapeutic graft for treating wounds where there is a total loss of tissue and cells as in burn injury.

PEGylated starch acetate nanoparticles and its potential use for oral insulin delivery.

A novel controlled release formulation has been developed with PEGylated starch acetate nanoparticles. Biodegradable polymers, such as starch, have been studied for various pharmaceutical applications because of their biocompatibility and biodegradability. Starch acetate is one of the hydrophobic biodegradable polymers currently being used or studied for controlled drug delivery. Polyethylene glycol was conjugated with starch acetate, to obtain an amphiphilic polymeric derivative. On its incubation with insulin solution at the critical micelle concentration, self-aggregated nanoparticles with mean particle size of 32 nm are formed. These self-aggregated nanoparticles with associated insulin have enhanced encapsulation efficiency. The mean particle size of these nanoparticles increased with the increase in the molecular weight of PEG. Present study indicated that PEGylated starch acetate nanoparticles are highly bioadhesive and can be utilized as a carrier system for controlled delivery of insulin or other proteins for various therapeutic applications.

Blood compatibility studies of Swarna bhasma (gold bhasma), an Ayurvedic drug.

Swarna bhasma (gold bhasma) preparations are widely utilized as therapeutic agents. However, in vitro biological evaluations of bhasma preparations are needed along with the physicochemical characterization for present day standardization of metallic bhasma preparations to meet the criteria that supports its use. Therefore, an attempt has been made to evaluate the protein adsorption, blood compatibility and complement activation potential of two batches of Swarna bhasma preparation, along with its physicochemical characterization. The particle size, morphology, elemental analysis, and in vitro cytotoxicity were evaluated initially. Red blood cell hemolysis, aggregation studies with blood cells, protein adsorption, complement C3 adsorption, platelet activation and tight junction permeability in Caco-2 cell line were investigated. The Swarna bhasma preparations with a crystallite size of 28-35 nm did not induce any blood cell aggregation or protein adsorption. Activation potential of these preparations towards complement system or platelets was negligible. These particles were also non-cytotoxic. Swarna bhasma particles opened the tight junctions in Caco-2 cell experiments. The results suggest the application of Swarna bhasma preparations as a therapeutic agent in clinical medicine from the biological safety point of view.

Nature vs. nurture: gold perpetuates "stemness".

Adult tissues contain quiescent reservoirs of multipotent somatic stem cells and pluripotent embryonic-like stem cells (ELSCs). Credited with regenerative properties gold is used across both -contemporary and -ancient medicines. Here, we show that gold exerted these effects by enhancing the pool of pluripotent ELSC while improving their stemness. We used hESCs as an in-vitro model to understand if gold could enhance self-renewal and pluripotency. Swarna-bhasma (SB), an ancient Indian gold microparticulate (41.1 nm), preparation, reduced spontaneous-differentiation, improved self-renewal, pluripotency and proliferation of hESCs. Colloidal gold-nanoparticles (GNP) (15.59 nm) were tested to confirm that the observations were attributable to nanoparticulate-gold. SB and GNP exposure: maintained -stemness, -karyotypic stability, enhanced pluripotency till day-12, increased average colony-sizes, and reduced the number of autonomously-derived differentiated FGFR1 positive fibroblast-niche-cells/colony. Particulate-gold induced upregulation of FGFR1 and IGF2 expression, and decrease in IGF1 secretion indicates IGF1/2 mediated support for enhanced pluripotency and self-renewal in hESCs.

Enhanced delivery of lopinavir to the CNS using Compritol-based solid lipid nanoparticles.

BACKGROUND: Protease inhibitors such as lopinavir have negligible permeability to the CNS due to blood-brain and blood-cerebrospinal fluid interfaces. An attempt has been made to develop solid lipid nanoparticles to increase the availability of lopinavir in the CNS. RESULTS/DISCUSSION: Solid lipid nanoparticle formulations exhibited a Cmax and T(max) of 632.86 +/- 81.61 ng/ml and 25 +/- 7.75 min, respectively, with a significant increase in bioavailability in a rat model compared with a free-drug suspension. An appreciable increase in cerebrospinal fluid concentration was detected with solid lipid nanoparticle formulations. CONCLUSION: Compritol-based solid lipid nanoparticles with a poloxamer coating can be effectively absorbed through the lymphatic system, prolong the circulation of drug in blood by acting as a reservoir and can effectively target the drug to the CNS due to the combined effect of lipophilicity and surface charge. The high biocompatibility, biodegradability and nontoxicity of compritol make the compritol-based solid lipid nanoparticles an excellent carrier for enhanced CNS delivery of lopinavir.

Copper complexed polymer carriers for IgG adsorption.

Cost effective adsorption matrix is recently, a much sought after alternative to the normal and expensive forms of matrices that are being used for the separation and purification of therapeutic molecules like immunoglobulins. A study therefore, has been focussed on developing copper complexed poly (vinyl alcohol) [PVA] and poly (styrene allyl alcohol) [PSA] gel beads for the separation of immunoglobulin G (IgG) from aqueous solutions. The copper-immobilized PVA and PSA gel beads were prepared, characterized and the copper content was estimated using EDX analysis. Further compatibility tests such as erythrocyte aggregation, lyses and cell counting were also investigated. An increase in the IgG adsorption capacities was achieved with the copper immobilized micro particles, when compared with the unmodified particles.

Translational regulation mechanisms of AP-1 proteins.

The activator protein 1 (AP-1) transcription factor is assembled from jun-jun, jun-fos, or jun-atf family protein homo- or heterodimers. AP-1 belongs to the class of basic leucine zipper (bZIP) transcription factors. It binds to promoters of its target genes in a sequence-specific manner, and transactivates or represses them. AP-1 proteins are implicated in the regulation of a variety of cellular processes including proliferation and survival, differentiation, growth, apoptosis, cell migration, and transformation. The decision if a given AP-1 factor is positively or negatively regulating a specific target gene is made upon abundance of dimerization partners, dimer-composition, post-translational regulation, and interaction with accessory proteins. In this review we describe translational control mechanisms that can regulate the abundance of AP-1 proteins. The Atf4/5, and JunD (mRNAs) are regulated by upORF dependent mechanisms. JUNB (mRNA) translation is controlled via mTOR. Translation efficiency of the unstable c-Fos (mRNA) can be decreased by the miRNA mir7B, while its perinuclear translation might facilitate efficient nuclear c-fos protein import. c-Jun (mRNA) appears to be regulated by both, m7G cap (CAP)-dependent and CAP-independent translational control mechanisms, via putative internal ribosome entry segments (IRES). IRES elements were also proposed to play a role in the regulation of JunD (mRNA). We conclude that in addition to transcriptional and post-translational control mechanisms translational regulation contributes to the balanced production of AP-1 proteins, in order to maintain physiological cellular conditions.

Synthesis and characterization of PEGylated calcium phosphate nanoparticles for oral insulin delivery.

The inconvenience of subcutaneous insulin delivery leads to low patient compliance with the dosage regimens. The most desirable form of administration seems to be through the oral route. This work investigates the utility of PEGylated calcium phosphate nanoparticles as oral carriers for insulin. Calcium phosphate nanoparticles (CaP) with an average particle size of 47.9 nm (D50) were synthesized and surface modified by conjugating it with poly(ethylene glycol) (PEG). These modified nanoparticles were having a near zero zeta potential. Protection of insulin from the gastric environment has been achieved by coating the nanoparticles with a pH sensitive polymer that will dissolve in the mildly alkaline pH environment of the intestine. The release profiles of coated nanoparticles exhibited negligible release in acidic (gastric) pH, i.e., only 2% for CaP and 6.5% for PEGylated CaP. However, a sustained release of insulin was observed at neutral (intestinal) pH for over 8 h. The conformation of the released insulin, studied using circular dichroism, was unaltered when compared with native insulin. The released insulin was also stable as it was studied using dynamic light scattering. Radioimmunoassay was performed and the immunoreactivity of the released insulin was found to be intact. These results suggest PEGylated calcium phosphate nanoparticles as an excellent carrier system for insulin toward the development of an oral insulin delivery system.

Tricalcium phosphate delayed release formulation for oral delivery of insulin: a proof-of-concept study.

Several attempts have been made for delivering insulin orally utilizing several polymers with varying degrees of effectiveness. A major obstacle associated with polymeric delivery system for protein or polypeptide drugs is the poor retention of the structure and its biological activity of encapsulated proteins particularly for the unstable insulin. Calcium phosphate ceramic is considered highly compatible to protein or peptide drugs, particularly insulin. Therefore, an attempt has been made to load insulin in tricalcium phosphate (TCP) microspheres and coat with pH sensitive polymer of methacrylate derivative, and to study the stability and conformational variations of loaded insulin, and finally the biological activity of the formulation in diabetic rats. TCP microspheres were prepared by a standard procedure. Human insulin was loaded in to these porous microspheres by diffusion filling and coated with Eudragit S100. This was subjected to in vitro release studies in simulated fluids and the stability and conformational variations of the released insulin were studied using photon correlation spectroscopy and circular dichroism (CD). Biological activity of the formulation was studied on induced diabetic rats. Insulin released in the intestinal fluid (SIF) maintained the native conformation without showing any aggregation. A dose dependent reduction of blood glucose level (BGL) was achieved in streptozotocin induced diabetic Wistar rats, demonstrating its biological activity. It has been established from this preliminary study that insulin loaded in to TCP microspheres is highly compatible with no degradation or loss of biological activity of loaded insulin. The TCP microsphere based delayed release formulation of insulin has effected a decrease in elevated glucose level in induced diabetic rats, establishing its feasibility towards the development of a noninvasive delivery device.

Effect of calcium, zinc and magnesium on the attachment and spreading of osteoblast like cells onto ceramic matrices.

Calcium phosphate ceramic has been widely used as bone substitute materials. Neumerours approaches have been investigated to develop tissue-engineered scaffold from hydroxyapatite because of its advantages like osteoconduction. We have developed porous ceramic matrices from nanoparticles of calcium phosphate containing zinc and magnesium. Mimicking the grain size of natural bone enhances the bone forming function of cells. Osteoblast-like MG63 cells were cultured on to these porous ceramic matrices. Cell adhesion and spreading onto these matrices were studied for 24 h and 5 days in vitro. It was observed that on calcium phosphate matrix, containing a combination of zinc and magnesium, the osteoblast adhesion and spreading was significant on 5th day. This appeared to be comparable to the hydroxyapatite control. This makes it a promising candidate as a bone tissue-engineering scaffold.

Ceramic drug delivery: a perspective.

Different ceramic substances are offered in the market as bone substitute materials. These include monophasic calcium phosphate ceramics of tricalciumphosphate (TCP) or hydroxyapatite (HA), biphasic calcium phosphate ceramics and multiphasic bio-glasses synthetic calcium phosphate cements. Ceramics with appropriate three-dimensional geometry are able to bind and concentrate bone morphogenetic proteins in circulation and may become osteoinductive (capable of osteogenesis) and can be effective carriers of bioactive peptide or bone cell seeds and are therefore potentially useful in tissue engineering and drug delivery. An attempt has been made to review various drug delivery applications of ceramics.