Pharmaceutical Nanotechnology (v.1, #1)

Editorial-It's here! by Ijeoma Uchegbu (1-1).

Spotlight - Professor Lisbeth Illum by Stanley (Bob) Davis (4-5).

Cyclodextrins for Non-Viral Gene and siRNA Delivery by Aoife M. O'Mahony, Martin J. O'Neill, Bruno M.D.C. Godinho, Raphael Darcy, John F. Cryan, Caitriona M. O'Driscoll (6-14).
Considerable research is focused on the development of non-viral vectors for gene and RNA interference therapies, with significant advancements in this field over the past number of years. Cationic lipids and polymers have been extensively investigated for these purposes, but there still remains a need for alternative vectors. Cyclodextrins (CDs) are cyclic oligosaccharides derived from starch and are well characterised pharmaceutical excipients. They offer many advantages as potential non-viral vectors for gene and siRNA delivery, in particular the ease with which they can be chemically modified and their lack of toxicity. In recent years, there has been a surge in the number of publications concerning CDs in this field. In this paper, we will review the two main approaches to the use of CDs for gene and siRNA delivery. In the first instance, CDs are used as a scaffold, to which various chemical groups can be grafted, yielding monodisperse functionalised CDs which can self-assemble in the presence of oligonucleotides. CDs are particularly amenable to chemical modification and this approach enables specific and precise design of CD vectors for targeting to various cell and tissue types. In the second approach, CDs can be included as a component of a delivery system, for example, as part of a polymer backbone, appended to a dendrimeric vector, or in polyrotaxane systems. Here, the inclusion of CDs facilitates post-modification of the vector through the formation of inclusion complexes with adamantane and, in some instances, reduces toxicity of the vector. Lastly, we will consider the development of in vivo CD vectors for therapeutic use and other novel applications including siRNA delivery in neurons and the CNS.

Hydrophobic drugs are problematic to deliver. We have previously shown that poly(ethylenimine) (PEI) amphiphile nanoparticles facilitate the oral absorption of the model drug cyclosporine A (CsA). We hypothesised that polymer molecular features (molecular weight, a branched or linear polymer architecture, polymer hydrophobicity) would drive this oral absorption enhancement. To test this hypothesis, we synthesised five linear PEI amphiphiles (Mn ~ 1.3 kDa or 13 kDa) and two branched PEI amphiphiles (Mn ~ 4 kDa or 10 kDa) by cetylation of PEI, followed by methylation to give N-cetyl, N-methyl, N,N-dimethyl, N,N,N-trimethyl poly(ethylenimines). Polymer aggregation, CsAencapsulation, polymer in vitro cytotoxicity and polymer enabled oral drug absorption were all studied. The polymers aggregated in aqueous media, with critical micellar concentrations (CMCs) ranging from 0.2 – 0.9 mg mL-1. The polymer CMCs, CsA encapsulation (up to 0.36 g CsA per g of PEI amphiphile) within the 60 – 200 nm nanoparticles and rat oral drug absorption after a 7.5 mg kg-1 dose of CsA, polymer nanoparticles (polymer, drug weight ratio – 5: 1) all increased with polymer hydrophobicity, although these parameters were not affected by polymer molecular weight or polymer branching. Release of CsA from a tablet dosage form was enhanced in the presence of the more hydrophilic branched polymer, however, as the more hydrophobic polymers endowed the tablets with longer disintegration times. Cell cytotoxicity (A431 cells and rat erythrocytes) was similar for all polymers (A431 IC50 = 10 – 50 μg mL-1 and 50% haemolysis = 30 – 55 μg mL-1).We conclude that the most important parameter controlling amphiphilic polymer nanoparticle enabled oral drug absorption is the hydrophobicity of the polymer amphiphile.

Effect of Formulations of Nanosized Quercetin Liposomes on COX-2 and NF-κB in MCF-10A Cells by Aroonsri Priprem, Saengrawee Sutthiparinyanont, Hye-Kyung Na, Young-Joon Surh, Malyn Chulasiri (26-34).
The effect of formulations of quercetin, a dose-dependent bioflavonoid, on COX-2 expression and NF-!B activity in human epithelial MCF-10A cells was investigated. To protect quercetin from exposure to aqueous environment, four formulations of quercetin-encapsulated liposomes were developed and tested with the MCF-10A cell line. Phospholipids, cholesterol and Span 60 at a molar ratio of 1:1:1 were used to form liposomes with or without either piperine or polyethylene glycol 400 (PEG) or alginate as additional ingredients. Overall, the liposomal particles averaged 200 nm in diameter with negative zeta potentials and encapsulation efficiencies of higher than 85%. Using Western blot analysis, pretreatment of MCF-10A cells with these liposomes containing 7.5 and 15 μM of quercetin were compared with controls. COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was significantly reduced when PEG was the additional ingredient of the quercetin liposomes. Also, quercetin at 7.5 μM was found to be effective in COX-2 inhibition provided that it was delivered in a liposomal encapsulation with PEG coating. Electrophoretic mobility shift assay for NF- !B DNA binding activity confirmed the anti-inflammatory activity of quercetin in the nuclei after it was delivered into the cells by this liposomal formulation. Therefore, some anti-inflammatory activities of quercetin in MCF-10A cells were influenced by the physicochemical properties of its nanosized liposomes.

Primaquine Loaded Chitosan Nanoparticles For Liver Targeting by Nidhi Gupta, Rampal Rajera, Manju Nagpal, Sandeep Arora (35-43).
Primaquine (PQ) loaded chitosan (CS) nanoparticles were prepared by ionic gelation technique using different concentrations of chitosan and sodium tripolyphosphate (TPP) and the potential of using these nanoparticles for liver targeting was investigated. Various factors such as concentration of chitosan and the amount of the cross-linking agent (TPP) were investigated to reveal their influences on the zeta potential, particle size, entrapment efficiency and in vitro release of the resulted nanoparticles. The formulation conditions were optimized to obtain a positive charged spherical nanoparticles with zeta potential of 29.0mV, polydispersity index of 0.213 and particle size in the range of 100-200nm. The entrapment efficiency of best batch of cross-linked chitosan nanoparticles encapsulated with primaquine (PQ-CS-NP) (i.e. 0.6mg/ml TPP into 0.1% chitosan solution) was found to be 93.21±0.17. It was observed that the in vitro release of primaquine from CS NPs was 96.52±0.54 within 24 h demonstrating sustained drug release effect. TEM showed that the prepared nanoparticles were smooth and spherical in shape. DSC and FTIR revealed no significant interactions between primaquine and CS after encapsulation and cross-linking. XRD data indicated that primaquine creates a non crystalline dispersion into the chitosan nanoparticles. The optimized batch of nanoparticles was tested in vivo to study the liver targeting effect. Primaquine loaded chitosan nanoparticles showed a much higher distribution of drug into liver as compared to that in blood. In comparison, primaquine solution showed lesser concentration in both liver and blood after IV administration into mice. The results concluded that chitosan nanoparticles were efficient carriers for the targeting of drug into liver.

Risperidone Microemulsion for Transnasal Delivery: Pharmacodynamic and Pharmacokinetic Evaluation by R. B. Patel, M. R. Patel, K. K. Bhatt, B. G. Patel, A. Mishra, K. Chutani, R. V. Gaikwad, A. Samad (44-53).
The present study describes development and evaluation of risperidone (RPD) microemulsions (RME) for transnasal delivery in the treatment of schizophrenia. The RME was prepared by the titration method and characterized for physicochemical parameters. Pharmacodynamic evaluations (apomorphine – induced compulsive behavior and spontaneous locomotor activity) were performed using mice. All formulations were radiolabeled with 99mTc (technetium) and biodistribution of drug in the brain was studied using Swiss albino rats. Brain scintigraphy imaging in rabbits was also performed to ascertain the uptake of the drug into the brain. RME were transparent and stable with mean globule size of 15- 30 nm and zeta potential of !30mV to !40mV. In pharmacodynamic studies, significant (P < 0.05) variation in parameters estimated, were found between the treated and control groups. 99mTc-labeled risperidone solution (RS)/RME/RPD mucoadhesive microemulsion (RMME) 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 RMME compared to intravenous RME was found to be 6 to 8 fold higher indicating larger extent of distribution of the drug in brain. Drug targeting efficiency, and direct drug transport were found to be highest for RMME post-intranasal administration compared to intravenous RME. Rabbit brain scintigraphy also showed higher intranasal uptake of the drug into the brain. This investigation demonstrates a more rapid and larger extent of transport of risperidone into the brain with intranasal RMME, which may prove useful for treatment of schizophrenia.

In the present work amphotericin B (AmB) was formulated in tristearin-based solid lipid nanoparticles (SLNs) stabilized by soya phosphatidylcholine (PC), for macrophage targeting against visceral leishmaniasis (VL). SLNs were modified by coating them with macrophage-specific ligand, O-palmitoyl mannan (OPM). The surface modified and unmodified SLNs were characterised for size, shape, zeta potential and entrapment efficiency. The antileishmanial activity of free and SLNs entrapped AmB was tested in vitro in Leishmania donovani infected macrophage-amastigote system (J774A.1 cells), which showed higher efficacy of OPM grafted AmB-SLNs (SLNs-OPM) over plain AmB-SLNs and free AmB (AmB-Doc). The in-vivo organ distribution studies in albino rats confirmed that degree of accretion of SLNs entrapped AmB in macrophage rich organs, particularly liver, spleen and lungs was considerably high when compared against AmB-Doc. The rate and degree of accretion were found to increase further on ligand anchoring. The in vivo antileishmanial activity of the AmB encapsulated SLNs-OPM was found to be better as compared to SLNs and AmB-Doc against VL in L. donovani infected hamsters. The proposed formulations, SLNs and SLNs-OPM demonstrated tremendous potential for passive and active intramacrophage targeting respectively and the strategy could be a booming alternative to the presently existing drug regimens of VL and systemic fungal infections.

Preparation, Characterization and In-Vivo Evaluation of Raloxifene Hydrochloride Solid Lipid Nanoparticles by Vijaykumar Nekkanti, Vobalaboina Venkateshwarlu, Raviraj Pillai (68-77).
Raloxifene hydrochloride is a relatively new selective estrogen receptor modulator. Clinical studies revealed that absolute bioavailability of raloxifene in humans is 2%. The major problems that have limited the therapeutic efficacy of raloxifene as an oral dosage form are its low solubility in physiological pH conditions, extensive first pass metabolism, high inter-individual and intra-individual variability (30%) following oral administration. The objective of the present investigational study was to improve the bioavailability and minimize variability of raloxifene hydrochloride by preparing solid lipid nanoparticles (SLN) using Tristearin (TS), Trimyristin (TM) and Tripalmitin (TP), by hot homogenization technique. Formulation and process variables were studied and evaluated. Particle size and zeta potential were measured by photon correlation spectroscopy using Malvern Zetasizer (ZS-90). The solid state properties of the drug in SLN's were characterized by DSC and X-ray powder diffraction (XRPD) analysis. In-vitro release studies were performed in physiologically relevant media using modified franz diffusion cell. Systemic exposure of nanoparticle formulation was evaluated in male Wistar rats for increase in the rate and extent of drug absorption. The results demonstrated that stable raloxifene SLN formulations having a mean size range of 154 to 175 nm with a zeta potential range of –17 to –22 mV were developed. The entrapment efficiency of drug was more than 98% indicating homogeneous dispersion of drug in the lipid matrix. The in-vitro release studies showed that SLN (TM) formulation has significantly higher rate of drug dissolution as compared to other SLN formulations. Systemic exposure studies in rats indicated a significant increase in the rate and extent of drug absorption.

Fabrication of Silver Nanoparticles with Cotton for Antibacterial Wound Dressing by MubarakAli, D, ArunKumar, J, Rahuman Sheriff, M, Pandiaraj, SheikSyedIshack, K.A, N. Thajuddin (78-82).
Nosocomial infections are presently an important health issue which is caused by the transfer of infection by patient, fomites and surgical materials in hospital. In solving these problems, silver nanoparticles play an inevitable role in the development of antibacterial and dressings materials by nanofabrication to minimize the risk factor in nosocomial infections. Nanofabrication can be defined as a bottom-up process to fabricate the material of interest from ionic level to nanoscale level with superior property. In the present study, silver nanoparticles have been fabricated into cotton fabrics for antibacterial wound dressing materials. Fabrication of silver nanoparticles into cotton fabrics was analyzed using SEM, EDAX, FTIR and UV-Vis spectroscopy. It was found that 20-nm -sized fine particles incorporated in cotton fiber without damaging the texture of the fiber. Antibacterial property of fabrics was tested against clinically isolated pathogens, Escherichia coli and Staphyloccocus aureus by disc diffusion and colony forming count method. The results proved that antibacterial activity was found against tested pathogens. This technology may apply for the development of silvernanoparticle- incorporated surgical materials and wound dressing materials to overcome nosocomial infections, secondary infection and drug storing articles.