International Journal of Pharmaceutics (v.340, #1-2)

This paper describes the effect on Sun Protection Factor (SPF) of the combination of inorganic and organic filters in sunscreen products as determined by an in vitro method. O/W emulsions containing inorganic filters, such as titanium dioxide and zinc oxide, combined with 18 EU-authorized UV-B organic filters were tested. SPF measurements were carried out using a spectrophotometer equipped with an integrating sphere.This study observed a synergic effect when titanium dioxide was combined with either anisotriazine or octyldimethylPABA. The combination of zinc oxide with 11 UV-B organic filters also exhibited a similar synergy; however, the measured SPF was systematically lower than the protection factor achieved with titanium dioxide.
Keywords: Inorganic; Organic; Filter; Combination; Synergy; SPF in vitro;

Bioerodible devices for intermittent release of simvastatin acid by Ju Hyeong Jeon; Mark V. Thomas; David A. Puleo (6-12).
The association polymer system of cellulose acetate phthalate (CAP) and Pluronic F-127 (PF-127) was used to create intermittent release devices for mimicking the daily injection of simvastatin that has been reported to stimulate bone formation. To enhance solubility in water, prodrug simvastatin was modified by lactone ring opening, which converts the molecule to its hydroxyacid form. CAP/PF-127 microspheres incorporating simvastatin acid were prepared by a water–acetone–oil–water (W/A/O/W) triple emulsion process. Devices were then fabricated by pressure-sintering UV-treated blank and drug-loaded microspheres. Using a multilayered fabrication approach, pulsatile release profiles were obtained. Delivery was varied by changing loading, number of layers, blend ratio, and incubation conditions. To determine the cellular effects of intermittent exposure to simvastatin acid, MC3T3-E1 cells were cultured with either alternating or sustained concentrations of simvastatin acid in the medium, and DNA content, alkaline phosphatase activity, and osteocalcin secretion were measured. For all three cell responses, cultures exposed to simvastatin acid showed higher activity than did control cultures. Furthermore, cell activity was greater for cells cultured with intermittent concentrations of simvastatin acid compared to cells that were constantly treated. These results imply that devices intermittently releasing simvastatin acid warrant further study for locally promoting osteogenesis.
Keywords: Intermittent release; Controlled release; Pulsatile release; CAP/PF-127 system; Simvastatin acid;

Cationic transfersomes based topical genetic vaccine against hepatitis B by Sunil Mahor; Amit Rawat; Praveen K. Dubey; Prem N. Gupta; Kapil Khatri; Amit K. Goyal; S.P. Vyas (13-19).
DNA vaccines have been shown to elicit both cellular and humoral immune responses and to be effective in a variety of preclinical bacterial, viral, and parasitic animal models. We have recently described a needle-free method of vaccination, transcutaneous immunization, based on topical application of vaccine antigens on intact skin using a novel carrier system, namely transfersomes. In the present study, a novel modified version of transfersomes, i.e., cationic transfersomes for topical DNA vaccine delivery has been developed. Cationic transfersomes composed of cationic lipid DOTMA and sodium deoxycholate as constitutive lipids were prepared and optimized for their size, shape, zeta potentials, deformability and loading efficiency. Plasmid DNA encoding hepatitis B surface antigen (HBsAg) was loaded in the cationic transfersomes using charge neutralization method. The immune stimulating activity was studied by measuring serum anti-HBsAg titer and cytokines level (IL-2 and IFN-γ) following topical applications of plasmid DNA loaded cationic transfersomes in Balb/c mice and results were compared with naked DNA applied topically as well as naked DNA and pure recombinant HBsAg administered intramuscularly. Results revealed that DNA loaded cationic transfersomes elicited significantly (*P  < 0.05) higher anti-HBsAg antibody titer and cytokines level as compared to naked DNA. It was also observed that topical application of DNA loaded cationic transfersomes elicited a comparable serum antibody titer and endogenous cytokines levels as produced after intramuscular recombinant HBsAg administration. The study signifies the potential of cationic transfersomes as DNA vaccine carriers for effective topical immunization.
Keywords: DNA vaccine; Non-invasive immunization; Cationic transfersomes; Topical vaccine; Hepatitis B;

Avidin bioconjugate with a thermoresponsive polymer for biological and pharmaceutical applications by Stefano Salmaso; Sara Bersani; Sivanand S. Pennadam; Cameron Alexander; Paolo Caliceti (20-28).
A thermoresponsive polymer, N-isopropylacrylamide-co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 °C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein–co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85 ± 3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.
Keywords: Polymer conjugation; Thermoresponsive polymers; Avidin; Protein delivery; Poly-N-isopropylacrylamide;

Effect of injection routes on pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin in rats by Jun Chen; Qineng Ping; Jianxin Guo; Minling Liu; Baochang Cai (29-33).
Pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin (9-NC) were compared after intravenous (i.v.) and intramuscular (i.m.) injection at a dose of 1.5 mg/kg 9-NC solution. The concentrations of three different forms of 9-NC, namely lactone, carboxylate and total 9-NC, were measured by HPLC analysis. Injection routes were demonstrated to have significant effect on pharmacokinetics of 9-NC. Compared with i.v. injection route, mean residence time (MRT) of 9-NC three forms was significantly prolonged following i.m. route (p  < 0.05). The AUC0–∞ ratios of i.m. to i.v. route were calculated to be 102 ± 43%, 273 ± 221% and 150 ± 62% for lactone, carboxylate and total 9-NC, respectively. Compared with i.v. injection route, although AUC0–∞ was barely changed, MRT of lactone 9-NC was dramatically prolonged 4.5-fold after i.m. injection, which may account for the reported improved antitumor efficacy. However, the results of the present study also demonstrated that i.m. injection route increased both AUC0–∞ and MRT of carboxylate 9-NC more significantly. Since the carboxylate form of CPT analogs including 9-NC is associated with their unwanted toxicity, i.m. injection route might lead to severe toxicity compared with i.v. route. Lactone/carboxylate equilibrium was also significantly influenced by injection routes. Based on the AUC0–∞ measurements, the lactone 9-NC constituted 50 ± 8% and 32 ± 7% of circulating total 9-NC after i.v. or i.m. administration, respectively (p  < 0.01).
Keywords: 9-Nitrocamptothecin; Pharmacokinetics; Rats; Lactone; Intravenous injection; Intramuscular injection;

Detection of lot-to-lot variations in the amorphous microstructure of lyophilized protein formulations by Yutaka Hirakura; Hideto Yamaguchi; Masayasu Mizuno; Hideo Miyanishi; Satoshi Ueda; Satoshi Kitamura (34-41).
Reconstitution of lyophilized protein formulations sometimes results in a cloudy solution, depending on the compositions and manufacturing conditions, which causes quality concerns. In this study, the lyophilized protein formulation of recombinant human Interleukin-11 (rhIL-11) was investigated using different lots with varying dissolution behaviors upon reconstitution due to differing processing conditions. In an attempt to distinguish the solid structures in the different lots, relatively new techniques such as inverse gas chromatography (IGC) and thermally stimulated depolarized current (TSDC) as well as powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) were adopted for analysis. PXRD, DSC, and IGC all failed to distinguish between the solid structures, but TSDC was able to discern the differences. Interestingly, TSDC suggested that the variations in dissolution behavior were attributable to the differences in molecular mobility and the micro heterogeneity of amorphous components in the solid structures. Since even the cloudiest reconstituted solutions became transparent in several minutes, it was likely that the differences in the solid structures of the different lots of lyophilized cakes were slight. This study demonstrates the usefulness of TSDC in the analysis of lot-to-lot variations in amorphous pharmaceuticals.
Keywords: Lot-to-lot variation; TSDC; TSC; Amorphous; Heterogeneity; Lyophilized formulation;

Protease inhibition by oleic acid transfer from chronic wound dressings to albumin by J. Vincent Edwards; Phyllis Howley; Rachel Davis; Andrew Mashchak; Steven C. Goheen (42-51).
High elastase and cathepsin G activities have been observed in chronic wounds to inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid is a non-toxic elastase inhibitor. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of oleic acid by albumin under conditions mimicking chronic wounds. The mechanism of oleic acid uptake from cotton and binding by albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin–oleic acid complexes under liquid equilibrium conditions revealed fully saturated albumin–oleic acid complexes with a 1:1 weight ratio of albumin:oleic acid. Liquid–solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of oleic acid per mole albumin. Comparing oleic acid formulated wound dressings for dose dependent ability to lower elastase activity, we found cotton gauze > hydrogel > hydrocolloid. In contrast, the cationic serine protease cathepsin G was inhibited by oleic acid within a narrow range of oleic acid–cotton formulations. 2% albumin was sufficient to transfer quantities of oleic acid necessary to achieve a significant elastase-lowering effect. Oleic acid bound to cotton wound dressings may have promise in the selective lowering of cationic serine protease activity useful in topical application for chronic inflammatory pathogenesis.
Keywords: Occlusive wound dressings; Oleic acid; Cationic proteases; Cotton; Albumin; Micelles; Chronic wounds;

Liquid crystalline phases that are stable in excess water, formed using lipids such as glyceryl monooleate (GMO) and oleyl glycerate (OG), are known to provide a sustained release matrix for poorly water soluble drugs in vitro, yet there has been no report of the use of these materials to impart oral sustained release behaviour in vivo. In the first part of this study, in vitro lipolysis experiments were used to compare the digestibility of GMO with a second structurally related lipid, oleyl glycerate, which was found to be less susceptible to hydrolysis by pancreatic lipase than GMO. Subsequent oral bioavailability studies were conducted in rats, in which a model poorly water soluble drug, cinnarizine (CIN), was administered orally as an aqueous suspension, or as a solution in GMO or OG. In the first bioavailability study, plasma samples were taken over a 30 h period and CIN concentrations determined by HPLC. Plasma CIN concentrations after administration in the GMO formulation were only sustained for a few hours after administration while for the OG formulation, the plasma concentration of cinnarizine was at its highest level 30 h after dosing, and appeared to be increasing. A second study in which CIN was again administered in OG, and plasma samples taken for 120 h, revealed a T max for CIN in rats of 36 h and a relative oral bioavailability of 344% when compared to the GMO formulation (117%) and the aqueous suspension formulation (assigned a nominal bioavailability of 100%). The results indicate that lipids that form liquid crystalline structures in excess water, may have application as an oral sustained release delivery system, providing they are not digested rapidly on administration.
Keywords: Poorly water soluble drug; Lipid formulation; Liquid crystal; Cinnarizine; Bioavailability;

The objective of this study was to develop a capsular drug delivery system, which releases the drug when a sensor element is activated by gastrointestinal fluids. The sensor element consists of a microswitch and a control membrane. After disintegration or dissolution of the control membrane the gastrointestinal fluid switches on an electric circuit. Via a gas producing cell the drug reservoir of the capsule is emptied within 6 min.In vitro experiments with pH-sensitive polymethacrylic sensor membranes (Eudragit S 100) show that prednisolone dihydrogenphosphate is released within a few minutes when the capsule is transferred from gastric juice to artificial intestinal fluid of pH 6.8. Experiments with sensor elements prove that the membrane thickness influences the response time of the sensor. When 32 μm membranes are used, the electric circuit is switched on with a delay of about 70–80 min at pH 6.8. The developed systems are intended to be used as pump systems to deliver drugs into specific areas of the gastrointestinal (GI) tract.
Keywords: Sensor elements; Controlled drug release; Drug delivery systems; Capsule;

The relative importance of solubility parameters and other solvent properties on membrane diffusion processes has not been fully elucidated in the literature. Previously, we have studied the effect of different vehicles on the permeation of caffeine, benzoic acid (BA) and salicylic acid (SA) through silicone membranes. The present paper investigates diffusion of the selected permeants from different saturated solutions through human epidermis.The permeation of caffeine was strongly affected by the vehicle chosen and the maximum enhancement observed for the permeation of caffeine was 288-fold. A maximum of 12-fold enhancement in the flux was observed for the permeation of SA and a maximum of 10-fold enhancement was observed for the permeation of BA. The diffusion profiles obtained for SA in the different solvents were very similar when compared with those obtained for BA but the permeation rates were higher for BA than for SA. This similarity results from the similar chemical structure and lipophilicity.
Keywords: Solubility parameter; Skin; Vehicle; Flux;

The aim of this study was to investigate the influence of two types of chlorhexidine species, chlorhexidine base and its salts, on the physico-chemical features of liquid crystalline systems and on drug transport through lipophilic membranes.A non-ionic surfactant, Synperonic A7 (PEG7-C13-15) was selected for the preparation of the liquid crystalline systems. Mixtures of different ratios of Synperonic A7 and water were prepared. The liquid crystalline systems were characterized using polarizing microscopy and dynamic oscillatory test. Membrane transport was also examined. The addition of chlorhexidine species to the liquid crystalline system modified the structure of the liquid crystalline system. As a result of the changes of liquid crystalline structures, the drug release of various types of chlorhexidine could be also modified. The combination of the base and salt forms of the drug in one dosage form could eliminate the drug release changes from liquid crystalline systems of dynamically changeable structures.
Keywords: Liquid crystals; Lamellar and hexagonal structure; Chlorhexidine; Chlorhexidine digluconate; Drug release; Polarizing microscopy; Dynamic oscillatory test;

The aim of the present study was to experimentally examine whether poorly water-soluble drugs dispersed in a polymeric matrix exist as amorphous nanodispersions or molecularly dispersed compounds. Felodipine (Felo) dispersed in PVP matrix (solid dispersion) was used as a model drug in this study. Drug/polymer ratios have an impact on the drug average particle size, morphology and dissolution profile while solid dispersions containing up to 50 wt% Felo are completely amorphous. SEM, TEM micrographs, and micro-Raman mapping reveal that Felo is dispersed in the form of nanoparticles into the PVP matrix. Due to the high spatial resolution of TEM, it was established that these nanoparticles are not uniform particles, but rather agglomerates of individual particles with sizes smaller than 5–10 nm. Moreover, micro-Raman mapping allowed us to observe the size and spatial distribution of domains where the drug existed as molecularly or nanodispersed. Experimental evidence presented in this work contradicts the common belief that amorphous poorly water-soluble drugs exist only in the state of molecular dispersion inside a polymer matrix by showing that both types of dispersions (molecular-level and nanodispersions) can coexist.
Keywords: Poor water-soluble drugs; Solid dispersions; Amorphous nanodispersions; SEM; TEM; Raman;

Xibornol is a lipophilic drug mainly used in Italy and Spain in spray dosage forms for the local treatment of infection and inflammation of the throat. Its poor water solubility makes difficult the development of aqueous formulations of the drug, thus giving rise to a limited number of stable and pharmaceutically accepted preparations. In fact, xibornol is actually marketed only as spray aqueous suspension. The aim of this work was to evaluate the possibility of developing a stable liquid formulation of the drug intended for oral spray administration using a self-microemulsifying drug delivery system (SMEDDS). These systems are able to adequately improve the drug solubility, allowing the introduction of relatively high concentration of drugs in the form of solution. Labrafil M1944, Labrafil M2125 and Labrafac CC were screened as oil phases, Labrasol and Labrafac PG as surfactants and Transcutol as co-surfactant. Pseudo-ternary phase diagrams were constructed, by titration with the aqueous phase of different oil phases and surfactant/co-surfactant mixtures in order to identify the self-microemulsification region and the optimal micro-emulsion composition. Then, complete pharmaceutical formulations were prepared and evaluated for stability and viscosity properties. The final selected formulations, containing Labrafil M1944, Transcutol, Labrafac PG and a hydrophilic co-solvent (propylene glycol or PEG 200) allowed complete solubilization of the required xibornol concentration (3%, w/v) and showed physical good stability up to 2 months at 25 and 4 °C, suitable viscosity and organoleptic properties.
Keywords: Xibornol; Liquid spray formulation; Self-microemulsifying delivery systems; Pseudo-ternary diagrams; Viscosity;

An in vitro assessment of bioadhesive zinc/carbomer complexes for antimicrobial therapy within the oral cavity by Gemma Keegan; John Smart; Matthew Ingram; Lara Barnes; Gareth Rees; Gary Burnett (92-96).
The effectiveness of antimicrobial agents in the oral cavity is limited by their retention at the site of action. In this work the antimicrobial cation zinc was complexed with the bioadhesive agent Carbopol 971P, in order to allow an extended antimicrobial effect. Zinc ions were shown to form stable complexes with the polymer, and were not released into distilled water. However, in the presence of other cations, it was possible to displace zinc over an extended period. A low pH was seen to enhance zinc release. The complexes were found to have similar bioadhesive properties to the polymer alone when tested using a buccal cell adsorption model and texture probe analysis. It was concluded that this complex shows promise as a means of allowing the extended delivery of zinc ions locally within the oral cavity.
Keywords: Oral cavity; Antimicrobial; Zinc; Carbomer; Poly(acrylic acid); Bioadhesion;

Applications of NIR in early stage formulation development by Weiyong Li; Abraham Bashai-Woldu; John Ballard; Mark Johnson; Maria Agresta; Henrik Rasmussen; Steven Hu; John Cunningham; Denita Winstead (97-103).
This article describes a semi-quantitative reflectance near infrared (SQ-NIR) method for blend uniformity (BU) and content uniformity (CU) analyses in early stage formulation development. Applicability of the method depends upon three factors: separation of NIR signals of the active pharmaceutical ingredient (API) from placebo; strength of the signal; and a quantitative relationship between API concentration and NIR signal. Based on these three criteria, suitable NIR signals of the API, separated from those of placebo through suitable pretreatment of the spectra, can be used for BU and CU calculations without calibration models. The method was applied to an early stage formulation development project. Multiple batches of tablets were prepared and analyzed using the SQ-NIR method and a validated UV–VIS reference method. The SQ-NIR method was able to distinguish between batches that had satisfactory and unsatisfactory content uniformity and potency. In addition, effects of compression force and API particle size on the SQ-NIR results are discussed. It is proposed that the SQ-NIR method may be used as an independent test in early stage formulation development. The advantages and limitations of the method compared with traditional HPLC or UV–VIS methods are also discussed.
Keywords: Near infrared spectroscopy; Semi-quantitative; Reflectance; Blend uniformity; Content uniformity;

A ‘biorelevant’ system to investigate in vitro drug released from a naltrexone implant by Sunil S. Iyer; William H. Barr; Mario E. Dance; Peter R. Coleman; H. Thomas Karnes (104-118).
This research is based on the recognized need for an in vitro release method for drug implants that better simulate physiological conditions at the site of implantation (‘biorelevance’). In this paper, we describe the evaluation of a ‘biorelevant’ approach for in vitro drug release testing of a biodegradable implant of naltrexone in a pre-clinical stage of development.A miniature, capillary cell culture device was modified and tested as a biorelevant alternative for a standard commercially available flow-through cell. The real-time data generated through 90 days indicated a 48% lower rate of release for the capillary system. The profiles using both systems followed zero-order kinetics after an initial period of burst release. In vitro release data from the capillary device resulted in a 1-to-1 correlation with dog plasma pharmacokinetic data, and furthermore, the capillary device potentially simulated the lag-time in absorption more effectively than the flow-through cell. Scanning electron micrographs revealed that the sheath was continuous with no signs of cracks at the end of in vitro and in vivo studies. However, at the interface of the sheath and the core, intercalating, “finger-like” projections were observed consistent with penetration of the medium. No macroscopic or clinical toxicity signs were observed during the in vivo implantation study.
Keywords: Naltrexone implant; Modified Hanks’ Balanced Salts Solution; Capillary and flow-through devices; In vitro release testing;

The development of a ‘biorelevant’ approach for accelerating drug release from an implant is described. A miniature, capillary system has been shown previously to be suitable for real-time release tests for a biodegradable, naltrexone implant. Whereas the real-time study under physiological condition was essential for evaluation of the system, the accelerated (short-term) method provides for a faster assessment of in vitro drug release that would be useful in product development and quality control.Increased temperature was employed as the mechanism for accelerating drug release. Release rates were investigated and compared using modifications of two devices: the flow-through cell and the new, potentially more ‘biorelevant’ capillary device. The data generated for accelerated release using both devices through 45 days indicated approximately two-fold and four-fold increases in release rates at 45 and 55 °C, respectively, as compared to the real-time release rate. The similar activation energy values for both devices obtained from Arrhenius plots demonstrated that the release mechanism had been consistent; and that the rates of release could be used for long-term prediction. The rate of release reverted to that observed in real-time data, however, upon a reduction of temperature to 38 °C. The results demonstrated that temperature was the sole factor involved in modification of the release rate in vitro. The profiles using both systems followed zero-order kinetics after an initial period of burst release.
Keywords: Naltrexone implant; Modified Hank's balanced salts solution; Capillary and flow-through devices; Accelerated in vitro release testing;

Nanosuspension as an ophthalmic delivery system for certain glucocorticoid drugs by M.A. Kassem; A.A. Abdel Rahman; M.M. Ghorab; M.B. Ahmed; R.M. Khalil (126-133).
Poorly-water-soluble compounds are difficult to develop as drug products using conventional formulation techniques. The use of nanotechnology to formulate poorly-water-soluble drugs as nanosuspensions offers the opportunity to address many of the deficiencies associated with this class of molecules. In the present study, the high pressure homogenization method used to prepare nanosuspensions of three practically insoluble glucocorticoid drugs; hydrocortisone, prednisolone and dexamethasone. The effect of particle size in the micron and nano-size ranges as well as the effect of viscosity of the nanosuspension on the ocular bioavailability was studied by measuring the intraocular pressure of normotensive Albino rabbits using shiØetz tonometer. The results show that compared to solution and micro-crystalline suspensions it is a common feature of the three drugs that the nanosuspensions always enhance the rate and extent of ophthalmic drug absorption as well as the intensity of drug action. In the majority of cases nanosuspensions extend the duration of drug effect to a significant extent. The data presented confirms that nanosuspensions differ from micro-crystalline suspensions and solution as ophthalmic drug delivery systems and that the differences are statistically, highly to very highly significant. The results confirm also the importance of viscosity of nanosuspension especially in increasing the duration of drug action.
Keywords: Nanosuspension; Particle size; Ophthalmology; Glucocorticoids;

Chitosan/cyclodextrin nanoparticles as macromolecular drug delivery system by Alexander H. Krauland; María José Alonso (134-142).
The aim of this study was to generate a new type of nanoparticles made of chitosan (CS) and carboxymethyl-β-cyclodextrin (CM-β-CD) and to evaluate their potential for the association and delivery of macromolecular drugs. CS and CM-β-CD or mixtures of CM-β-CD/tripolyphosphate (TPP) were processed to nanoparticles via the ionotropic gelation technique. The resulting nanoparticles were in the size range of 231–383 nm and showed a positive zeta potential ranging from +20.6 to +39.7 mV. These nanoparticles were stable in simulated intestinal fluid pH 6.8 at 37 °C for at least 4 h. Elemental analysis studies revealed the actual integration of CM-β-CD to CS nanoparticles. Insulin and heparin used as macromolecular model drugs, could be incorporated into the different nanocarriers with association efficiencies of 85.5–93.3 and 69.3–70.6%, respectively. The association of these compounds led to an increase of the size of the nanoparticles (366–613 nm), with no significant modification of their zeta potentials (+23.3 to +37.1 mV). The release profiles of the associated macromolecules were highly dependent on the type of molecule and its interaction with the nanomatrix: insulin was very fast released (84–97% insulin within 15 min) whereas heparin remained highly associated to the nanoparticles for several hours (8.3–9.1% heparin within 8 h). In summary, CS-CD (cyclodextrin) nanoparticles may be considered as nanocarriers for the fast or slow delivery of macromolecules.
Keywords: Nanoparticles; Chitosan; Cyclodextrins; Insulin; Heparin;

Permeability of the anti-human immunodeficiency virus (HIV) agents, including stavudine (D4T), delavirdine (DLV), and saquinavir (SQV), across the in vitro blood–brain barrier (BBB) was studied. Here, the anti-HIV agents were incorporated with polybutylcyanoacrylate (PBCA) nanoparticles (NPs), methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) NPs, and solid lipid nanoparticles (SLNs). Transport of the anti-HIV agents across BBB is a key factor in their applications to the therapy of the acquired immunodeficiency syndrome (AIDS). Experimental results revealed that the drug order of the loading efficiency (LE) on PBCA and MMA-SPM was D4T > DLV > SQV. For the entrapment efficiency (EE) in SLNs, this order was reversed. Also, LE of D4T on MMA-SPM was larger than that on PBCA; however, the reverse was true for DLV and SQV. As the particle size increased, LE decreased and EE increased. For a fixed drug carrier, an increase in the particle size yielded a decrease in the BBB permeability coefficient of the anti-HIV agents. Moreover, enhancement in the BBB permeability was on the carrier order of PBCA > MMA-SPM > SLNs for D4T, and for DLV and SQV, the order became PBCA > SLNs > MMA-SPM. PBCA, MMA-SPM, and SLNs were efficacious carriers of D4T, DLV, and SQV to meliorate BBB permeability by 3–16 folds, indicating the clinical potential of the present NP formulations for the AIDS treatment.
Keywords: Polybutylcyanoacrylate; Methylmethacrylate-sulfopropylmethacrylate; Solid lipid nanoparticle; Blood–brain barrier; Anti-HIV agent; AIDS;

Solid lipid nanoparticles (SLNs) loaded with insulin-mixed micelles (Ins-MMs) were prepared by a novel reverse micelle-double emulsion method, in which sodium cholate (SC) and soybean phosphatidylcholine (SPC) were employed to improve the liposolubility of insulin, and the mixture of stearic acid and palmitic acid were employed to prepare insulin loaded solid lipid nanoparticles (Ins-MM-SLNs). Some of the formulation parameters were optimized to obtain high quality nanoparticles. The particle size and zeta potential measured by photon correlation spectroscopy (PCS) were 114.7 ± 4.68 nm and −51.36 ± 2.04 mV, respectively. Nanospheres observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed extremely spherical shape. The entrapment efficiency (EE%) and drug loading capacity (DL%) determined with high performance liquid chromatogram (HPLC) by modified ultracentrifuge method were 97.78 ± 0.37% and 18.92 ± 0.07%, respectively. Differential scanning calorimetry (DSC) of Ins-MM-SLNs indicated no tendency of recrystallisation. The core-shell drug loading pattern of the SLNs was confirmed by fluorescence spectra and polyacrylamide gel electrophoresis (PAGE) which also proved the integrity of insulin after being incorporated into lipid carrier. The drug release behavior was studied by in situ and externally sink method and the release pattern of drug was found to follow Weibull and Higuchi equations. Results of stability evaluation showed a relatively long-term stability after storage at 4 °C for 6 months. In conclusion, SLNs with small particle size, excellent physical stability, high entrapment efficiency, good loading capacity for protein drug can be produced by this novel reverse micelle-double emulsion method in present study.
Keywords: Insulin; Solid lipid nanoparticle; Mixed micelle; Double emulsion; Recrystallisation; Drug loading pattern;

10-Hydroxycamptothecin (HCPT) is insoluble in both water and physiological acceptable organic solvents and tends to change into its carboxylate form, which shows minimal anticancer activity and several unpredictable side effects. The goal of this study is to exploit an appropriate delivery system for HCPT to improve the stability of its lactone form. Bovine serum albumin (BSA) nanoparticles entrapping HCPT were prepared by reformative emulsion-heat stabilization technique. During this process, HCPT transformed from lactone to carboxylate and finally back to lactone form successfully. A simple reversed-phased HPLC method was developed to analyze both lactone and carboxylate forms of HCPT synchronously. Mean particle size and the ratio of lactone and carboxylate forms of HCPT were evaluated to investigate the effects of the formulations and preparation conditions. It was indicated the percentage of lactone form of HCPT in resultant BSA nanoparticles could be improved over 95% through adjusting the concentration of NaOH solution and the stirring time after high-speed emulsification. This drug delivery system was also characterized by dynamic light scattering (DLS) and light microscopy. The investigations on drug loading, in vitro release and body distribution in rats after intravenous (i.v.) administration were also carried out. It was found that the obtained nanoparticles showed spherical shape with the mean particle size of around 600 nm, and drug loading content, encapsulation efficiency and yield achieved 2.21%, 57.5% and 90.5% with the optimal preparation conditions, respectively. The in vitro release behavior exhibited a sustaining release manner and was affected by the trypsin in medium. HCPT could release more than 90% within 20 h in the medium of pH 7.4 PBS containing 750 U/ml trypsin, but only 25% within 40 h in the pure pH 7.4 PBS. The results of body distribution study in rats showed the liver targeting potential of HCPT–BSA nanoparticles that 59.6%, 52.9% and 55.3% of the examined amount of lactone HCPT accumulated in livers at 1, 4 and 24 h after injection, respectively. These results suggest that the HCPT–BSA nanoparticles seem to be a stable delivery system for poorly soluble HCPT or its derivatives.
Keywords: 10-Hydroxycamptothecin (HCPT); Bovine serum albumin (BSA); Nanoparticles; Lactone form; Drug delivery system;

Characterization of chitosan thiolation and application to thiol quantification onto nanoparticle surface by I. Bravo-Osuna; D. Teutonico; S. Arpicco; C. Vauthier; G. Ponchel (173-181).
The objective of the present work was to establish a simple and appropriated method for the quantification of thiol groups standing on the surface of core–shell nanoparticles elaborated with poly(isobutyl cyanoacrylates) and thiolated chitosan.A critical analysis of the widely used Ellman's method for the determination of thiol groups in various compounds was made. The reduced solubility of the thiolated polymer at the optimal pH of the Ellman's assay (pH 8–8.5) made difficult the accessibility of the Ellman's reagent to thiol groups in the cross-linked polymer. Furthermore, the lack of stability of the Ellman's reaction with time lead to the conclusion that the Ellman's method was of limited value to evaluate thiol groups in thiolated polymers like thiolated chitosan.An alternative and very simple thiol quantification method was developed on the bases of the classical iodine titration. The new method allowed the determination of thiol groups in small amount of samples at acidic pH, and the monitoring of the thiol determination kinetic with time. It was successfully applied to the quantification of active thiol groups on the surface of poly(isobutyl cyanoacrylates) nanoparticles coated with thiol chitosan.
Keywords: Surface modified nanoparticles; Thiolated chitosan; Ellman's reagent; DNTB; Iodine titration;

Anti-adherent and antifungal activities of surfactant-coated poly(ethylcyanoacrylate) nanoparticles by Paul A. McCarron; Ryan F. Donnelly; Waleed Marouf; Deborah E. Calvert (182-190).
Application of non-drug-loaded poly(ethylcyanoacrylate) nanoparticles (NP) to buccal epithelial cells (BEC) imparted both anti-adherent and antifungal effects. NP prepared using emulsion polymerisation and stabilised using cationic, anionic and non-ionic surfactants decreased Candida albicans blastospore adhesion, an effect attributable to the peripheral coating of surfactant. Cetrimide and Pluronic® P123 were shown to be most effective, producing mean percentage reductions in blastospore adherence of 52.7 and 37.0, respectively. Resultant zeta potential matched the polarity of the surfactant, with those stabilised using cetrimide being especially positive (+31.3 mV). Preparation using anionic surfactants was shown to be problematic, with low yield and wide particle size distribution. Evaluation of the antifungal effect of the peripheral coat was evaluated using zones of inhibition and viable counts assays. The former test revealed poor surfactant diffusion through agar, but did show evidence of limited kill. However, the latter method showed that cationic surfactants associated with NP produced high levels of kill, in contrast to those coated with anionic surfactants, where kill was not evident. Non-ionic surfactant-coated NP produced intermediate kill rates. Results demonstrate that surfactant-coated NP, particularly the cationic types, form the possible basis of a prophylactic formulation that primes the candidal target (BEC) against fungal adhesion and infection.
Keywords: Candida albicans; Blastospores; Poly(ethylcyanoacrylate) nanoparticles; Adherence; Antifungal;

The characteristics of various pharmaceutical dosage forms are influenced by surface properties such as the friction behavior. For example, die wall friction is a key issue in developing a solid dosage form. However, the friction properties are not completely understood mainly because of the lack of fundamental measurements. Herein, the friction behavior of pharmaceutical materials was investigated and compared with their adhesion behavior using atomic force microscopy. The sliding speed causes significant variations in the frictional force. Compared with other materials, lubricant materials showed less distinct differences in friction tests than in adhesion tests, indicating the dependence of the lubricant efficiency on the stress state. The three parameters obtained from the modified Amonton's law, i.e., absolute frictional force, friction coefficient and residual force, showed consistent trends. Overall, the friction behavior was not a direct reflection of the adhesion forces. The intrinsic friction behavior of a single pharmaceutical particle can be quantified using atomic force microscopy.
Keywords: Atomic force microscopy (AFM); Adhesion; Flowability; Acetaminophen;

Encapsulation of ascorbyl palmitate in nanostructured lipid carriers (NLC)—Effects of formulation parameters on physicochemical stability by Veerawat Teeranachaideekul; Rainer Helmut Müller; Varaporn Buraphacheep Junyaprasert (198-206).
Enhancement of the chemical stability of ascorbyl palmitate (AP) after incorporation into nanostructured lipid carriers (NLC) has been reported. However, the formulation parameters of AP-loaded NLC have not been completely investigated. Moreover, the long-term chemical stability of AP in any colloidal systems has not been yet achieved. Therefore, in this study the formulation parameters affecting the stability of AP after incorporation into NLC were evaluated including types of lipids, types of surfactants, storage conditions, i.e. temperature and nitrogen gas flushing, the effects of drug loading as well as types of antioxidants. After storage for 90 days, the mean particle size analyzed by photon correlation spectroscopy (PCS) was lower than 350 nm. The zeta potential measured by the Zetasizer IV was higher than −30 mV in all developed AP-loaded NLC formulations which varied according to the types of lipid and surfactant. Concerning the chemical stability of AP, it was found that AP-loaded NLC prepared and stored in non-degassing conditions, a higher percentage of AP loading in NLC, lower storage temperature (4 °C), addition of antioxidants as well as selection of suitable surfactants and solid lipids improved the chemical stability of AP. Moreover, an improvement of long-term chemical stability of AP was achieved by addition of antioxidants with nitrogen gas flushing as compared to those without antioxidant. The percentage of drug remaining at both 4 °C and room temperature (25 °C) was higher than 85% during 90 days of storage.
Keywords: Nanostructured lipid carriers; NLC; Ascorbyl palmitate; Chemical stability; Antioxidant;

UEA I-bearing nanoparticles for brain delivery following intranasal administration by Xiaoling Gao; Jun Chen; Weixing Tao; Jianhua Zhu; Qizhi Zhang; Hongzhuan Chen; Xinguo Jiang (207-215).
Surface engineering of nanoparticles with lectins opened a novel pathway to improve the brain uptake of agents loaded by biodegradable PEG-PLA nanoparticles following intranasal administration. Ulex europeus agglutinin I (UEA I), specifically binding to l-fucose, which is largely located in the olfactory epithelium, was selected as a promising targeting ligand and conjugated onto the PEG-PLA nanoparticles surface with an optimized protocol relying on maleimide-mediated covalent binding technique. The in vivo results in rats suggested that UEA I modification at the nanoparticles surface facilitated the absorption of a fluorescent marker—6-coumarin associated with the nanoparticles into the brain following intranasal administration with significant increase in the area under the concentration–time curve (about 1.7 times) in different brain tissues compared with that of coumarin incorporated in the unmodified ones. UEA I-conjugation also elevated the brain-targeting efficiency of nanoparticles. Inhibition experiment of specific sugar suggested that the interactions between the nasal mucosa and the lectinised nanoparticles were due to the immobilization of carbohydrate-binding pockets on the surface of the nanoparticles. Distribution profiles of UEA I-modified nanoparticles indicated their higher affinity to the olfactory mucosa than to the respiratory one. Therefore, the UEA I-modified nanoparticles might serve as potential carriers for brain drug delivery, especially for mental therapeutics with multiple biological effects.
Keywords: Brain targeting; Nanoparticles; Ulex europeus agglutinin I; Intranasal administration;

Cationic supported lipid bilayers for antigen presentation by N. Lincopan; N.M. Espíndola; A.J. Vaz; A.M. Carmona-Ribeiro (216-222).
Polystyrene sulfate (PSS) particles (301 nm mean diameter) were covered with single cationic dioctadecyldimethylammonium bromide (DDA) bilayers and used for antigen adsorption and presentation. The antigen was a mixture of purified 18/14 Taenia crassiceps proteins (18/14-Tcra). Firstly, the DDA/PSS assembly was characterized at 1 mM NaCl and 5 × 109 PSS particles/mL over a range of DDA concentrations (0.001–1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.01 mM DDA is enough to produce homodisperse and cationic bilayer-covered particles. Secondly, under these experimental conditions, 18/14-Tcra adsorption isotherms onto biomimetic particles or aluminium hydroxide (Al(OH)3) yield limiting adsorption of 0.36 and 1.32 mg protein/mg biomimetic particles or Al(OH)3, respectively. Finally, in mice, superior humoral and cellular immunoresponse from serum IgG and footpad swelling was obtained for antigen/biomimetic particles in comparison to conventional Al(OH)3. Cationic bilayer-covered particles are a novel, highly organized and, possibly, general immunoadjuvant for antigen presentation and subunit vaccine design.
Keywords: Polymeric particles; Cationic lipid; Dioctadecyldimethylammonium bromide; Bilayer and protein adsorption; Antigen presentation; Taenia crassiceps antigens;

New analytical techniques to facilitate preformulation screening in propellant systems by Erik Mogalian; Philip J. Kuehl; Paul B. Myrdal (223-229).
The objective of these studies was to investigate the applicability of an online direct inject HPLC method for the preformulation screening of pharmaceutical agents in pressurized metered dose inhalers (MDIs). The technique was initially utilized for the solubility determination of solid solutes. This study explores the extension of the online direct inject method for the evaluation of drug stability in propellant systems as well as for the analysis of MDI vials crimped with metered valves. Through-life content analysis confirmed that a single vial may be repeatedly sampled, thus facilitating the stability evaluation of a single unit over time. The method was successfully used for evaluating the stability of a model drug, as a function of several different formulation configurations, with minimal sample numbers. Additionally, studies determined that after modifications were made to the injection coupler, the technique was also feasible for use with 50 and 100 μL metered valves, however further modifications are necessary for 25 μL valves.
Keywords: MDI; Preformulation; MDI stability; Metered valves; Direct inject; HFA;

Noticeboard (230).