International Journal of Pharmaceutics (v.369, #1-2)

Our new USA editor: Prof. Diane Burgess by Alexander T. Florence (1).

Use of a static eliminator to improve powder flow by Kalyana C. Pingali; Stephen V. Hammond; Fernando J. Muzzio; Troy Shinbrot (2-4).
Glidants and lubricants have long been used to improve the flow and processing of pharmaceutical and other powder blends. In this letter, we find that similar improvements can be attained, without additives, by using a simple static eliminator. These results indicate, first, that electrostatic effects on powder blends may be a significant cause of powder aggregation and flow instabilities, and second, that common additives such as magnesium stearate, colloidal silica, and talc may have as their chief effect the reduction of static. This suggests both that intelligent placement of static eliminators can eliminate the need for some of these additives and that judicious engineering of ionic and cationic additives may be effective in improving flow of “clingy” materials.
Keywords: Static; Flow; Eliminator; Additives; Electrostatics;

Studies on coumestrol/β-cyclodextrin association: Inclusion complex characterization by Camila Franco; Liege Schwingel; Ivana Lula; Rubén D. Sinisterra; Letícia Scherer Koester; Valquiria Linck Bassani (5-11).
Coumestrol is an estrogenic and antioxidant agent, characterized by its low solubility in aqueous and lipophilic media, once in the aglicone form. In order to improve its solubility in water, coumestrol was associated with β-cyclodextrin in aqueous media followed by freeze-drying and characterized by SEM, 1H NMR and molecular modeling. The analysis proved the existence of an inclusion complex, with higher probability of inclusion of the coumestrol B-ring into the wider rim of the β-cyclodextrin molecule.
Keywords: Coumestrol; Cyclodextrin; Complexation; Modeling;

Reevaluation of solubility of tolbutamide and polymorphic transformation from Form I to unknown crystal form by Gen Hasegawa; Takao Komasaka; Rui Bando; Yasuo Yoshihashi; Etsuo Yonemochi; Kotaro Fujii; Hidehiro Uekusa; Katsuhide Terada (12-18).
Thermodynamic stability order of tolbutamide between polymorphs was evaluated using calorimetry and spectroscopic analysis. The heat of solution (ΔH) of Forms I–III measurements were carried out in dimethylsulfoxide between 298.2 K and 319.2 K. It was found that the ΔH of Forms II and III was increased nearly parallel with a temperature rise. However, change of the ΔH of Form I with a temperature rise was not in correspondence with that of other forms. Solubility data confirmed the change in ΔH of Form I around 308.2 K. XRD–DSC measurement of Form I detected a polymorphic transformation (Form IL  → unknown form) at 311 K. Obtained data suggested that the new crystal form (Form IH) would exist above 311 K, and the order of thermodynamic stability was “Form III < Form IL  < Form II” below the transition temperature, on the other hand, the order was changed in “Form IL and Form IH  < Form III < Form II” above the transition temperature. The crystal structure of Form IH was determined by measurement of PXRD pattern on BL19B2 at SPring-8, and the change in the solid-state NMR spectrum confirmed the transformation of the crystal structure of Form IL.
Keywords: Tolbutamide; Polymorphic transformation; Structure determination from PXRD data; Solid-state NMR;

A new tyrosine kinase inhibitor (denoted as CH331) and its poly(ɛ-caprolactone) (PCL) microspheres were studied. The CH331 particles were pretreated with ethanol and then used to prepare CH331 loaded PCL microspheres by S/O/W solvent evaporation technique. Solubility values of CH331 in several organic and aqueous media were measured. The amount of ethanol and CH331 solubility play a significant role in drug loading, encapsulation efficiency, mean diameter and morphology of the microspheres, crystallinity and in vitro drug release. The treatment with a suitable amount of ethanol leads to more uniform sizes, better appearance and higher encapsulation efficiency for the microspheres. Compared with 0.5% PVA phosphate buffer solution (pH 7.4), 0.5% PVA aqueous solution as outer aqueous phase lowers encapsulation efficiency of microspheres, however, improves the drug release behavior.
Keywords: Tyrosine kinase inhibitor; Poly(ɛ-caprolactone); Microspheres; S/O/W;

Transcutaneous sampling of ciprofloxacin and 8-methoxypsoralen by electroporation (ETS technique) by Srinivasa M. Sammeta; Siva Ram K. Vaka; S. Narasimha Murthy (24-29).
The novel technique of transcutaneous sampling of drugs by electroporation was developed to study the dermatokinetics of ciprofloxacin and 8-methoxypsoralen. The selected drugs differ in their aqueous solubility and also with respect to the extent of protein binding. Ciprofloxacin (15 mg/kg) was administered i.v. through tail vein, whereas 8-methoxypsoralen (5 mg/kg) was given by oral administration, in hairless rats and the time course of drug concentration in the plasma was determined. Drug concentration in the dermal extracellular fluid (ECF) was determined by ETS and microdialysis sampling techniques. The extent of penetration into dermal ECF for ciprofloxacin and 8-methoxypsoralen was found to be ∼19–32% and ∼13–23%, respectively. The drug concentration in the dermal ECF determined by ETS and microdialysis did not differ significantly from each other and so as were the pharmacokinetic parameters. The results show that ETS can be utilized as a potential technique for sampling of drugs from the dermal ECF.
Keywords: Electroporation; Transcutaneous sampling; Hairless rats; Microdialysis; Pharmacokinetics; Extracellular fluid;

Plastic bags and tubes are increasingly used for the storage and application of pharmaceutical formulations. The most common polymer material for drug application sets is plasticized poly(vinylchloride) (PVC). During application of pharmaceutical drug solution through PVC tubes, the polymer and the contact media interact which leads to leaching out of polymer additives or sorption of ingredients of the drug solution. Whereas the discussion of leaching of plasticizers is focussed on the toxicological properties of a drug packaging system, the sorption of drug formulation compounds has an influence on the dosage of the active pharmaceutical ingredient resulting in a reduced drug delivery to the patient. Therefore sorption has an influence on the effectiveness and success of the therapy. Within the study, the concentration profiles of nitroglycerin and diazepam solutions were determined after pumping the solutions through infusion administration sets. The study includes plasticized PVC tubes with different plasticizers (DEHP, DEHA, DEHT, TEHTM, DINCH, poly adipate), PVC (DEHP) tubes with different shore hardness as well as alternative polymer materials like EVA, TPE, PUR, silicone, LDPE and PP. From the experimental concentration curves it could be shown, that in the first minutes of the application of the drug solution the sorption of the active compound is at its maximum, resulting in the lowest concentration in the applied pharmaceutical solution. For a PVC tube with DEHP as plasticizer and a shore hardness of 80 only about 57% of the initial nitroglycerin concentration in the solution is applied to the patient in the first minutes of the application. For PVC tube (DEHP, shore 80) the experimental data were simulated using mathematical diffusion models. The concentration profiles during application could be well simulated using the partition coefficient K  = 50 (nitroglycerin) and K  = 300 (diazepam), respectively. However, the experimental results indicate, that the sorption of nitroglycerin into the PVC tube alters the diffusion behaviour of the polymer over flow time, which results in an increase of the diffusion coefficient during application. On the other hand, the other investigated alternative tube materials like PE or PP show a significantly lower sorption compared to PVC plasticizer systems. Due to the fact that the amount of sorption is varying over time, the concentration of the active pharmaceutical compound in the solution after passing the infusion administration set is not constant which makes the application of a constant concentration of a certain active ingredient to the patient very difficult. The simulated partition and diffusion coefficients for given PVC(DEHP) tubes were therefore used to simulate the initial concentrations profile of the feeding drug solution to assure a constant concentration flow profile after passing the administration set. The proposed methodology of this study represents a straight forward approach for the assessment of the drug sorption in dynamic flow systems based on experimental data as well as mathematical diffusion modelling. From the results a non-constant initial concentration profile for the active ingredient in a pharmaceutical drug solution can be established in order to compensate the loss of the pharmaceutical compound by sorption during infusion.
Keywords: Sorption; Diffusion; Nitroglycerin; Diazepam; Infusion administration set; PVC tubing; Mathematical modelling;

Monte Carlo simulations for the study of drug release from cylindrical matrix systems with an inert nucleus by Lizbeth Martínez; Rafael Villalobos; Marisol Sánchez; Jeny Cruz; Adriana Ganem; Luz María Melgoza (38-46).
In this work, drug release from matrices with an inert nucleus using Monte Carlo simulation was studied. Drug-excipient systems were simulated, where the drug is a soluble material while the excipient is a non-soluble material. In the center of these devices, an inert nucleus was placed. The release of the drug was unidirectional and the results were fitted to the square root of time law (Higuchi law), the power law and the Weibull equation. The percolation threshold of the drug was found to be near 0.35 close to the expected value for the cubic lattice, the difference is due to the finite and rather small size of the systems in study as well as to the fact that the lattice in use is not exactly cubic. Near the percolation threshold, the parameters of the different release models presented a drastic change; this was due to a phase transition of the system. On the other hand, it was found that the size of the matrix system modifies the transport properties of the release platform. In general, the release kinetics was adequately described by the Weibull equation.
Keywords: Percolation theory; Drug release; Matrix systems; Monte Carlo simulation; Anomalous diffusion;

Use of 1H NMR to facilitate solubility measurement for drug discovery compounds by Melissa Lin; Marc Tesconi; Mark Tischler (47-52).
The use of 1H NMR experiments to determine the solubility of potential drug candidates using a panel of solubilizing agents is proposed as an alternative to an HPLC-UV method. The advantages of using this approach will be discussed and results comparing the two methodologies will be presented. This effort highlights the importance of a simple method for determining a suitable formulation for discovery compounds for studies using a minimal amount of material in support of early in vivo studies.
Keywords: 1H NMR; Solubility studies; HPLC-UV; In vivo studies; Formulation;

Effects of process parameters on the properties of biocompatible Ibuprofen-loaded microcapsules by P. Valot; M. Baba; J.-M. Nedelec; N. Sintes-Zydowicz (53-63).
The objective of this study was to obtain an optimum formulation for microencapsulating Ibuprofen. This was achieved by investigating various factors which influenced the microcapsule size. Considering Ibuprofen as a lipophilic model drug, biocompatible Ibuprofen-loaded microcapsules in the size range of 20–60 μm were prepared by the water in oil emulsion-solvent evaporation method. An aqueous surfactant phase was used as the continuous external phase (W), a biocompatible organic solvent dissolving Ibuprofen was used as oil phase (O), in addition with a low boiling solvent. The biocompatible polymeric microcapsule membrane was composed of Eudragit RSPO or Ethylcellulose. The influence of various process parameters, such as the volatile organic solvent, the oily core, the stirring rate, on the characteristics of microcapsules was investigated. The encapsulation yield of Ibuprofen close to 100%, whatever the polymer type, was determined by UV–vis experiments, in accordance with the results obtained by 13C NMR spectroscopy. An innovative technique, DSC-based thermoporosimetry, was used for the estimation of the loading rate of Ibuprofen. The results indicated that this developed analytical method had to be improved since DSC-transitions accounted to free and enclosed Ibuprofen were observed and altered the accuracy of the results.
Keywords: Ibuprofen; Biocompatible microcapsules; Emulsion-solvent evaporation method; Process parameters; Encapsulation efficiency;

Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine by Leonardo Sáenz; Andrónico Neira-Carrillo; Rodolfo Paredes; Marlies Cortés; Sergio Bucarey; José L. Arias (64-71).
Peptide vaccines using specific antigens with poor immunogenicity like GnRH-I are unable to develop an effective adaptive immune response and require the presence of adjuvants, essential to lymphocytic activation.Three chitosan formulations were evaluated for their ability as adjuvant of a poor immunogenic peptide vaccine against GnRH-I. Male Sprague–Dawley rats were immunized subcutaneously with recombinant His-GnRH-tandem-repeat peptide in high, low and phosphorylated high molecular weight chitosan solution at 0.5% (w/v). Freund's complete adjuvant was used as a positive control of immune response. Our results suggest that different chitosan formulations as adjuvant, with high or low viscosity degree allow inducing a high and persistent immune response against a poor immunogenic recombinant peptide. We found that the immune response was mediated by a increasing of IgG isotype 1, which were significantly greater than levels presented by the animals immunized with Freund's complete adjuvant. Nevertheless, chitosan with low molecular weight and highest acetylation degree was able to induce an immune response mediated by IgG isotype 2a. Additionally, high molecular weight phosphorylated chitosan, in which the phosphate groups were linked to N-acetyl-d-glucosamine unit, the immune response was reduced. All the immune responses obtained with chitosan as adjuvant were able to neutralize effectively the GnRH hormone proves by reducing of animal steroidogenesis and spermatogenesis demonstrating its capacity to improve immunogenicity in peptide vaccine.
Keywords: Chitosan; Adjuvant; GnRH-I vaccine; Peptide vaccine;

SEM/EDX and confocal microscopy analysis of novel and conventional enteric-coated systems by Fang Liu; Rosario Lizio; Uwe J. Schneider; Hans-Ulrich Petereit; Peter Blakey; Abdul W. Basit (72-78).
A novel double coating enteric system (comprising an inner layer of neutralised EUDRAGIT® L 30 D-55 and organic acid, and an outer layer of standard EUDRAGIT® L 30 D-55) was developed to provide fast dissolution in proximal small intestinal conditions. The mechanisms involved in the dissolution of the double coating were investigated and compared with a conventional single layer enteric coating and an hypromellose (HPMC) sub-coated enteric system. Rates of drug release from coated prednisolone pellets were established using USP II dissolution methods (0.1 M HCl for 2 h and subsequently pH 5.5 phosphate buffer) and the coating dissolution process was illustrated using confocal laser scanning microscopy (CLSM). The distribution of sodium, as a representative ion, in the double-coating system during dissolution was determined using scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX). The double-coating system showed faster dissolution compared to the single coating and the HPMC sub-coated system in pH 5.5 buffer. The dissolution process of the double-coating was unusual; the inner coat dissolved before the outer coat and this accelerated the dissolution of the outer coat. During dissolution, sodium ions diffused from the inner coat to the outer coat. This migration of ions and the increased ionic strength and buffer capacity of the inner coat contribute to the rapid dissolution of the double-coating system.
Keywords: Modified release; Double coating; Enteric coatings; Enteric polymers; pH-sensitive polymers; Methacrylic acid-ethyl acrylate copolymer; Adipic acid; Sub-coating; Polymer dissolution;

The intracellular processing and the apoptotic activity of conjugates of oligoarginine and cytochrome c (Cyt c) were studied. Disulfide and thioether linked conjugates were prepared by coupling Cyt c to cysteinyl-nonaarginine, C(R)9, through SPDP and SMPB cross-linkers, respectively. Internalization of the radiolabeled conjugates was measured, and biological activity via induction of apoptosis was determined using the annexin V and the acridine orange assays in HeLa cells. The internalization of both conjugates is increased when compared to that of Cyt c alone. However, the biological activity of the internalized Cyt c, indicated by apoptosis in HeLa cells, was expressed only in the thioether (SMPB) conjugate, but not the disulfide (SPDP) conjugate or free Cyt c. The addition of the proteasomal inhibitor MG132 increased the apoptotic activity of both the disulfide conjugate and free Cyt c, but not the thioether conjugate. Our results suggest that the intracellular cleavage of the linker in cell penetrating peptide conjugates is critical in determining the fate and activity of biologically degradable cargo molecules.
Keywords: Cytochrome c; Cell penetrating peptides; Oligoarginine; Apoptosis; Intracellular degradation;

Preparation of cefpodoxime proxetil fine particles using supercritical fluids by Junho Chu; Guanghua Li; Kyung Ho Row; Hwayong Kim; Youn-Woo Lee (85-91).
Fine particles of cefpodoxime proxetil (CPD) were prepared using an Aerosol Solvent Extraction System (ASES) with supercritical CO2. The resulting primary particles were approximately 0.1–0.2 μm in size and were almost spherical in shape. The secondary particles were approximately 0.2–0.6 μm in size and had irregular shapes. The larger particle size and irregular shapes were due to the agglomeration of the primary particles. The effects of solvent type, CO2-to-CPD solution weight ratio, and CPD solution concentration on the extent of agglomeration were investigated. As a result, the use of ethyl acetate and acetone as solvents also reduced the degree of agglomeration. The degree of agglomeration was reduced with the use of a high CO2-to-solution weight ratio, and a low solution concentration. In particular, spherical particles, approximately 0.1–0.4 μm in size, were obtained when a 10.0 wt% CPD solution was used. As a result of dissolution study, almost 90% of the processed CPD had dissolved within 10 min. The recovery yield of the CPD powder reached approximately 80% using a membrane filter.
Keywords: Cefpodoxime proxetil; Aerosol Solvent Extraction System; Supercritical carbon dioxide; Particle formation;

OcuDrain-E™—A noninvasive technique for reduction of intraocular pressure by S. Narasimha Murthy; Srinivasa M. Sammeta; Siva Ram K. Vaka (92-95).
OcuDrain-E™ is a noninvasive technique in which electrical pulses are applied across the cornea to enhance the rate of transcorneal water evaporation (TCWE). In vitro studies were carried out with rabbit cornea mounted on a Franz diffusion cell. Application of 30 pulses each of 1 millisecond (ms) duration at ≥40 V/cm2 decreased the corneal resistivity ∼80% indicating permeabilization of the cornea. The corneal resistivity was almost completely recovered within 6 h when the pulse voltage was <40 V/cm2. The average TCWE at 40 V/cm2 was significantly (∼39-fold) higher than the control (t-test, p  < 0.0001). Application of electrical pulses (40 V–30 pulses–1 ms–1 Hz) across the cornea resulted in significant decrease in the intraocular pressure (IOP) in rabbits. The electrical protocol was well tolerated by the rabbits. Microscopic studies revealed that the applied electrical protocol did not cause any edema or detachment of the epidermal layers. The results of current investigation suggest that OcuDrain-E™ could be developed as a potential technique for the treatment of glaucoma in patients who respond poorly to drugs.
Keywords: Glaucoma; Intraocular pressure; Transcorneal water evaporation rate; Noninvasive;

Characteristics of Carbopol-containing pellets have been shown to be dependent on the form of the weakly basic drug, propranolol, when the drug forms are fixed as masses in the formulations. To further investigate the effect of the drug forms on pellet and drug release characteristics, the drug forms were incorporated as a fixed number of moles in the formulation. Forms of propranolol, viz. the free base and the hydrochloride and maleate salt forms, resulted in different yield, roundness, smoothness, and friability, but the average pellet diameter was not affected. The free base form was released more slowly than the other two forms. Mathematical analysis of the release data revealed that Fickian diffusion and polymer relaxation contributed to the release mechanism in each case, although polymer relaxation was more influential with the free base form.
Keywords: Pellets; Carbopol; Drug release profiles; Extrusion; Spheronization;

Ion-exchange membrane assisted transdermal iontophoretic delivery of salicylate and acyclovir by Qingfang Xu; Sarah A. Ibrahim; William I. Higuchi; S. Kevin Li (105-113).
The presence of endogenous competing counterions is a main reason for the generally low efficiency of transdermal iontophoretic drug delivery. The objective of the present study was to test the hypothesis that the incorporation of an ion-exchange membrane (Ionac) in an iontophoresis system to hinder transdermal transport of these counterions can enhance iontophoretic delivery. The properties of Ionac were characterized in passive and iontophoretic transport experiments. Iontophoretic transport across human epidermal membrane (HEM) and across HEM in series with Ionac was then studied. To assess the effect of HEM electrical resistance upon Ionac-assisted iontophoresis, HEM resistance was reduced in the iontophoresis experiments with alternating current (AC). Salicylate (SA) was the negatively charged permeant first tested in this study. Mannitol was the model permeant to examine the effects of electroosmosis. At the completion of the SA study, experiments were performed with acyclovir (ACV), an antiviral drug with limited water solubility. When Ionac was used to enhance SA transdermal fluxes, higher SA fluxes were observed with HEM of lower resistances in Ionac-assisted iontophoresis. Up to a four-fold flux enhancement was achieved when the electrical resistance of HEM was reduced using an AC iontophoresis method. For ACV, two-fold flux enhancement was observed in Ionac-assisted iontophoresis compared with the conventional iontophoresis baseline. In all experiments, the contribution of electroosmosis to drug transport was less than 10%. The present study has demonstrated the potential of a new approach using a positively charged ion-exchange membrane to enhance transdermal iontophoretic transport of negatively charged drugs.
Keywords: Iontophoresis; Transdermal; Ion-exchange membrane; Salicylate; Acyclovir; Transport enhancement;

Novel porous matrix of hyaluronic acid for the three-dimensional culture of chondrocytes by Jeong Yeon Kang; Chung Wook Chung; Jong-Hyuk Sung; Byung-Soon Park; Je-Yong Choi; Seung Jin Lee; Byung-Chul Choi; Chang-Koo Shim; Suk-Jae Chung; Dae-Duk Kim (114-120).
A novel three-dimensional (3D) scaffold of chemically unmodified hyaluronic acid (HA) with minimum cross-linkage was developed for the culture of chondrocytes, thereby to promote cartilage repair. The porous structure of the scaffold was observed by scanning electron microscopy (SEM), and the pore size was controlled by fabrication conditions including swelling time and composition of the HA matrix. Rabbit primary chondrocytes and human chondrocytic cell lines (C-20/A4) were cultured in the HA matrix to investigate whether they can be applied to construct the cartilage tissue in vitro. The chondrocytes retained chondrocytic spherical morphology in this HA matrix. Moreover, results from the MTT assay showed good cellular viability within the HA matrix; optical density increased for up to 28 days, demonstrating that the cells continued to proliferate inside the HA matrix. Phenotypic analysis (RT-PCR, Alcian blue staining and quantification of s-GAG) showed that chondrocytes, when three-dimensionally cultured within the HA matrix, expressed transcripts encoding collagen type II and aggrecan, and produced sulfated glycosaminoglycans (s-GAG), indicating chondrogenic differentiation. The new HA matrix therefore appears as a potentially promising scaffold for the three-dimensional culture of chondrocytes for cartilage tissue engineering.
Keywords: Hyaluronic acid; Scaffold; Chondrocytes; Cartilage tissue engineering; Differentiation;

Interferon alpha delivery systems for the treatment of hepatitis C by Sumalee Thitinan; Jason T. McConville (121-135).
Hepatitis C virus (HCV) infections are the most common chronic blood-borne viral infections in the world. The prevalence of HCV infections varies significantly by race or ethnicity, with a high prevalence of the disease displayed in the Hispanic population. Additionally, Hispanics with chronic HCV have also more advanced hepatic fibrosis and faster liver fibrosis progression rates than either African Americans or Caucasians. Furthermore, a higher prevalence of cirrhosis and extent of mortality from liver cirrhosis is also observed in the Hispanic population compared with other groups. Current recommendations for treatment of hepatitis C are interferon alpha (IFNα)-based monotherapy and combination of IFNα preparations with ribavirin. Future treatment regimens will still be based on IFNα therapy with or without other effective antiviral agents, currently under investigation. However, there are some inherent limitations, mainly their relative short systemic circulation lifespan, and their unwanted effects on some non-target tissues. New research focuses on the development of novel modified interferon molecules which demonstrate reduced side effects and extended systemic circulation time, which can ultimately provide greater efficacy. Alternative routes for IFNα delivery, such as oral delivery, demonstrate challenging but promising areas of research for improving future patient compliance.
Keywords: Hepatitis C; ADME; Pegylation; Liposomes; Microspheres; Oral drug delivery;

Nifedipine nanoparticle agglomeration as a dry powder aerosol formulation strategy by Carl Plumley; Eric M. Gorman; Nashwa El-Gendy; Connor R. Bybee; Eric J. Munson; Cory Berkland (136-143).
Efficient administration of drugs represents a leading challenge in pulmonary medicine. Dry powder aerosols are of great interest compared to traditional aerosolized liquid formulations in that they may offer improved stability, ease of administration, and simple device design. Particles 1–5 μm in size typically facilitate lung deposition. Nanoparticles may be exhaled as a result of their small size; however, they are desired to enhance the dissolution rate of poorly soluble drugs. Nanoparticles of the hypertension drug nifedipine were co-precipitated with stearic acid to form a colloid exhibiting negative surface charge. Nifedipine nanoparticle colloids were destabilized by using sodium chloride to disrupt the electrostatic repulsion between particles as a means to achieve the agglomerated nanoparticles of a controlled size. The aerodynamic performance of agglomerated nanoparticles was determined by cascade impaction. The powders were found to be well suited for pulmonary delivery. In addition, nanoparticle agglomerates revealed enhanced dissolution of the drug species suggesting the value of this formulation approach for poorly water-soluble pulmonary medicines. Ultimately, nifedipine powders are envisioned as an approach to treat pulmonary hypertension.
Keywords: Drug delivery; Nanoparticles; Nifedipine; Dry powder aerosol;

Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Polyethyleneimine (PEI) has been extensively studied as a vector for nucleic acids and incorporation of PEI into poly(d,l-lactide-co-glycolide) (PLGA) particles has been shown to be useful in the development of gene delivery. PEI was incorporated into the PLGA particles by spontaneous modified emulsification diffusion method. Incorporation of PEI into PLGA particles with the PLGA to PEI weight ratio 29:1 was found to produce spherical and positively charged nanoparticles where type of polymer, type and concentration of surfactant could affect their physical properties. Particle size of around 100 nm was obtained when 5% (m/v) PVA was used as a stabiliser. PLGA-PEI nanoparticles were able to completely bind siRNA at N/P ratio 20:1 and to provide protection for siRNA against nuclease degradation. In vitro cell culture studies subsequently revealed that PLGA-PEI nanoparticles with adsorbed siRNA could efficiently silence the targeted gene in mammalian cells, better than PEI alone, with acceptable cell viability. PLGA-PEI nanoparticles have been found to be superior to its cationising parent compound; PEI polymer.
Keywords: Poly(d,l-lactide-co-glycolide); Polyethyleneimine; Nanoparticles; siRNA; Gene silencing;

DNA loaded carrier preferential extravasation from tumor blood vessel by Ge Jiang; Yingzhi Jiang; Yuanyuan Shen; Kweon-Ho Nam; Donhaeng Lee; Zhonggao Gao (155-161).
Non-viral gene delivery carriers were prepared by using DNA/polyethylenimine/polymethacrylic acid (DPP) polyplexes and its extravasation from tumor blood vessel was evaluated with mouse dorsal skin fold window chamber model. The DNA/PEI (DP) complex with a ratio of N to P (10/1) was coated with polymethacrylic acid, and the ratio of PMA to DNA complex in DNA/PEI/PMA (DPP) polyplex was fixed 0.03 (w/w). The surface charges of the DP and DPP polyplex were positive 26 and 15, respectively. The size of DP and DPP polyplex were 161 nm and 195 nm. The transfection efficiencies in HepG2 cells were about 30-fold and 20-fold higher than that in HeLa and L/C cells in the presence of 50% serum, respectively. The DPP polyplex showed a reduced erythrocyte aggregation activity and a decreased cytotoxicity in cancer cells. After being incubated 30 min, Fluorescently labelled DPP polyplex uptaken by cancer cells decreased, compared with DP by measuring flowcytometry. DPP polyplex penetrating through tumor blood vessel appeared fast and stayed longer in tumour interstitial, this fact was observed from mouse dorsal skin fold window chamber model.
Keywords: DNA delivery; Polymethacrylic acid; Dorsal skin fold window chamber; Cancer cells;

The effect of the structure of small cationic peptides on the characteristics of peptide-DNA complexes by Geetha N. Goparaju; C. Satishchandran; Pardeep K. Gupta (162-169).
A series of transcriptional activator (TAT)–protein transduction domains (PTDs) modified with hydrophobic amino acids were used as model cationic amphiphilic peptides to study the effect of hydrophobicity on interaction of such peptides with plasmid DNA. The peptide–DNA complexes were analyzed by dynamic light scattering and gel electrophoresis to determine their size and electrokinetic properties at various +/− charge ratios. Peptides in solution were found to have a tendency to aggregate and the hydrodynamic size of the aggregates depends on the structure of peptide. Peptides with smaller hydrophobic residues at the N-terminal formed smaller complexes with DNA compared to the ones with larger hydrophobic tails. DNA complexes having peptides with more than one hydrophobic moiety at the N-terminal had a tendency to aggregate. Among the peptides having single hydrophobic amino acid at the N-terminal, DNA complexes of Tyr-TAT and Phe-TAT were found to be stable in solution. The size of the hydrophobic domain and the type of hydrophobic amino acid at the N-terminal of cationic amphiphilic peptides play an important role not only in the complex formation but also in stabilizing the system. The studies presented here indicate that there is a potential for strategic development of these peptides into potential non-viral gene delivery vectors.
Keywords: Non-viral gene delivery; Cationic amphiphilic peptides; Particle size; Zeta potential;

Self-assembled honokiol-loaded micelles based on poly(ɛ-caprolactone)-poly(ethylene glycol)-poly(ɛ-caprolactone) copolymer by XiaWei Wei; ChangYang Gong; Shuai Shi; ShaoZhi Fu; Ke Men; Shi Zeng; XiuLing Zheng; MaLing Gou; LiJuan Chen; LiYan Qiu; ZhiYong Qian (170-175).
Self-assembled polymeric micelles are widely applied in drug delivery system (DDS). In this study, honokiol (HK) loaded micelles were prepared from biodegradable poly(ɛ-caprolactone)-poly(ethylene glycol)-poly(ɛ-caprolactone) (PCEC) copolymers. Micelles were prepared by self-assembly of triblock copolymer PCEC in distilled water triggered by its amphiphilic character without any organic solvent. Drug loading and encapsulation efficiency were determined by adjusting the weight ratio of HK and PCEC. The particle size and zeta potential distribution of obtained micelles were determined using Malvern laser particle sizer, and spherical geometry were observed on atomic force microscope (AFM). Otherwise, the thermo-sensitivity of honokiol-loaded micelles was monitored. And the cytotoxicity results of drug loaded micelles showed that the encapsulated honokiol remained potent antitumor effect. Moreover, in vitro release profile demonstrated a significant difference between rapid release of free honokiol and much slower and sustained release of HK-loaded micelles. These results suggested that we have successfully prepared honokiol-loaded micelles in an improved method which is safer and more efficient. The prepared micelles might be potential carriers for honokiol delivery in cancer chemotherapy.
Keywords: Self-assembly; Honokiol; Micelle; Thermo-sensitive; In vitro release behavior;

Reverse aqueous emulsions and microemulsions in HFA227 propellant stabilized by non-ionic ethoxylated amphiphiles by Udayan Chokshi; Parthiban Selvam; Lionel Porcar; Sandro R.P. da Rocha (176-184).
In this work we use in situ high-pressure tensiometry to screen non-ionic ethoxylated surfactants at the 1,1,1,2,3,3,3-heptafluoropropane (HFA227) propellant|Water (HFA227|W) interface. The EO n PO 30 EO n series, where EO stands for ethylene oxide and PO for propylene oxide, and n the number of repeat EO units, was selected for this study based on the favorable interactions reported between HFA propellants and the PO moiety. The surfactants used in FDA-approved pressurized metered-dose inhaler formulations were also investigated. Tension measurements provide not only information on the relative activity of the different surfactants in the series, but they also serve as a guide for selecting an appropriate candidate for the formation of reverse aggregates based on the surfactant natural curvature. Moreover, the effect of ethanol and the chemistry of the surfactant tail group on the surfactant activity were also investigated. Surfactants with hydrogenated tails are not capable of forming stable water-in-HFA227 microemulsions. This is true even at very low tensions observed when in the presence of ethanol, indicating the lack of affinity between HFA227 and hydrogenated moieties—the surfactant does not tend to curve about water. On the other hand, PO-based amphiphiles can significantly reduce the tension of the HFA227|W interface. Small angle neutron scattering (SANS) and UV–vis spectroscopy results also reveal that a selected ethoxylated amphiphile (EO 13 PO 30 EO 13 at 1 mM concentration), when in the presence of ethanol, is capable of forming stable cylindrical reverse aqueous microemulsions. EO 13 PO 30 EO 13 is also capable of forming emulsions of water-in-HFA227 that are fairly stable against coalescence. Such dispersions are potential candidates for the delivery of small polar solutes and larger therapeutic biomolecules to and through the lungs in the form of pMDI formulations, and in other medical sprays.
Keywords: Inhalation; pMDI; HFAs; Novel Formulations; Microemulsions; Emulsions; SANS;

Characteristics and properties of nanospheres co-loaded with lipophilic and hydrophilic drug models by T. Hammady; A. El-Gindy; E. Lejmi; R.S. Dhanikula; P. Moreau; P. Hildgen (185-195).
The biphasic nature of polymeric nanospheres prepared by the double emulsion method was exploited to co-encapsulate lipophilic and hydrophilic molecules. All-trans retinoic acid (RA) was selected as a lipophilic drug model whereas calf thymus DNA was chosen as a water-soluble model. Simultaneous quantification of the loaded ingredients was achieved by a second derivative spectrophotometric technique. In addition, prepared batches were fully characterized by atomic force microscopy, porosity measurement, and thermal analysis. Finally, the angiosuppressive action of loaded RA was assessed in a tissue culture model. A blend of either polycaprolactone-multiblock copolymer or the microemulsion technique improved DNA-loading, whereas RA-loading was decreased. DSC data were helpful in explaining the initial phase of RA release from the nanospheres. Along with affinity for the polymeric matrix, the microporosity of nanospheres seemed to play an important role in the diffusion rate and release profiles of both loaded drug models in aqueous medium. The anti-angiogenic effect of microencapsulated RA was generally more pronounced than that of the free drug, and its inhibitory action was maintained for the 14-day study period. Moreover, a relationship was observed between the release profiles and anti-angiogenic properties of the batches tested.
Keywords: Co-encapsulation; Lipophilic; Hydrophilic; Derivative spectrophotometry; Microemulsion; Multiblock copolymer;

Solubilisation of griseofulvin in aqueous micellar solutions of diblock copolymers of ethylene oxide and 1,2-butylene oxide with lengthy B-blocks by Maria Elenir N.P. Ribeiro; Igor Marques Cavalcante; Nágila M.P.S. Ricardo; Shao-Min Mai; David Attwood; Stephen G. Yeates; Colin Booth (196-198).
The influence of hydrophobic-block length on solubilisation capacity was examined for micelles of E m B n copolymers (E = oxyethylene, B = oxybutylene, subscripts denote number-average block lengths in repeat units) with B-block lengths in the range of 30–76 and with E-blocks of sufficient length to ensure the formation of spherical micelles. Griseofulvin was used as a model poorly-water-soluble drug known to be almost exclusively solubilised in the micellar core. Combination of solubilisation data with those of a previous study has shown that the amount of drug solubilised per gram of hydrophobe is essentially independent of B-block length when this exceeds about 15 B units, suggesting that core size is not a major influence on solubilisation.
Keywords: Block copolymer micelles; Drug solubilisation;

Noticeboard (199).