International Journal of Pharmaceutics (v.458, #1)

HSA nanocapsules functionalized with monoclonal antibodies for targeted drug delivery by Alexandra Rollett; Tamara Reiter; Anna Ohradanova-Repic; Christian Machacek; Artur Cavaco-Paulo; Hannes Stockinger; Georg M. Guebitz (1-8).
The chronic autoimmune disorder rheumatoid arthritis (RA) affects millions of adults and children every year. Chronically activated macrophages secreting enzymes and inflammatory cytokines play a key role in RA. Distinctive marker molecules on the macrophage surface could be used to design a targeted drug delivery device for the treatment of RA without affecting healthy cells and tissues. Here, different methods for covalent attachment of antibodies (mAb) recognizing MHC class II molecules found on macrophages onto human serum albumin (HSA) nanocapsules were compared. HSA nanocapsules were prepared with a hydrodynamic diameter of 500.7 ± 9.4 nm and a narrow size distribution as indicated by a polydispersity index (PDI) of 0.255 ± 0.024. This was achieved by using a sonochemical process avoiding toxic cross linking agents and emulsifiers. Covalent binding of mAb on the surface of HSA nanocapsules was realized using polyethyleneglycol (PEG)3000 as spacer molecule. The presence of mAb was confirmed by confocal laser scanning microscopy (CLSM) and enzyme-linked immunosorbent assay (ELISA). Specific binding of mAb-HSA nanocapsules to MHC class II molecules on antigen-presenting cells was demonstrated by flow cytometry analysis.
Keywords: Targeted; Drug delivery; Albumin nanocapsules; Cross linking; Antibody;

A novel approach to monitor coating amount by short-wavelength near-infrared spectroscopy using a tracer with a long-chain hydrocarbyl group by Takahiro Ozawa; Makoto Yokoyama; Tetsuya Hosono; Takuya Nagato; Kohei Tahara; Hirofumi Takeuchi (9-14).
Investigation into the use of near-infrared (NIR) as a Process Analytical Technology has been conducted for in-process monitoring of coating amounts for oral pharmaceutical products. However, the low specificity of NIR spectra has made it time consuming and costly to establish quantitative calibration models for commercial production. Here we revealed that long-chain hydrocarbyl group compounds containing saturated hydrocarbon chains, such as cetyl and stearyl, exhibit specific and strong absorption in the short wavelength (SW)-NIR region (800–1100 nm) with limited interference from peaks corresponding to other components. To simplify the quantitative model, we used cetanol as a model tracer of coating amount to enhance detection sensitivity and analytical precision. The coating amount on crystalline cellulose granules was determined only from the intensity of NIR absorption at a single wavelength, which was attributed to the tracer. The results showed close agreement with quantitative analyses from gas chromatography and measurement of weight gain. In conclusion, we determined coating amount with considerable accuracy from NIR absorption at a single wavelength in the SW-NIR region using the long-chain hydrocarbyl containing compound as a tracer, thereby eliminating the need for complicated statistics.
Keywords: Process Analytical Technology (PAT); Near-infrared (NIR); In-process monitoring; Film coating; Short wavelength near-infrared (SW-NIR); Long-chain hydrocarbyl;

PAT tools for the control of co-extrusion implants manufacturing process by Fabrice Krier; Jérôme Mantanus; Pierre-Yves Sacré; Pierre-François Chavez; Justine Thiry; Aude Pestieau; Eric Rozet; Eric Ziemons; Philippe Hubert; Brigitte Evrard (15-24).
Hot melt extrusion is a novel pharmaceutical manufacturing process technique. In this study, we identified four Critical Quality Attributes (CQAs) of the implant manufacturing process by hot melt extrusion: the implant diameter, the quantity of the Active Pharmaceutical Ingredient (API), the homogeneity distribution of API and the thickness of the membrane. We controlled the implant diameter and the quantity of API in-line with a laser measurement, NIR and Raman spectroscopy, respectively. These two different spectroscopic techniques provided comparable results. In fact, the RMSEC and RMSECV were very close in each PAT technique but NIR spectroscopy was easier to use and less sensitive to external changes. For the control of the homogeneity of API distribution and the thickness of the membrane, we used successfully Raman spectroscopy imaging. These PAT tools help reducing analysis time.
Keywords: Implant; NIR spectroscopy; Raman spectroscopy; Hot melt extrusion; Process Analytical Technology; Raman imaging;

Cationic quaternized aminocalix[4]arenes: cytotoxicity, haemolytic and antibacterial activities by Elena V. Ukhatskaya; Sergey V. Kurkov; Martha A. Hjálmarsdóttir; Vladimir A. Karginov; Susan E. Matthews; Roman V. Rodik; Vitaly I. Kalchenko; Thorsteinn Loftsson (25-30).
This study reports the characterization of three cationic amphiphillic aminocalix[4]arenes as potential antimicrobial agents in vitro. In cytotoxicity tests on mouse macrophage RAW 264.7 cells aminocalix[4]arenes 1 and 3 showed no toxicity up to 200 and 100 μM concentrations, respectively, while 2 was non-toxic only up to 50 μM. With regard to the haemolytic activity on rabbit red blood cells, 1 was not active at concentrations up to 100 μM in contrast to the other two studied macrocycles. Compounds showed negligible ability to protect either mouse macrophage RAW 264.7 cells from anthrax lethal toxin of Bacillus anthracis (B. anthracis) or rabbit red blood cells from α-haemolysin of Staphylococcus aureus (S. aureus) in comparison to amino-β-cyclodextrins. However, all aminocalix[4]arenes showed potential as antimicrobials. Their minimum inhibitory concentrations (MIC) against Escherichia coli (E. coli) and S. aureus were in the 16–32 μg/ml concentration range, while minimum lethal concentrations (MLC) varied from 16 to 256 μg/ml depending on the bacteria and aminocalix[4]arene considered. Macrocycle 1 showed partial synergism against S. aureus in tandem with a model antibacterial drug, fusidic acid, at certain concentration combinations.
Keywords: Antibacterial; Calix[n]arene; Cytotoxicity; Haemolytic; Solubilization;

Self-assembled nanoparticles based on galactosylated O-carboxymethyl chitosan-graft-stearic acid conjugates for delivery of doxorubicin by Hejian Guo; Dianrui Zhang; Caiyun Li; Lejiao Jia; Guangpu Liu; Leilei Hao; Dandan Zheng; Jingyi Shen; Tingting Li; Yuanyuan Guo; Qiang Zhang (31-38).
A novel polymer, i.e. galactosylated O-carboxymethyl chitosan-graft-stearic acid (Gal-OCMC-g-SA) was synthesized for liver targeting delivery of doxorubicin. The chemical structure was characterized by FT-IR, 1H NMR and elemental analysis. Gal-OCMC-g-SA could self-assemble into nanoparticles with diameter of 160 nm by probe sonication in aqueous medium and exhibited a low critical aggregation concentration of 0.047 mg/mL. The DOX-loaded Gal-OCMC-g-SA (Gal-OCMC-g-SA/DOX) self-assembled nanoparticles were almost spherical in shape with an average diameter of less than 200 nm and zeta potential of around −10 mV. In vitro release revealed that the Gal-OCMC-g-SA/DOX nanoparticles exhibited a sustained and pH-dependent drug release manner. Furthermore, the hemolysis test demonstrated the good safety of Gal-OCMC-g-SA in blood-contacting applications. These results indicated that Gal-OCMC-g-SA/DOX nanoparticles were highly potential to be applied in cancer therapy.
Keywords: O-carboxymethyl chitosan; Stearic acid; Liver targeting; Doxorubicin;

Further experimentation of inhaled; Lantus, Actrapid and Humulin with todays’ production systems by Paul Zarogoulidis; Dimitris Petridis; Christos Ritzoulis; Qiang Li; Haidong Huang; Yunye Ning; Kaid Darwiche; Lutz Freitag; Konstantinos Zarogoulidis (39-47).
Several aerosol production systems have been used for aerosol insulin production. However; since the first studies several new models of jet-nebulizers and ultrasound nebulizers have been introduced in the market.Three different models of jet-nebulizers (different brands, same properties) and three different ultrasound nebulizers (different brands, same properties). Six residual cups (2 small ≤ 6 ml and 3 large ≤ 8 ml) were used for the jet-nebulizers. The ultrasound nebulizers were used with their facemasks or with their inlets which were included in the purchase package.Ultrasound nebulizers; LANTUS produces by far the lowest mean droplets (2.44) half the size of the other two drugs (4.43 = 4.97). GIMA nebulizer is the most efficient producing one third of the droplet size of SHIMED and one second of EASYNEB (2.06 < 3.15 < 6.62). Finally, the 4 ml loading concentration is more suitable for supporting the production of smaller droplets (3.65 < 4.24). Drugs and nebulizers act interactively yielding very large droplets when ACTRAPID and HUMULIN are administered in joint with SHIMED nebulizer (9.59 = 7.72). Jet-nebulizers; HUMULIN again is the least preferred insulin since it hardly reaches the low but equal performance of others at the loading level of 6 ml. Residual cups E and B produce uniquely lower mean droplets at loading level 6.Ultrasound nebulizers; the best suggested combination should be LANTUS insulin, GIMA nebulizer administered at loading dose of 4 ml jet-nebulizers. A global review can give the best combination: the lowest mean droplets are produced when the drugs LANTUS (mostly) and ACTRAPID are administered, applying the SUNMIST nebulizer in concert with residual cup B at loading levels of 6 ml.
Keywords: Aerosol insulin; Jet-nebulizers; Ultrasound;

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex by Jing Wang; Yang Lei; Cao Xie; Weiyue Lu; Zhiqiang Yan; Jie Gao; Zuoxu Xie; Xiaoyu Zhang; Min Liu (48-56).
Safe and efficient systems capable of specifically targeting brain tumour cells represent a promising approach for the treatment glioblastoma multiforme. Neuropilin-1 (NRP-1) is over-expressed in U87 glioma cells. In the current study, the tumour specific peptide RGERPPR, which binds specifically to NRP-1, was used as a targeting ligand in a gene delivery strategy for glioblastoma. The RGERPPR peptide was coupled to branched polyethylenimine (PEI, 25 kDa) using heterobifunctional Mal–PEG–NHS, resulting in a novel gene delivery polymer. Polymer/plasmid DNA (pDNA) complexes were formed and their sizes and zeta potentials were measured. Compared with the unmodified mPEG–PEI/pDNA complexes, the RGERPPR–PEG–PEI/pDNA complex led to a significant enhancement in intracellular gene uptake and tumour spheroid penetration. Furthermore, the RGERPPR–PEG–PEI/pDNA complex facilitated enhanced transfection efficiency levels, as well as a reduction in cytotoxicity when tested in U87 glioma cells in vitro. Most significantly of all, when complexes formed with pDsRED-N1 were injected into the tail vein of intracranial U87 tumour-bearing nude mice, the RGERPPR–PEG–PEI complexes led to improved levels of red fluorescence protein expression in the brain tissue. Taken together, the results show that RGERPPR–PEG–PEI could be used as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene delivery.
Keywords: Glioma targeting; C-end rule peptide; Neuropilin-1; Gene delivery; Polyethylenimine;

Quaternary polymethacrylate–magnesium aluminum silicate films: Molecular interactions, mechanical properties and tackiness by Thitiphorn Rongthong; Srisagul Sungthongjeen; Juergen Siepmann; Thaned Pongjanyakul (57-64).
The aim of this study was to investigate the impact of the addition of magnesium aluminum silicate (MAS), a natural clay, on the properties of polymeric films based on quaternary polymethacrylates (QPMs). Two commercially available aqueous QPM dispersions were studied: Eudragit® RS 30D and Eudragit® RL 30D (the dry copolymers containing 5 and 10% quaternary ammonium groups, respectively). The composite QPM-MAS films were prepared by casting. Importantly, QPM interacted with MAS and formed small flocculates prior to film formation. Continuous films were obtained up to MAS contents of 19% (referred to the QPM dry mass). ATR-FTIR and PXRD revealed that the positively charged quaternary ammonium groups of QPM interacted with negatively charged ―SiO groups of MAS, creating nanocomposite materials. This interaction led to improved thermal stability of the composite films. The puncture strength and elongation at break of dry systems decreased with increasing MAS content. In contrast, the puncture strength of the wet QPM-MAS films (upon exposure to acidic or neutral media) increased with increasing MAS content. Furthermore, incorporation of MAS into QPM films significantly decreased the latter's tackiness in the dry and wet state. These findings suggest that nanocomposite formation between QPM and MAS in the systems can enhance the strength of wet films and decrease their tackiness. Thus, MAS offers an interesting potential as novel anti-tacking agent for QPM coatings.
Keywords: Quaternary polymethacrylate; Magnesium aluminum silicate; Molecular interaction; Tackiness; Mechanical properties;

Ozonated oils as functional dermatological matrices: Effects on the wound healing process using SKH1 mice by G. Valacchi; I. Zanardi; Y. Lim; G. Belmonte; C. Miracco; C. Sticozzi; V. Bocci; V. Travagli (65-73).
Wound tissue repair is a complex and dynamic process of restoring cellular structures and tissue layers. Improvement of this process is crucial for several pathologies characterized by chronic delayed wound closure such as diabetes, and the investigation of new approaches aimed to ameliorate the wound healing process is under continuous evolution. Recently, the usage of vegetable matrices in the form of ozonated oils has been proposed and several researchers have shown a positive effect in the wound, based on their bactericidal, antiviral, and antifungal properties. The present study was undertaken to compare the effect that different ozonated oils (olive, sesame and linseed) with the same level of ozonation have on wound healing rate in SKH1 mice. Several histological parameters and the level of key proteins such as VEGF and PCNA have been analyzed. Only treatment with ozonated sesame oil shows a faster wound closure in the first 7 days. This effect paralleled with the increased VEGF and PCNA levels, NFκB nuclear translocation and 4-HNE formation. The present study shows that not only the ozonation grade is of importance for the improvement of wound healing process but also the typical composition of the oil.
Keywords: Wound healing; 1,2,4-Trioxolane moiety; Prodrugs; Vegetable matrices;

An innovative matrix controlling drug delivery produced by thermal treatment of DC tablets containing polycarbophil and ethylcellulose by Gabriele Caviglioli; Sara Baldassari; Paola Cirrincione; Eleonora Russo; Brunella Parodi; Paolo Gatti; Giuliana Drava (74-82).
SEM micrograph of a cross section of a tablet: matrix structure produced by thermal treatment.An innovative matrix, produced by thermal treatment on direct compression (DC) tablets containing polycarbophil (POL) and ethylcellulose (EC), identified as matrix forming polymers, and able to control the release of diltiazem hydrochloride, was developed. At pH 7.2, 72 ± 1.2% (w/w) of drug loaded was released in 25 h, mostly at constant rate. This swellable and unerodible matrix controls drug release by an anomalous transport mechanism. The modifications induced by the thermal treatment are irreversible and can be used to control and characterize the matrix. A 3-component constrained mixture design allowed the investigation of the experimental domain in which the matrix forms and the computation of a mathematical model that can be used to optimize the formulation properties. The release rate can be modulated (0.032–0.064% drug released/min) through the choice of suitable treatment conditions and tablet composition. The maximum amount of diltiazem hydrochloride released by zero-order kinetics, at the lowest release rate, occurs for POL:EC ratio in the range of 1:1–2:3 with 20–30% of diluent. The tablets are able to load up to 50% (w/w) of diltiazem hydrochloride without losing their properties. A stability study performed on a selected formulation containing DTZ showed stability for at least 2.7 years at RT conditions.
Keywords: DC tablet; Polycarbophil; Ethylcellulose; Matrix tablets; Thermal treatment; Controlled release;

In vivo evaluation of poly-l-asparagine nanocapsules as carriers for anti-cancer drug delivery by Gustavo R. Rivera-Rodriguez; Giovanna Lollo; Tristan Montier; Jean Pierre Benoit; Catherine Passirani; Maria José Alonso; Dolores Torres (83-89).
Here, we report the in vivo proof of-concept of a novel nanocarrier, poly-l-asparagine (PASN) nanocapsules, as an anticancer targeted drug delivery system. The nanocapsules were loaded with the fluorescent marker DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate) and also with the model drug docetaxel to evaluate the biodistribution and efficacy profiles in healthy and glioma-bearing mice, respectively. Regardless of their cargo, the nanocapsules presented a size close to 180 nm, a surface charge around −40 mV and an encapsulation efficiency of 75–90%. The biodistribution study in healthy mice showed that PASN nanocapsules led to a two- and three-fold increment in the mean residence time (MRT) and area under the curve (AUC) values, respectively, compared to those of a non-polymeric nanoemulsion. Finally, the efficacy/toxicity study indicated that the encapsulated drug was as efficacious as the commercial formulation (Taxotere®), with the additional advantage of being considerably less toxic. Overall, these results suggest the potential of PASN nanocapsules as drug nanocarriers in anticancer therapy.
Keywords: Polyaminoacids; Poly-l-asparagine; Nanocapsules; Docetaxel; Cancer therapy; Tumor targeting;

Quantification of the types of water in Eudragit RLPO polymer and the kinetics of water loss using FTIR by Chompak Pirayavaraporn; Thomas Rades; Keith C. Gordon; Ian G. Tucker (90-98).
Coalescence of polymer particles in polymer matrix tablets influences drug release. The literature has emphasized that coalescence occurs above the glass transition temperature (T g) of the polymer and that water may plasticize (lower T g) the polymer. However, we have shown previously that nonplasticizing water also influences coalescence of Eudragit RLPO; so there is a need to quantify the different types of water in Eudragit RLPO. The purpose of this study was to distinguish the types of water present in Eudragit RLPO polymer and to investigate the water loss kinetics for these different types of water. Eudragit RLPO was stored in tightly closed chambers at various relative humidities (0, 33, 56, 75, and 94%) until equilibrium was reached. Fourier transform infrared spectroscopy (FTIR)-DRIFTS was used to investigate molecular interactions between water and polymer, and water loss over time. Using a curve fitting procedure, the water region (3100–3700 cm−1) of the spectra was analyzed, and used to identify water present in differing environments in the polymer and to determine the water loss kinetics upon purging the sample with dry compressed air. It was found that four environments can be differentiated (dipole interaction of water with quaternary ammonium groups, water cluster, and water indirectly and directly binding to the carbonyl groups of the polymer) but it was not possible to distinguish whether the different types of water were lost at different rates. It is suggested that water is trapped in the polymer in different forms and this should be considered when investigating coalescence of polymer matrices.
Keywords: FTIR; DRIFTs; Methacrylates; Eudragit; Water; Environment;

Polymer structure and antimicrobial activity of polyvinylpyrrolidone-based iodine nanofibers prepared with high-speed rotary spinning technique by István Sebe; Barnabás Szabó; Zsombor K. Nagy; Dóra Szabó; László Zsidai; Béla Kocsis; Romána Zelkó (99-103).
Poly(vinylpyrrolidone)/poly(vinylpyrrolidone-vinylacetate)/iodine nanofibers of different polymer ratios were successfully prepared by a high-speed rotary spinning technique. The obtained fiber mats were subjected to detailed morphological analysis using an optical and scanning electron microscope (SEM), while the supramolecular structure of the samples was analyzed by positron annihilation lifetime spectroscopy (PALS). The maximum dissolved iodine of the fiber samples was determined, and microbiological assay was carried out to test their effect on the bacterial growth. SEM images showed that the polymer fibers were linear, homogenous, and contained no beads. The PALS results, both the o-positronium (o-Ps) lifetime values and distributions, revealed the changes of the free volume holes of fibers as a function of their composition and the presence of iodine. The micro- and macrostructural characterisation of polymer fiber mats enabled the selection of the required composition from the point of their applicability as a wound dressing.
Keywords: High-speed rotary spinning; PVP-iodine; Fiber composite; Positron annihilation lifetime spectroscopy (PALS); Antimicrobial activity;

Nanosuspension improves tretinoin photostability and delivery to the skin by Francesco Lai; Rosa Pireddu; Francesco Corrias; Anna Maria Fadda; Donatella Valenti; Elena Pini; Chiara Sinico (104-109).
The aims of this work were to improve cutaneous targeting and photostability of tretinoin by using nanosuspension formulation. Tretinoin is a drug widely used in the topical treatment of various dermatological diseases. The tretinoin nanosuspension was prepared by precipitation method and then characterized by photo correlation spectroscopy for mean size and size distribution, and by transmission electron microscopy for morphological studies. An oil in water tretinoin nanoemulsion was also prepared and used as a control. Dermal and transdermal delivery of both tretinoin nanosuspension and nanoemulsion were tested in vitro by using Franz diffusion cells and newborn pig skin. Photodegradation studies were carried out by UV irradiation (1 h, λ  = 366 nm) of the tretinoin nanosuspension in comparison with the nanoemulsion and a methanolic solution of the drug. During 8 h percutaneous experiments, the nanosuspesion was able to localize the drug into the pig skin with a very low transdermal drug delivery, whereas the nanoemulsion greatly improved drug permeation. UV irradiation of the nanosuspension showed a great improvement of tretinoin stability in comparison with both controls. Overall results show that nanosuspension might be a useful formulation for improving tretinoin dermal delivery and stability.
Keywords: Tretinoin; Nanosuspension; Nanoemulsion; Dermal delivery; Photostability;

A novel lactoferrin-modified β-cyclodextrin nanocarrier for brain-targeting drug delivery by Yajing Ye; Yi Sun; Hongli Zhao; Minbo Lan; Feng Gao; Chao Song; Kaiyan Lou; Hao Li; Wei Wang (110-117).
Schematic diagram of IR loaded-Lf-CD nanoparticles.The blood–brain barrier (BBB) restricts the transfer and delivery of most drug substances to brain. In this study, a novel nano-drug delivery system for brain-targeting was developed and investigated in vitro and in vivo. Lactoferrin (Lf) was selected as a brain-targeting ligand and conjugated to β-cyclodextrin (β-CD) via the heterobifunctional polyethyleneglycol (PEG) linker NHS-PEG-MAL, yielding Lf conjugated β-cyclodextrin (Lf-CD). UV–vis, FTIR, NMR and transmission electron microscopy (TEM) techniques clearly demonstrated the successful synthesis of Lf-CD nanoparticles with the average diameter of 92.9 ± 16.5 nm. Using near-infrared fluorescent dye IR-775 chloride (IR) as a model compound of poorly water-soluble drugs, IR-loaded Lf-CD nanoparticles (Lf-CD/IR) were successfully prepared with a high entrapment efficiency of 98.1 ± 4.8%. Biodistribution and pharmacokinetics of Lf-CD/IR were evaluated in KM mice after intravenous administration. The results of tissue distribution studies revealed that Lf-CD/IR treatment showed greatly improved BBB transport efficiency. In addition, AUC0–2h of IR in brain after Lf-CD/IR treatment was seven fold higher compared with that of IR treatment without Lf-CD nano-carriers, demonstrating that the introduction of Lf-CD drug-delivery system positively resulted in a higher AUC located in brain tissue. These results provide evidence that Lf-CD nanoparticles could be exploited as a potential brain-targeting drug delivery system for hydrophobic drugs and diagnostic reagents which normally fail to pass through the BBB.
Keywords: β-cyclodextrin derivatives; Lactoferrin; Transferrin; Brain-targeting; Nano-drug delivery system;

The influence of salt formation on electrostatic and compression properties of flurbiprofen salts by Enes Šupuk; Muhammad U. Ghori; Kofi Asare-Addo; Peter R. Laity; Pooja M. Panchmatia; Barbara R. Conway (118-127).
Specific charge and polarity for flurbiprofen and the salts against stainless steel container at saturation point of tribo-charging.Salt formation is an effective method of improving physicochemical properties of acidic and basic drugs. The selection of a salt form most suitable for drug development requires a well-designed screening strategy to ensure various issues are addressed in the early development stages. Triboelectrification of pharmaceutical powders may cause problems during processing such as segregation of components due to the effects of particle adhesion. However, very little work has been done on the effect of salt formation on triboelectrification properties. In this paper, salts of flurbiprofen were prepared by combining the drug with a selection of closely related amine counter ions. The aim of the work was to investigate the impact of the counter ion on electrostatic charge of the resultant salts to inform the salt selection process. The experimental results show the magnitude of charge and polarity of the flurbiprofen salts to be highly dependent on the type of counter ion selected for the salt formation. Furthermore, particle adhesion to the stainless steel surface of the shaking container and the salts’ compression properties were measured. The formed salts had lower electrostatic charges, improved tabletability, and resulted in reduced adhesion of these powders compared with the parent drug.
Keywords: Triboelectrification; Electrostatic; Salt formation; Crystal structure; Compaction;

Caco-2 based cell models have been the gold standard in vitro method to study intestinal drug permeability, despite the absence of many important features with major influence in the drug absorption mechanism. In the present work, a triple co-culture comprising Caco-2, HT29-MTX and Raji B cells was established to mimic in a closely way the human intestinal epithelium, presenting the main components in the process of drug absorption, namely the absorptive cells that resemble enterocytes, mucus producers cells and cells able to induce M-cell phenotype on Caco-2 cells. All the three cell lines maintained their function when cultured together with each other being, thus, an asset to new orally administrated drugs development. The seeding ratio of 90:10 between Caco-2 and HT29-MTX showed to be the best to achieve physiological proportions after cells maturation and differentiation in culture. The formation of M-cells phenotype from enterocytes was identified for the first time in a co-culture system comprising Caco-2 and HT29-MTX cells through immunocytochemical techniques. Thus, the triple co-culture model presented in the herein work is a good and reliable alternative to the in vitro methods already existents for the study of drugs permeability.
Keywords: In vitro model; Caco-2 cells; HT29-MTX cells; Raji B cells; Co-culture; Intestinal permeability;

Effects of particle size on the pharmacokinetics of puerarin nanocrystals and microcrystals after oral administration to rat by Liangxing Tu; Yueneng Yi; Wei Wu; Fuqiang Hu; Kaili Hu; Jianfang Feng (135-140).
With the decreasing of particle size, an increasing of absolute bioavailability (Fa) and a decreasing of clearance rate (CL/F) were observed for puerarin nanocrystal, which reveals that nanocrystal technique can be used to improve the bioavailability of puerarin and the decrease of clearance rate might be an important way for nanocrystals to improve the oral bioavailability.Puerarin, which is extracted from traditional Chinese medicine, is widely used in clinic in China and mainly used as a therapeutic agent to cardiovascular diseases. Owing to its poor water solubility and adverse drug reactions caused by cosolvents after intravenous administration, the development of oral formulation is urgently needed. Nowadays, nanocrystals technique has become a preferred way to develop oral dosage form. In this study, we used high pressure homogenization (HPH) to prepare puerarin nanocrystals and microcrystals with different sizes ranged from 525.8 nm to 1875.6 nm and investigated the influence of particle size on pharmacokinetics. The nanocrystals and microcrystals prepared were characterized using DLS, DSC, XRD and SEM, and we found that the crystalline state of puerarin was changed during the preparation process and the drug was dispersed into HPMC. In the pharmacokinetic study, we observed an increasing of C max and AUC and a decreasing of CL/F with the decreasing of particle size. The AUC of the puerarin nanocrystals (525.8 nm) was 7.6-fold of that of raw puerarin suspension, with an absolute bioavailability of 21.44%. From the above results, we can conclude that nanocrystal technique is an efficient technology to improve the oral bioavailability of puerarin.
Keywords: Puerarin; Nanocrystals; Microcrystals; Bioavailability; Particle size;

Physico-chemical properties of permeation enhancers like molecular weight/size, hydrophobicity/hydrophilicity, co-solvency, etc. are necessary during their selection for pharmaceutical product development. Chemical permeation enhancers modulate the viscoelastic properties of pressure sensitive adhesives. The extent of this modulation depends upon the molecular size and branching of the polymeric chains. The functional nature of this branching additionally changes the peel and tack properties of PSA's. Chemical permeation enhancers alone are not able to modify viscoelastic properties of aqueous based PSA's as compared with their solvent based counterparts. These modulated mechanical aspects need to be maintained throughout development of transdermal patch along with other pharmaceutical aspects like drug release and drug stability.
Keywords: Dynamic mechanical analysis (DMA); Permeation enhancer; Transdermal; Pressure sensitive adhesive (PSA); Acrylate;

Mucoadhesive hybrid gel improves intraperitoneal platinum delivery by Sungpil Cho; Yongen Sun; Elke A. Jarboe; Andrew P. Soisson; Mark K. Dodson; David K. Gaffney; C. Matthew Peterson; Margit M. Janát-Amsbury (148-155).
Intraperitoneal cisplatin (CDDP) delivery with chitosan–alginate-CDDP hybrid gel system enhanced the accumulation of CDDP to gDNA from applied tissues.A leading cause of death and suffering in patients with abdominal or pelvic malignancies is progression of peritoneal surface disease. Changes in the use of chemotherapy have shown significant survival benefits for intraperitoneal or combined intraperitoneal and intravenous treatment following optimal surgical cytoreduction. However, broader clinical use of intraperitoneal therapy has not reached its full potential due to limited efficacy, accessibility and nonspecific toxicity. To overcome these problems, we developed a mucoadhesive hybrid gel (HG) for a local, intraperitoneal drug delivery. In vivo studies confirmed reliable adherence and residence of the gel to the peritoneal sidewall for at least 72 h exhibiting no signs of tissue toxicity. Functionally active CDDP was released from HG within 2 h and was equal to free CDDP in vitro. Moreover, intraperitoneal application of HG-CDDP significantly enhanced CDDP accumulation in the genomic DNA of peritoneal tissues compared to the same CDDP dose administered intravenously. These findings indicate the potential application of this hybrid gel as a mucoadhesive drug carrier amendable to use for intraperitoneal drug delivery and possible expansion for use on other mucosal surfaces of the female reproductive tract.
Keywords: Intraperitoneal delivery; cis-Diaminedichloroplatinum (II) (CDDP); Chitosan; Hybrid gel; Mucoadhesion;

A physiologically based pharmacokinetics model for melatonin—Effects of light and routes of administration by Henry T. Peng; Fethi Bouak; Oshin Vartanian; Bob Cheung (156-168).
Physiologically based pharmacokinetic (PBPK) models were developed using MATLAB Simulink® to predict diurnal variations of endogenous melatonin with light as well as pharmacokinetics of exogenous melatonin via different routes of administration. The model was structured using whole body, including pineal and saliva compartments, and parameterized based on the literature values for endogenous melatonin. It was then optimized by including various intensities of light and various dosage and formulation of melatonin. The model predictions generally have a good fit with available experimental data as evaluated by mean squared errors and ratios between model-predicted and observed values considering large variations in melatonin secretion and pharmacokinetics as reported in the literature. It also demonstrates the capability and usefulness in simulating plasma and salivary concentrations of melatonin under different light conditions and the interaction of endogenous melatonin with the pharmacokinetics of exogenous melatonin. Given the mechanistic approach and programming flexibility of MATLAB Simulink®, the PBPK model could provide predictions of endogenous melatonin rhythms and pharmacokinetic changes in response to environmental (light) and experimental (dosage and route of administration) conditions. Furthermore, the model may be used to optimize the combined treatment using light exposure and exogenous melatonin for maximal phase advances or delays.
Keywords: PBPK; MATLAB Simulink®; Melatonin; Light; Circadian rhythms; Pharmacokinetics;

Multifunctional nanomedicine platform for concurrent delivery of chemotherapeutic drugs and mild hyperthermia to ovarian cancer cells by Olena Taratula; Raj Kumar Dani; Canan Schumann; Hong Xu; Andrew Wang; Han Song; Pallavi Dhagat; Oleh Taratula (169-180).
Nanocarrier for concurrent delivery of chemotherapeutic drug and heat.A multifunctional tumor-targeting delivery system was developed and evaluated for an efficient treatment of drug-resistant ovarian cancer by combinatorial therapeutic modality based on chemotherapy and mild hyperthermia. The engineered iron oxide nanoparticle (IONPs)-based nanocarrier served as an efficient delivery vehicle for doxorubicin and provided the ability to heat cancer cells remotely upon exposure to an alternating magnetic field (AMF). The nanocarrier was additionally modified with polyethylene glycol and LHRH peptide to improve its biocompatibility and ability to target tumor cells. The synthesized delivery system has an average size of 97.1 nm and a zeta potential close to zero, both parameters favorable for increased stability in biological media and decreased elimination by the immune system. The nanocarrier demonstrated faster drug release in acidic conditions that mimic the tumor environment. It was also observed that the LHRH targeted delivery system could effectively enter drug resistant ovarian cancer cells, and the fate of doxorubicin was tracked with fluorescence microscope. Mild hyperthermia (40 °C) generated by IONPs under exposure to AMF synergistically increased the cytotoxicity of doxorubicin delivered by the developed nanocarrier to cancer cells. Thus, the developed IONPs-based delivery system has high potential in the effective treatment of ovarian cancer by combinatorial approach.
Keywords: Iron oxide nanoparticles; Combinatorial treatment; Mild hyperthermia; Alternating magnetic field (AMF); Doxorubicin; Ovarian cancer;

A novel bi-layer ascending release osmotic pump tablet: In vitro investigation and in vivo investigation in pharmacokinetic study and IVIVC evaluation by Heming Xu; Zhao Li; Hao Pan; Zhihong Zhang; Dandan Liu; Baocheng Tian; Shilin Ma; Shilong Song; Weisan Pan (181-187).
This study was aimed to develop an ascending release push–pull osmotic pump (APOP) system with a novel mechanism and an easy manufacture process.Theoretical analysis showed that the key to obtain the non-zero order drug release was to break the balance between the drug suspension release rate in the drug layer and the swelling rate of the core, and an ascending drug release rate was achieved when the former was slower than the latter. A polymer (Polyox WSR N-12K) was introduced as a suspension agent in drug layer to slow down the hydration rate of drug layer. Influence of the composition of drug layer (PEO category, total amount, drug loading and fraction of NaCl), push layer (NaCl amount), and also the level of coating weight gain on the drug release profiles was investigated. Observation of hydration state was estimated by taking photos, and also was confirmed by the theories. Paliperidone was delivered successfully by APOP at an ascending release rate up to 20 h in vitro. The in vivo plasma concentration of paliperidone in beagle dogs increased gradually up to 19 h.The APOP with an easy manufacture process was a promising strategy to deliver drug at an ascending rate.
Keywords: Ascending release osmotic pump; Rate balance; Mechanism verification; Paliperidone;

Validation protocol of analytical procedures for quantification of drugs in polymeric systems for parenteral administration: Dexamethasone phosphate disodium microparticles by Cristina Martín-Sabroso; Daniel Filipe Tavares-Fernandes; Juan Ignacio Espada-García; Ana Isabel Torres-Suárez (188-196).
In this work a protocol to validate analytical procedures for the quantification of drug substances formulated in polymeric systems that comprise both drug entrapped into the polymeric matrix (assay:content test) and drug released from the systems (assay:dissolution test) is developed. This protocol is applied to the validation two isocratic HPLC analytical procedures for the analysis of dexamethasone phosphate disodium microparticles for parenteral administration. Preparation of authentic samples and artificially “spiked” and “unspiked” samples is described. Specificity (ability to quantify dexamethasone phosphate disodium in presence of constituents of the dissolution medium and other microparticle constituents), linearity, accuracy and precision are evaluated, in the range from 10 to 50 μg mL−1 in the assay:content test procedure and from 0.25 to 10 μg mL−1 in the assay:dissolution test procedure. The robustness of the analytical method to extract drug from microparticles is also assessed. The validation protocol developed allows us to conclude that both analytical methods are suitable for their intended purpose, but the lack of proportionality of the assay:dissolution analytical method should be taken into account.The validation protocol designed in this work could be applied to the validation of any analytical procedure for the quantification of drugs formulated in controlled release polymeric microparticles.
Keywords: Validation protocol; Polymeric drug delivery systems; Analytical procedure; Microparticles; Dexamethasone phosphate disodium;

Rapid and soft formulation of folate-functionalized nanoparticles for the targeted delivery of tripentone in ovarian carcinoma by J. Tomasina; L. Poulain; E. Abeilard; F. Giffard; E. Brotin; L. Carduner; F. Carreiras; P. Gauduchon; S. Rault; A. Malzert-Fréon (197-207).
We report the development of folate-functionalized nanoparticles able to target folate receptors, and to deliver a poorly water soluble cytotoxic agent, a tripentone, in ovarian carcinoma. The stability under incubation of lipid nanoparticles formulated by a low-energy phase inversion temperature method was investigated. Thanks to the presence of Labrasol®, a macrogolglyceride into the composition of the nanocarriers, the conjugation of different quantities of a folate derivate (folic acid-polyethylene glycol2000-distearylphosphatidylethanolamine) to nanoparticles was possible by a rapid, soft, very simple post-insertion process. As determined by dynamic light scattering, nanoparticles present a monodisperse diameter of about 100 nm, a spherical shape as attested by transmission electron micrographs, a weakly negative surface zeta potential, and are able to encapsulate the tripentone MR22388. The presence of folate receptors on SKOV3 human ovarian cancer cells was identified by fluorescent immunocytochemistry. Cellular uptake studies assessed by flow cytometry indicated that these nanoparticles reached the SKOV3 cells rapidly, and were internalized by a folate-receptor mediated endocytosis pathway. Moreover, nanoparticles allowed the rapid delivery of the antitumor agent tripentone into cells as shown in vitro by real-time cellular activity assay. Such folate-lipid nanoparticles are a potential carrier for targeted delivery of poorly water soluble compounds into ovarian carcinoma.
Keywords: Nanoparticles; Folate; Active targeting; Tripentone; MR22388; Ovarian cancer;

Sponges carrying self-microemulsifying drug delivery systems by Elinor Josef; Havazelet Bianco-Peled (208-217).
Self-microemulsifying drug delivery systems (SMEDDS) increase the solubility of lipophilic drugs. One barrier to their wide application is their liquid nature. We report on a new method to solidify SMEDDS—their incorporation in sponges made from a hydrophilic natural polymer. Using different freeze-drying schemes, sponges were prepared from alginate gels containing microemulsions. The sponges’ structures were studied with scanning electron microscopy and small angle X-ray scattering. The oil droplets survived the drying process, and SMEDDS were present as 9 nm-sized objects in the dried sponges. The sponges were rehydrated in water, and evidence of the presence of SMEDDS in the rehydrated sponges was found. A model hydrophobic molecule, Nile red, was soluble in all dry and rehydrated sponges. SMEDDS containing Nile red were gradually released from the sponges, at a rate that depended on the drying method. The equilibrium water uptake of the sponges was also found to be influenced by the drying scheme. The combination of SMEDDS and sponges may be a way to overcome the disadvantages of each component separately, provide a solid dosage form for SMEDDS that can sustain the release of drugs and also enable utilization of hydrophilic sponges for the delivery of hydrophobic drugs.
Keywords: Self-microemulsifying drug delivery systems; Alginate; Solubility; Sponge; Solid;

New insights on how to adjust the release profile from coated pellets by varying the molecular weight of ethyl cellulose in the coating film by Mariagrazia Marucci; Helene Andersson; Johan Hjärtstam; Gary Stevenson; Julia Baderstedt; Mats Stading; Anette Larsson; Christian von Corswant (218-223).
The major aims of this work were to study the effect of the molecular weight (Mw) of ethyl cellulose (EC) on the drug release profile from metoprolol succinate pellets coated with films comprising EC and hydroxypropyl cellulose (HPC) with a weight ratio of 70:30, and to understand the mechanisms behind the different release profiles. A broad range of Mws was used, and the kinetics of drug release and HPC leaching followed. The higher the Mw of EC, the slower the HPC leaching and the drug release processes. Drug release occurred by diffusion through the pores created in the coating by the HPC leaching. A novel method was used to explain the differences in the release profiles: the effective diffusion coefficient (D e ) of the drug in the coating film was determined using a mechanistic model and compared to the amount of HPC leached. A linear dependence was found between D e and the amount of HPC leached and, importantly, the value of the proportionality constant decreased with increasing Mw of EC. This suggests that the Mw of EC affects the drug release profile by affecting the phase separated microstructure of the coating and the hindrance it imparts to drug diffusion.
Keywords: Ethyl cellulose; Molecular weight; Pellets; Diffusion; Phase separation; Mathematical modeling;

We designed novel niosomes based on alkyl glucopyranoside surfactants and containing methotrexate as anticancer drug, to be used in the pharmaceutical field. The effects of surfactants with chains of different length on niosome size and their distribution, drug entrapment efficiencies and in vitro drug release were determined. Systems made of alkyl glucopyranosides and cholesterol form vesicles whose average size scales with the alkyl chains length of such surfactants. Vesicles size ranges between 300 and 500 nm, with low polydispersity index. In addition, the hemolytic activity of alkyl glucopyranosides as surfactant solutions or vesicular formulations was studied and compared, to identify possible structure–activity relationships. High methotrexate entrapment efficiency was obtained, confirming significant interactions between the drug and the niosomal matrices. After 24 h the amount of methotrexate released from niosomal formulations is effectively delayed, compared to the free drug in solution. Hemolytic tests show that sugar-based surfactants are more hemolytic the longer is their alkyl chain. When the surfactants are in vesicular form, the reverse behavior holds. It was also inferred that vesicle formation reduces the surfactant toxicity. These niosomal formulations can be used as methotrexate delivery systems in anticancer therapy.
Keywords: Alkyl glucopyranoside surfactants; Niosomes; Methotrexate; Parenteral release; Hemolysis;

Polymer encapsulation of inorganic nanoparticles for biomedical applications by Rachid Ladj; Ahmad Bitar; Mohamed M. Eissa; Hatem Fessi; Yannick Mugnier; Ronan Le Dantec; Abdelhamid Elaissari (230-241).
Hybrid inorganic colloidal particles have attracted a great attention in the last years, and they have been largely used in various applications and more particularly in biomedical nanotechnology. Recently, they are used as carriers for biomolecules, and exploited for use in microsystems, microfluidics and in lab-on-a chip based bionanotechnology. Various kinds of hybrid particles can be listed starting from classical inorganic nanoparticles such as silica, gold, silver, iron oxide and those exhibiting intrinsic properties such as semiconducting nanoparticles (e.g. quantum dots). As a general tendency, to be conveniently used in biomedical applications, the encapsulation of the inorganic nanoparticles in a polymer matrix is incontestably needed. Consequently, various chemistry-based encapsulation processes have been developed and showed promising results as compared to the encapsulation using preformed polymers.
Keywords: Inorganic nanoparticles; Encapsulation; Polymer; Biomedical applications;