International Journal of Pharmaceutics (v.483, #1-2)

Display OmittedNanosimilars are considered as new medicinal outcomes combining the generic drugs and the nanocarrier as an innovative excipient, in order to evaluate them as final products. They belong to the grey area – concerning the evaluation process – between generic drugs and biosimilar medicinal products. Generic drugs are well documented and a huge number of them are in market, replacing effectively the off-patent drugs. The scientific approach for releasing them to the market is based on bioequivalence studies, which are well documented and accepted by the regulatory agencies. On the other hand, the structural complexity of biological/biotechnology-derived products demands a new approach for the approval process taking into consideration that bioequivalence studies are not considered as sufficient as in generic drugs, and new clinical trials are needed to support their approval process of the product to the market. In proportion, due to technological complexity of nanomedicines, the approaches for proving the statistical identity or the similarity for generic and biosimilar products, respectively, with those of prototypes, are not considered as effective for nanosimilar products. The aim of this note is to propose a complementary approach which can provide realistic evidences concerning the nanosimilarity, based on fractal analysis. This approach is well fit with the structural complexity of nanomedicines and smooths the difficulties for proving the similarity between off-patent and nanosimilar products. Fractal analysis could be considered as the approach that completely characterizes the physicochemical/morphological characteristics of nanosimilar products and could be proposed as a start point for a deep discussion on nanosimilarity.
Keywords: Biosimilars; Nanosimilars; Biotechnology-derived products; Fractal analysis; European Medicines Agency; Guidelines;

Formulation, stability and pharmacokinetics of sugar-based salmon calcitonin-loaded nanoporous/nanoparticulate microparticles (NPMPs) for inhalation by Maria Inês Amaro; Frederic Tewes; Oliviero Gobbo; Lidia Tajber; Owen I. Corrigan; Carsten Ehrhardt; Anne Marie Healy (6-18).
Display OmittedA challenge exists to produce dry powder inhaler (DPI) formulations with appropriate formulation stability, biological activity and suitable physicochemical and aerosolisation characteristics that provide a viable alternative to parenteral formulations. The present study aimed to produce sugar-based nanoporous/nanoparticulate microparticles (NPMPs) loaded with a therapeutic peptide – salmon calcitonin (sCT). The physicochemical properties of the powders and their suitability for pulmonary delivery of sCT were determined. Production of powders composed of sCT loaded into raffinose or trehalose with or without hydroxypropyl-β-cyclodextrin was carried out using a laboratory scale spray dryer. Spray dried microparticles were spherical, porous and of small geometric size (≤2 μm). Aerodynamic assessment showed that the fine particle fraction (FPF) less than 5 μm ranged from 45 to 86%, depending on the formulation. The mass median aerodynamic diameter (MMAD) varied between 1.9 and 4.7 μm. Compared to unprocessed sCT, sCT:raffinose composite systems presented a bioactivity of approximately 100% and sCT:trehalose composite systems between 70–90% after spray drying. Storage stability studies demonstrated composite systems with raffinose to be more stable than those containing trehalose. These sugar-based salmon calcitonin-loaded NPMPs retain reasonable sCT bioactivity and have micromeritic and physicochemical properties which indicate their suitability for pulmonary delivery. Formulations presented a similar pharmacokinetic profile to sCT solution. Hence the advantage of a dry powder formulation is its non-invasive delivery route and ease of administration of the sCT.
Keywords: Salmon calcitonin; Inhalation; Non-reducing sugars; Stability; Pharmacokinetics;

Polyethylene imine-6-phosphogluconic acid nanoparticles – a novel zeta potential changing system by Sonja Bonengel; Felix Prüfert; Glen Perera; Johanna Schauer; Andreas Bernkop-Schnürch (19-25).
Display OmittedThe aim of the study was to develop nanoparticles with the ability to change their zeta potential. By covalent attachment of 6-phosphogluconic acid to polyethylene imine, a charged, enzymatically removable moiety was introduced into the polymer. The novel conjugate displayed 400 μmol phosphate per gram polymer, as determined by malachite green assay. Studies evaluating the cleavage by intestinal alkaline phosphatase revealed that 69 % of the coupled phosphate could be released from the polymer. Furthermore, nanoparticles generated by polyelectrolyte complexation technique using carboxymethyl cellulose as negatively charged component exhibited a zeta potential of –6 mV and an average particle size of 300 nm. Enzymatic cleavage of the phosphate ester moiety by isolated intestinal alkaline phosphatase on these nanoparticles caused shift of the zeta potential from negative to positive value of +3 mV whereby 58 % of the total amount of phosphate were released. Studies on Caco-2 cells revealed the capability of a living system to hydrolyze the phosphate ester in the novel conjugate as well as on the nanoparticles via their intestinal alkaline phosphatase. Based on these results, polymeric nanoparticles comprising an enzymatically degradable phosphate ester moiety can provide a promising strategy for zeta potential changing systems.
Keywords: Nanoparticles; Mucus barrier; Zeta potential; Zeta potential changing systems; 6-Phosphogluconic acid; Intestinal alkaline phosphatase;

Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonum aviculare L. extract by Soon Sik Kwon; Sun Young Kim; Bong Ju Kong; Kyeong Jin Kim; Geun Young Noh; Na Ri Im; Ji Won Lim; Ji Hoon Ha; Junoh Kim; Soo Nam Park (26-37).
Display OmittedIn this study, Polygonum aviculare L. extract, which has superior antioxidative and cellular membrane protective activity, was loaded onto cell penetrating peptide (CPP) conjugated liposomes to enhance transdermal delivery. The physical characteristics of typical liposomes and CPP-conjugated liposomes containing P. aviculare extract were evaluated. The particle sizes of both liposomes were approximately 150 nm. Whereas the zeta potential of typical liposomes was −45 mV, that of CPP-conjugated liposomes was +42 mV. The loading efficiency of P. aviculare extract in both liposomes was calculated to be about 83%. Fluorescent-labeled liposomes were prepared to evaluate cellular uptake and skin permeation efficiency. Using flow cytometry, we found that CPP-conjugated liposomes improved cellular uptake of the fluorescent dye as compared with the typical liposomes. In addition, the skin permeation of CPP-conjugated liposomes was proved higher than that of typical liposomes by confocal laser scanning microscopy studies and Franz diffusion cell experiments. The improved cellular uptake and skin permeation of the CPP-conjugated liposomes were due to the cationic arginine-rich peptide. In vivo studies also determined that the CPP-conjugated liposomes were more effective in depigmentation and anti-wrinkle studies than typical liposomes. These results indicate that the CPP-conjugated liposomes could be effective for transdermal drug delivery of antioxidant and anti-aging therapeutics.
Keywords: Cell penetrating peptide; Liposome; Transdermal delivery; Polygonum aviculare L.; Depigmentation; Anti-wrinkle;

Display OmittedTumor-targeted drug delivery and microenvironment-responsive drug release are attractive strategies in cancer treatment. Our previous study demonstrated that redox-sensitive micelles based on hyaluronic acid-deoxycholic acid (HA-ss-DOCA) conjugates exhibited excellent drug-loading capacities (34.1%) for paclitaxel (PTX) and rapid drug release in response to reducing agent, glutathione. In the present study, the physicochemical and biological properties of PTX-loaded HA-ss-DOCA (PTX-HA-ss-DOCA) micelles were investigated further. The micelles have an average size of about 120 nm and a zeta potential of about −36 mV. Transmission electron microscopy and wide-angle X-ray diffraction analysis demonstrated redox-sensitive degradation of micelles in the presence of glutathione. Moreover, the encapsulated payload was effectively released from HA-ss-DOCA micelles into cytoplasm and then rapidly transported into nuclei. In vitro cytotoxicity and cell apoptosis assay further revealed that HA significantly improved the tumor-specific drug delivery of HA-ss-DOCA micelles via receptor-mediated endocytosis, while efficient intracellular drug release and transportation lead to marked inhibition of tumor cell growth, as compared to Taxol® and insensitive micelles. More importantly, PTX-HA-ss-DOCA micelles demonstrated superior in vivo antitumor activity compared with Taxol® and insensitive control, and decreased systemic toxicity. Herein we present data which provide valuable insight into the design and development of tumor-specific drug delivery systems.
Keywords: Paclitaxel; Redox-sensitive; Hyaluronic acid; Polymeric micelle; Drug delivery; Antitumor;

A newly developed lubricant, chitosan laurate, in the manufacture of acetaminophen tablets by Ahmad Bani-Jaber; Asuka Kobayashi; Kyohei Yamada; Dana Haj-Ali; Takeaki Uchimoto; Yasunori Iwao; Shuji Noguchi; Shigeru Itai (49-56).
Display OmittedTo study the usefulness of chitosan laurate (CS-LA), a newly developed chitosan salt, as a lubricant, lubrication properties such as the pressure transmission ratio and ejection force were determined at different concentrations of CS-LA in tableting. In addition, tablet properties such as the tensile strength, disintegration time, and dissolution behavior, were also determined. When CS-LA was mixed at concentrations of 0.1%–3.0%, the pressure transmission ratio was increased in a concentration-dependent manner, and the value at a CS-LA concentration of 3% was equal to that of magnesium stearate (Mg-St), a widely used lubricant. Additionally, a reduction in the ejection force was observed at a concentration from 1%, proving that CS-LA has good lubrication performance. A prolonged disintegration time and decreased tensile strength, which are known disadvantages of Mg-St, were not observed with CS-LA. Furthermore, with CS-LA, retardation of dissolution of the drug from the tablets was not observed. Conjugation of CS with LA was found to be quite important for both lubricant and tablet properties. In conclusion, CS-LA should be useful as an alternative lubricant to Mg-St.
Keywords: Chitosan laurate; Magnesium stearate; Lubricant properties; Tablet characteristics; Tableting;

Preferential targeting of human erythrocytes infected with the malaria parasite Plasmodium falciparum via hexose transporter surface proteins by Kajal Devi Heikham; Ankit Gupta; Ambrish Kumar; Chandan Singh; Juhi Saxena; Kumkum Srivastava; Sunil K. Puri; Anil K. Dwivedi; Saman Habib; Amit Misra (57-62).
Display OmittedGlucose uptake by Plasmodium-infected erythrocytes (RBC) is higher compared to uninfected RBC. Glucose is transported across the cell membrane by transporter proteins. Particles of median size 146.3 ± 18.7 nm, containing anti-malarial agents in corn starch were prepared for investigating: (a) whether the glucose moiety in starch targets RBC via hexose transporter(s), (b) whether there are differences in the extent of targeting to uninfected RBC versus infected RBC (iRBC) in view of higher cell surface density of these proteins on iRBC and (c) whether targeting provides enhanced efficacy against P. falciparum in comparison to drugs in solution. Binding of these particles to RBC was target-specific, since it could be blocked by phloretin, an inhibitor of glucose transporters (GLUT), or competed out in a dose-dependent manner with d-glucose in a flow cytometry assay. Significant (P=  0.048, t-test) differences in extent of targeting to iRBC versus RBC were observed in flow cytometry. CDRI 97/63 incorporated in particles was 63% more efficacious than its solution at 250 ng/ml, while quinine was 20% more efficacious at 6.25 ng/ml in a SYBR Green incorporation assay. Preferential targeting of iRBC using an inexpensive excipient promises advantages in terms of dose reduction and toxicity alleviation.
Keywords: Starch; Nanoparticles; GLUT1; PfHT-1; Malaria; Flow cytometry quinine; Trioxane;

Biological stimuli-responsive cyclodextrin-based host–guest nanosystems for cancer therapy by Zhaoling Dan; Haiqiang Cao; Xinyu He; Lijuan Zeng; Lili Zou; Qi Shen; Zhiwen Zhang (63-68).
Display OmittedStimuli-responsive nanosystems are of particular interest in cancer therapy, owing to their impressive capability to enable the on-demand drug release in response to specific biological stimuli in tumor microenvironments (such as pH, redox and enzyme, etc.). Cyclodextrin (CD)-based host–guest interactions provide a flexible and powerful platform for the development of multifunctional nanosystems. This article highlights the current progress of CD-based host–guest nanosystems (CHNs) with biological stimuli-responsive properties in cancer therapy. We summarize the composition, structure and design of various CHNs in response to specific stimuli in tumor, and focus on their performance in controlled drug delivery and cancer therapy. These recent advances make it a promising and intelligent drug delivery system to improve the anticancer efficacy.
Keywords: Stimuli-responsive; Cyclodextrin; Host–guest; Nanosystem; Drug delivery; Cancer therapy;

Display OmittedDrug association with isolated natural chylomicrons (nCM) can be used to predict the lymphatic transportation potential of highly lipophilic drugs. However, the nCM model is compromised by inter-group variations in isolated nCM samples and the need to sacrifice a large quantity of animals. In this study, reassembled chylomicrons (rCM) model was set up and evaluated with respect to mimicking the drug association capacity of nCMs. A thin-film dispersion method was used to prepare rCMs, whose compositions consisted of triglycerides, phospholipids, cholesterols and derivatives in a ratio similar to that of nCMs. Partial least squares (PLS) analysis was used to evaluate the influence of molecular descriptors on drug association with CMs and establish multivariable regression equations for prediction of drug association. Chemical descriptors affecting drug association with nCM are in the sequence of hydrogen binding acceptors (HBA) > polar surface area (PSA) > solubility in long-chain triglycerides (S LCT) > logP > melting point (MP) > logD > molar volume (MV) > density > pK a  > molar weight (MW) > freely rotatable bonds (FRB) > hydrogen binding donors (HBD). HBA, PSA, HBD, MP, density, pK a, FRB, and HBD were found to reduce the degree of drug association with nCM, whereas all other descriptors increased it. Sequences of chemical descriptors affecting drug association with rCM was in the order of pK a  >  S LCT  > FRB > HBA > MW > MV > HBD > logP > MP > PSA > logD > density. However, the degree of drug association with nCMs was closely correlated to that with rCMs. Drug association with both CMs could be predicted using pre-established equations and PLS. In conclusion, rCMs could be used as substitute for nCMs in prediction of lymphatic transportation of highly lipophilic drugs.
Keywords: Lymphatic transportation; Molecular descriptor; Chylomicrons; Reassembled; Oral;

Display OmittedPravastatin sodium (PVS) is a hydrophilic HMG-CoA reductase inhibitor that is mainly absorbed from duodenum. PVS has a short elimination half-life (1–3 h), suffers from instability at gastric pH, extensive hepatic first-pass metabolism and low absolute bioavailability (18%). The current work aimed to develop enteric surface-coated spanlastic dispersions as controlled-release duodenum-triggered systems able to surmount PVS drawbacks. PVS-loaded spanlastic dispersions were prepared by ethanol-injection method using span® 60. Tween® 60 and Tween® 80 were explored as edge activators. As a novel approach, the fine spanlastic dispersions were surface-coated with an enteric-polymer (Eudragit® L100-55) via freeze-drying. The systems were evaluated, before and after enteric-coating, for particle size, zeta potential, PVS entrapment efficiency (EE%), morphology and PVS release studies. PVS pharmacokinetics from the best achieved system and an aqueous solution were estimated in rats by UPLC–MS/MS. The best achieved enteric surface-coated spanlastic dispersion (E-S6) displayed spherical nanosized vesicles (647.60 nm) possessing negative zeta potential (−6.93 mV), promising EE% (63.22%) and a biphasic drug-release pattern characterized by a retarded-release phase (0.1 N HCl, 2 h) and a controlled-release phase (pH 6.8, 10 h). The higher C max, delayed T max, prolonged MRT(0−), longer elimination t 50% and enhanced oral bioavailability unravel E-S6 potential for oral PVS delivery.
Keywords: Duodenum-triggering; Pravastatin sodium; Spanlastic dispersions; Ethanol injection method; Enteric surface-coating; UPLC–MS/MS;

A polymeric prodrug of cisplatin based on pullulan for the targeted therapy against hepatocellular carcinoma by Yan Wang; Yuanyuan Liu; Yang Liu; Wen Zhou; Hemei Wang; Guoyun Wan; Duxin Sun; Ning Zhang; Yinsong Wang (89-100).
Display OmittedA polymeric prodrug of cisplatin (CP) with simple chemical structure was synthesized based on pullulan and its therapeutic effects on human hepatocellular carcinoma (HCC) were studied in vitro and in vivo. Briefly, CP was linked to pullulan monosuccinate (SUPA) via coordination bond to form prodrug of CP-SUPA with the CP weight content of 18.7%. CP-SUPA exhibited in vitro sustained releases and strong interactions with genomic DNA. CP-SUPA could effectively inhibit the proliferation of HCC HepG2 cells by promoting the cell apoptosis and arresting the cell cycle, and showed a significantly higher cytotoxicity on HepG2 cells than that on human lung epithelial A549 cells. Compared to free CP, CP-SUPA obviously prolonged the survival times of Balb/c mice after intravenous injection at CP doses of 15 and 30 μmol/kg, and no signs of toxic effects in main tissues were detected by histologic examination. In MHCC-97H tumor-bearing nude mice, CP-SUPA was mainly distributed in the liver and the tumor after 24 h post administration and evidently inhibited the tumor growth at CP doses of 3.5 and 7.0 μmol/kg. In conclusion, CP-SUPA was an ideal candidate of polymeric prodrug for the targeted therapy against HCC.
Keywords: Cisplatin; Pullulan; Prodrug; Hepatocellular carcinoma; Targeted therapy;

Display OmittedWe established an extremely high-shear melt pelletization technique using a mechanical powder processor to produce high-dose granules smaller than 300 μm with properties suitable for film coating. A mixture of ethenzamide and polyethylene glycol (used as a low-melting binder) at various weight ratios was mechanically treated under various jacket temperatures. When the jacket temperature was set to 50 °C or greater, the product temperature reached the melting point of the binder, resulting in pelletization. The drug powder were pelletized with a small amount of binder to yield pellets of approximately 150 μm with a drug content of more than 90%. The mechanism of melt pelletization through ultrahigh shearing involves a series of nucleation, consolidation, coalescence and breakage stages. The power consumption profile corresponding to each stage in the pelletization revealed that pellets between 75 and 300 μm were effectively obtained at a large power consumption peak. The resultant pellets showed comparative sphericity and smoothness, and higher durability than commercial core granules for film coating. In conclusion, this study demonstrates that the extremely high-shear melt pelletization technique can give drug pellets with desirable properties as core particles for the coating process.
Keywords: Mechanical powder processing; Melt pelletization; Extremely high shear; High drug content; Core granules; Polyethylene glycol;

Anti-cancer vaccination by transdermal delivery of antigen peptide-loaded nanogels via iontophoresis by Mao Toyoda; Susumu Hama; Yutaka Ikeda; Yukio Nagasaki; Kentaro Kogure (110-114).
Display OmittedTransdermal vaccination with cancer antigens is expected to become a useful anti-cancer therapy. However, it is difficult to accumulate enough antigen in the epidermis for effective exposure to Langerhans cells because of diffusion into the skin and muscle. Carriers, such as liposomes and nanoparticles, may be useful for the prevention of antigen diffusion. Iontophoresis, via application of a small electric current, is a noninvasive and efficient technology for transdermal drug delivery. Previously, we succeeded in the iontophoretic transdermal delivery of liposomes encapsulating insulin, and accumulation of polymer-based nanoparticle nanogels in the stratum corneum of the skin. Therefore, in the present study, we examined the use of iontophoresis with cancer antigen gp-100 peptide KVPRNQDWL-loaded nanogels for anti-cancer vaccination. Iontophoresis resulted in the accumulation of gp-100 peptide and nanogels in the epidermis, and subsequent increase in the number of Langerhans cells in the epidermis. Moreover, tumor growth was significantly suppressed by iontophoresis of the antigen peptide-loaded nanogels. Thus, iontophoresis of the antigen peptide-loaded nanogels may serve as an effective transdermal delivery system for anti-cancer vaccination.
Keywords: Iontophoresis; Anti-cancer vaccine; Transdermal delivery; Nanogel;

Controlled release of titanocene into the hybrid nanofibrous scaffolds to prevent the proliferation of breast cancer cells by Ashang Luwang Laiva; Jayarama Reddy Venugopal; Priyadharsini Karuppuswamy; Balchandar Navaneethan; Aleksander Gora; Seeram Ramakrishna (115-123).
Display OmittedElectrospun hybrid nanofibrous scaffolds have gained much importance in the field of tissue engineering and drug delivery applications owing to its multifaceted properties. In this study, the properties of composite polycaprolactone (PCL)/silk fibroin (SF) nanofibrous scaffolds was investigated as a potential scaffold for cell growth and also a drug eluting mat to control the proliferation of MCF-7 cells. Titanocene dichloride was chosen as the model drug to study its antitumor efficacy on MCF-7 cell lines. Fascinating properties relating to crystallization of silk fibroin and binding of drug has also been discussed for the controlled release of drugs. The presence of amino acid residues in silk fibroin plays a big role in the cell-scaffold interaction, the nature of drug binding and also its release characteristics to control the cell proliferation. Studies on material properties for the hybrid nanofibrous scaffolds showed interrelated changes in fiber diameter and mechanical behavior for the drug loaded nanofibers. Significant decrease in fiber diameters from 352 ± 52 nm to 281 ± 44.5 nm and sharp increase in tensile stress from 4.5 MPa to 50.3 MPa was observed for 0.03% drug loaded scaffolds with respect to PCL fibers. Cell viability and cell morphology study was performed to analyze the effect of different concentrations of titanocene dichloride loaded on PCL/silk fibroin nanofibrous scaffolds. Maximum cell viability inhibition percentage of change 26.93% was obtained for 0.03% titanocene with respect to 0.01% on day 3. The obtained results proved that the drug loaded hybrid mat to control the proliferation of MCF-7 cells at different time points and serve as a model for cancer therapy.
Keywords: Nanofibers; Silk fibroin; Titanocene dichloride; Anti-tumor activity;

Combined delivery of the adiponectin gene and rosiglitazone using cationic lipid emulsions by Enkhzaya Davaa; Bong-Seok Kang; Joo-Hui Han; Sang-Eun Lee; Choon Lian Ng; Chang-Seon Myung; Jeong-Sook Park (124-130).
Display OmittedFor the combined delivery of an insulin-sensitizing adipokine; i.e., the ADN gene, and the potent PPARγ agonist rosiglitazone, cationic lipid emulsions were formulated using the cationic lipid DOTAP, helper lipid DOPE, castor oil, Tween 20 and Tween 80. The effect of drug loading on the physicochemical characteristics of the cationic emulsion/DNA complexes was investigated. Complex formation between the cationic emulsion and negatively charged plasmid DNA was confirmed and protection from DNase was observed. The in vitro transfection efficiency and cytotoxicity were evaluated in HepG2 cells. The particle sizes of the cationic emulsion/DNA complex were in the range 230–540 nm and those of the rosiglitazone-loaded cationic emulsion/DNA complex were in the range 220–340 nm. Gel retardation of the complexes was observed when the complexation weight ratios of the cationic lipid to plasmid DNA exceeded 4:1 for both the drug-free and rosiglitazone-loaded complexes. Both complexes stabilized plasmid DNA against DNase. The ADN expression level increased dose-dependently when cells were transfected with the cationic emulsion/DNA complexes. The rosiglitazone-loaded cationic emulsion/DNA complexes showed higher cellular uptake in HepG2 cells depending on the rosiglitazone loading, but not depending on the type of plasmid DNA type such as pVAX/ADN, pCAG/ADN, or pVAX. The drug-loaded cationic emulsion/plasmid DNA complexes were less cytotoxic than free rosiglitazone. Therefore, a cationic emulsion could potentially serve as a co-delivery system for rosiglitazone and the adiponectin gene.
Keywords: Cationic emulsion; Gene delivery; Type 2 diabetes; Adiponectin; Rosiglitazone;

Stability of collapse lyophilized influenza vaccine formulations by Cihad Anamur; Gerhard Winter; Julia Engert (131-141).
Display OmittedA clear limitation of many liquid vaccines is the obligatory cold-chain distribution system. Therefore, distribution of a dried vaccine formulation may be beneficial in terms of vaccine stability, handling and transport. Collapse freeze-drying is a process which utilizes fairly aggressive but at the same time economic lyophilization cycles where the formulation is dried above its glass transition temperature.In this study, we used collapse freeze-drying for a thermosensitive model influenza vaccine (Pandemrix®). The dried lyophilizates were further cryo-milled to engineer powder particles in the size range of approximately 20–80 μm which is applicable for epidermal powder immunization.Vaccine potency and stability were neither affected by high temperature input during collapse lyophilization nor over a storage period of six months. Furthermore, cryo-milled vaccine lyophilizates showed good storage stability of up to three months at high storage temperature (40 °C).This technique can provide a powerful tool for the worldwide distribution of vaccine and for new application technologies such as engineered powder immunization.
Keywords: Powder immunization; Vaccination; Influenza vaccine; Collapse lyophilization; Cryo-milling;

Display OmittedA high drug-loading capacity is a critical factor for the clinical development of liposomal formulations. The accommodation of hydrophobic drugs within the liposomal membrane is often limited in saturated phosphatidylcholine (PC)-based liposomes owing to the rigidity of the lipid acyl chain. In the current study, we explored the possibility of improving the hydrophobic drug loading capacity of liposomes by incorporating triglyceride into liposomal membranes. Incorporation of Captex 300, a medium chain triglyceride, into liposomes composed of dimyristoylphosphatidylcholine and cholesterol greatly increased the fluidity and lamellarity of the resultant liposomes. Liposomal incorporation of medium or long chain, but not short chain, triglycerides greatly enhanced the concentration of loaded paclitaxel (PTX) in saturated PC-based liposomes. The enhancing effect of triglyceride saturated at a triglyceride content corresponding to the amount required to fluidize the liposome structure. In addition, the enhancing effect was not observed in unsaturated PC-based liposomes and was not associated with the solubility of PTX in each triglyceride. Triglycerides also enhanced the loading of docetaxel, another hydrophobic drug. Taken together, our results suggest that triglyceride incorporation in saturated PC-based liposomes provide an improved dosage form that enables increased hydrophobic drug loading by altering the fluidity and structure of liposomal membranes.
Keywords: Liposome; Multilamellar vesicle; Hydrophobic drug; Triglyceride; Fluidity;

Display OmittedMany natural products have the potential to modulate the dermal penetration of topically applied drugs and chemicals. We studied the effect of five natural compounds (hydroxycitronellal, limonene 1,2-epoxide, terpinyl acetate, p-coumaric acid, transferrulic acid) and ethanol on the transdermal penetration of two marker drugs (14C-caffeine and 14C-salicylic acid) in a flow through in vitro porcine skin diffusion system. The parameters of flux, permeability, diffusivity, and percent dose absorbed/retained were calculated and compared. The dermal absorption of 14C-caffeine was significantly higher with terpinyl acetate and limonene 1,2-epoxide as compared to ethanol; while dermal absorption of 14C-salicylic acid was significantly greater with hydroxycitronellal and limonene 1,2-epoxide as compared to ethanol. A 10-fold increase in flux and permeability of caffeine with terpinyl acetate was observed while limonene increased flux of caffeine by 4-fold and permeability by 3-fold. Hydroxycitronellal and limonene increased salicylic acid’s flux and permeability over 2-fold. The other natural compounds tested did not produce statistically significant effects on dermal penetration parameters for both caffeine and salicylic acid (p  ≥ 0.05). These results emphasize the differential effects of natural substances on the transdermal penetration of hydrophilic (caffeine) and hydrophobic (salicylic acid) drugs.
Keywords: Natural substance; Skin; Penetration; Drugs;

A novel biocompatible hyaluronic acid–chitosan hybrid hydrogel for osteoarthrosis therapy by S. Kaderli; C. Boulocher; E. Pillet; D. Watrelot-Virieux; A.L. Rougemont; T. Roger; E. Viguier; R. Gurny; L. Scapozza; O. Jordan (158-168).
Display OmittedA conventional therapy for the treatment of osteoarthrosis is intra-articular injection of hyaluronic acid, which requires repeated, frequent injections. To extend the viscosupplementation effect of hyaluronic acid, we propose to associate it with another biopolymer in the form of a hybrid hydrogel. Chitosan was chosen because of its structural similarity to synovial glycosaminoglycans, its anti-inflammatory effects and its ability to promote cartilage growth.To avoid polyelectrolyte aggregation and obtain transparent, homogeneous gels, chitosan was reacetylated to a 50% degree, and different salts and formulation buffers were investigated. The biocompatibility of the hybrid gels was tested in vitro on human arthrosic synoviocytes, and in vivo assessments were made 1 week after subcutaneous injection in rats and 1 month after intra-articular injection in rabbits.Hyaluronic acid–chitosan polyelectrolyte complexes were prevented by cationic complexation of the negative charges of hyaluronic acid. The different salts tested were found to alter the viscosity and thermal degradation of the gels. Good biocompatibility was observed in rats, although the calcium-containing formulation induced calcium deposits after 1 week. The sodium chloride formulation was further tested in rabbits and did not show acute clinical signs of pain or inflammation. Hybrid HA–Cs hydrogels may be a valuable alternative viscosupplementation agent.
Keywords: Hyaluronic acid; Chitosan; Hybrid hydrogel; Viscosupplementation; Biopolymers; Biocompatibility;

Display OmittedA new CD44-targeted gene delivery system, the star-shaped cationic polymer containing a β-cyclodextrin (β-CD) core and multiple branched oligoethylenimine (OEI) arms with conjugated oligomer of hyaluronic acid (HA), was synthesized by reductive amination between β-CD-OEI star polymer and HA, and was characterized for pDNA condensation and nanoparticle formation, followed by evaluation for targeted gene delivery of luciferase reporter gene and wild type p53 gene in CD44-positive and CD44-negative cell lines. The β-CD-OEI-HA polymer contained 6 arms of OEI (600 Da) and a short HA segment. It could fully condense pDNA to form nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The conjugation of HA reduced cytotoxicity of β-CD-OEI-HA/pDNA polyplexes. It was found that CD44 receptor was highly expressed and localized at the membrane of MDA-MB-231 breast cancer cell line, while no CD44 was found at the membrane of MCF-7 epithelial cell line. Compared with PEI (25 kDa) and β-CD-OEI star polymers, β-CD-OEI-HA demonstrated significant increased gene transfection efficiency in MDA-MB-231 cells, while such effect was absent in MCF-7 cells. The targeted delivery of wild type p53 gene by β-CD-OEI-HA in MDA-MB-231 cells resulted in an increased cell cycle arrest at sub-G1 phase.
Keywords: Cyclodextrin; Oligoethylenimine; Hyaluronic acid; CD44 receptor; Targeted gene delivery;

Bovine serum albumin nanoparticles for delivery of tacrolimus to reduce its kidney uptake and functional nephrotoxicity by Lei Zhao; Yanxia Zhou; Yajie Gao; Shujin Ma; Chao Zhang; Jinwen Li; Dishi Wang; Xueping Li; Chengwei Li; Yan Liu; Xinru Li (180-187).
Display OmittedThe purpose of the present study was to develop a new nanoparticulate formulation for delivery of tacrolimus to reduce its kidney distribution and functional nephrotoxicity. Tacrolimus (TAC)-loaded bovine serum albumin (BSA) nanoparticles (TAC-BSA-NPs) were prepared by emulsification-dispersion technique. The obtained TAC-BSA-NPs, with 189.50 ± 7.15 nm of diameter and −20.86 ± 0.45 mV of Zeta potential determined by DLS, were spherical in shape observed by TEM. The drug loading content and encapsulation efficiency were (1.7 ± 0.13)% and (85 ± 3.0)%, respectively. The in vitro release of TAC-BSA-NPs exhibited biphasic drug release pattern with an initial burst release and subsequently sustained release. Pharmacokinetic analysis displayed that TAC-BSA-NPs could enhance the drug blood level and prolong the circulation time in comparison to Prograf®. Meanwhile, compared with Prograf®, TAC-BSA-NPs could deliver less TAC to kidney and simultaneously reduce the functional nephrotoxicity of TAC to kidney. In conclusion, BSA nanoparticles might be a more safe carrier for delivery of hydrophobic drug TAC.
Keywords: Tacrolimus; Bovine serum albumin nanoparticles; Pharmacokinetics; Nephrotoxicity; Tissue distribution;

Zirconium phosphatidylcholine-based nanocapsules as an in vivo degradable drug delivery system of MAP30, a momordica anti-HIV protein by Guo Caizhen; Gao Yan; Chang Ronron; Yang Lirong; Chu Panpan; Hu Xuemei; Qiao Yuanbiao; Li Qingshan (188-199).
A schemed strategy on an essential drug delivery system of a momordica anti-HIV protien, MAP30, through the nanoencapsulation in layered zirconium phosphatidylcholine matrices realized a strong structure retention, a lowered toxicity and a prolonged in vivo life of the protein.Display OmittedAn essential in vivo drug delivery system of a momordica anti-HIV protein, MAP30, was developed through encapsulating in chemically synthesized matrices of zirconium egg- and soy-phosphatidylcholines, abbreviated to Zr/EPC and Zr/SPC, respectively. Matrices were characterized by transmission electron microscopy and powder X-ray diffractometry studies. Zr/EPC granule at an approximate diameter of 69.43 ± 7.78 nm was a less efficient encapsulator than the granule of Zr/SPC. Interlayer spacing of the matrices encapsulating MAP30 increased from 8.8 and 9.7 Å to 7.4 and 7.9 nm, respectively. In vivo kinetics on degradation and protein release was performed by analyzing the serum sampling of intravenously injected SPF chickens. The first order and biphasic variations were obtained for in vivo kinetics using equilibrium dialysis. Antimicrobial and anti-HIV assays yielded greatly decreased MIC50 and EC50 values of nanoformulated MAP30. An acute toxicity of MAP30 encapsulated in Zr/EPC occurred at a single intravenous dose above 14.24 mg/kg bw in NIH/KM/ICR mice. The folding of MAP30 from Zr/EPC sustained in vivo chickens for more than 8 days in high performance liquid chromatography assays. These matrices could protect MAP30 efficiently with strong structure retention, lowered toxicity and prolonged in vivo life.
Keywords: In vivo degradation and release; Nanoencapsulation; Therapeutic anti-HIV protein; Zirconium phosphatidylcholine; Protein structure stability;

Display OmittedNear-infrared chemical imaging (NIR-CI) is an attractive technique within the pharmaceutical industry, where tools are continuously in demand to assess the quality of the intermediate and final products. The present paper demonstrates how NIR-CI in combination with multivariate methods was utilized to spatially map physical properties and content of roll compacted ribbons and tablets. Additionally, extracted textural parameters from tablet images were correlated to the design parameters of the roll compaction process as well as to the physical properties of the granules. The results established the use of NIR-CI as a complementary nondestructive tool to determine the ribbon density and map the density distribution across the width and along the length of the ribbons. For the tablets, the compaction pressure developed during compression increased with the lateral distance from the center. Therefore, NIR-CI can be an effective tool to provide information about the spatial distribution of the compaction pressures on the surface of the tablet. Moreover, low roll compaction roll force correlated to a heterogeneous type of texture in the API chemical image. Overall, texture analysis of the tablets enabled efficient investigation of the spatial variation and could be used to advance process understanding. Finally, orthogonal projections to latent structures (O2PLS) model facilitated the understanding of the interrelationships between textural features, design parameters and physical properties data by separately joint and unique variations.
Keywords: Near-infrared chemical imaging; Roll compaction; Orthogonal projections to latent structures (O2PLS); Grey-level co-occurrence matrices; Texture analysis;

Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative by Giovanna Lollo; Pablo Hervella; Pilar Calvo; Pablo Avilés; Maria Jose Guillén; Marcos Garcia-Fuentes; Maria José Alonso; Dolores Torres (212-219).
Display OmittedThe focus of this study is to disclose a new delivery carrier intended to improve the pharmacokinetic characteristics of the anticancer drug plitidepsin and to favor its accumulation within the tumor. These nanocarriers named as nanocapsules, consist of an oily core surrounded by a highly PEGylated polyglutamic acid (PGA-PEG) shell loaded with plitidepsin. They showed a size of around 190 nm, a zeta potential of −24 mV and were able to encapsulate a high percentage (85%) of plitidepsin. In vivo studies, following intravenous injection in healthy mice, indicated that the encapsulation of the drug within PGA-PEG nanocapsules led to an important increase in its area under the curve (AUC) which is related to the important decrease of the clearance, as compared to the values observed for the drug dissolved in a Cremophor® EL solution. This improvement of the pharmacokinetic profile of the encapsulated plitidepsin was accompanied by a high increase (2.5-fold) of the maximum tolerated dose (MTD) in comparison to that of plitidepsin Cremophor® EL solution. The efficacy study performed in a xenograft tumor mice model evidenced the capacity of PGA-PEG nanocapsules to significantly reduce tumor growth. These promising results highlight the potential of PGA-PEG nanocapsules as an effective drug delivery system for cancer therapy.
Keywords: Nanomedicine; Antitumor drugs; Stealth properties; Polyglutamic acid; Plitidepsin; Cancer therapy;

Essential oils: From extraction to encapsulation by A. El Asbahani; K. Miladi; W. Badri; M. Sala; E.H. Aït Addi; H. Casabianca; A. El Mousadik; D. Hartmann; A. Jilale; F.N.R. Renaud; A. Elaissari (220-243).
Display OmittedEssential oils are natural products which have many interesting applications. Extraction of essential oils from plants is performed by classical and innovative methods. Numerous encapsulation processes have been developed and reported in the literature in order to encapsulate biomolecules, active molecules, nanocrystals, oils and also essential oils for various applications such as in vitro diagnosis, therapy, cosmetic, textile, food etc. Essential oils encapsulation led to numerous new formulations with new applications. This insures the protection of the fragile oil and controlled release. The most commonly prepared carriers are polymer particles, liposomes and solid lipid nanoparticles.
Keywords: Essential oil; Extraction; Encapsulation; Polymer; Liposomes; Particles;

Hyperspectral imaging in quality control of inkjet printed personalised dosage forms by Hossein Vakili; Ruzica Kolakovic; Natalja Genina; Mathieu Marmion; Harri Salo; Petri Ihalainen; Jouko Peltonen; Niklas Sandler (244-249).
Display OmittedThe aim of the study was to investigate applicability of near infra-red (NIR) hyperspectral imaging technique in quality control of printed personalised dosage forms. Inkjet printing technology was utilized to fabricate escalating doses of an active pharmaceutical ingredient (API). A solution containing anhydrous theophylline as the model drug was developed as a printable formulation. Single units solid dosage forms (SDFs) were prepared by jetting the solution onto 1 cm × 1 cm areas on carrier substrate with multiple printing passes. It was found that the number of printing passes was in excellent correlation (R 2  = 0.9994) with the amount of the dispensed drug (μg cm−2) based on the UV calibration plot. The API dose escalation was approximately 7.5 μg cm−2 for each printing pass concluding that inkjet printing technology can optimally provide solutions to accurate deposition of active substances with a potential for personalized dosing. Principal component analysis (PCA) was carried out in order to visualize the trends in the hyperspectral data. Subsequently, a quantitative partial least squares (PLS) regression model was created. NIR hyperspectral imaging proved (R 2  = 0.9767) to be a reliable, rapid and non-destructive method to optimize quality control of these planar printed dosage forms.
Keywords: Inkjet printing; Personalized medicine; Cellulose carrier substrates; Near infrared hyperspectral imaging; Quality control; Multivariate analysis;

Display OmittedThe effect of chitosan on the intestinal absorption of cyanocobalamin (VB12), a stable form of vitamin B12, was investigated in vivo in rats, with the aim of improving the oral bioavailability of VB12 for anemia treatment in patients with gastrectomy. The bioavailability was evaluated based on the plasma concentration profile of VB12 following intraintestinal administration of the VB12 solution containing chitosan at various concentrations. The bioavailability of VB12 was 0.6 ± 0.2% when the chitosan-free VB12 solution was administered, while it increased to 10.5 ± 3.3% when chitosan was dissolved in the VB12 solution at a concentration of 1%. The bioavailability of VB12 increases with the chitosan concentration, in which chitosan seems to augment the amount of VB12 absorbed without affecting the absorption rate constant of VB12. It was also shown that the bioavailability of VB12 does not increase further when the degree of chitosan deacetylation is increased from 83 to 100% by substitutively employing the fully deacetylated chitosan. These findings suggest that the oral administration of VB12 with readily available chitosan may be a practical approach for anemia treatment in patients with gastrectomy.
Keywords: Anemia; Bioavailability; Chitosan; Cyanocobalamin; Gastrectomy; Vitamin B12;

Identifying the mechanisms of drug release from amorphous solid dispersions using MRI and ATR-FTIR spectroscopic imaging by Kateřina Punčochová; Andrew V. Ewing; Michaela Gajdošová; Nina Sarvašová; Sergei G. Kazarian; Josef Beránek; František Štěpánek (256-267).
Display OmittedThe dissolution mechanism of a poorly aqueous soluble drug from amorphous solid dispersions was investigated using a combination of two imaging methods: attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopic imaging and magnetic resonance imaging (MRI). The rates of elementary processes such as water penetration, polymer swelling, growth and erosion of gel layer, and the diffusion, release and in some cases precipitation of drug were evaluated by image analysis. The results from the imaging methods were compared with drug release profiles obtained by classical dissolution tests. The study was conducted using three polymeric excipients (soluplus, polyvinylpyrrolidone – PVP K30, hydroxypropylmethyl cellulose – HPMC 100M) alone and in combination with a poorly soluble drug, aprepitant. The imaging methods were complementary: ATR-FTIR imaging enabled a qualitative observation of all three components during the dissolution experiments, water, polymer and drug, including identifying structural changes from the amorphous form of drug to the crystalline form. The comparison of quantitative MRI data with drug release profiles enabled the different processes during dissolution to be established and the rate-limiting step to be identified, which – for the drug–polymer combinations investigated in this work – was the drug diffusion through the gel layer rather than water penetration into the tablet.
Keywords: FT-IR spectroscopy; Magnetic resonance imaging; Solid dispersion; Dissolution rate; Water penetration; Spray drying;

Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions by Anna Lechanteur; Tania Furst; Brigitte Evrard; Philippe Delvenne; Pascale Hubert; Géraldine Piel (268-277).
Display OmittedCervical cancer induced by human papillomavirus (HPV) is the fourth highest mortality causing cancer in women despite the use of prophylactic vaccines. E6 targeting represents an attractive strategy to treat this cancer. Indeed, oncoprotein E6 is produced by keratinocytes infected by HPV and is partially responsible for carcinogenesis. E6 interferes with the apoptosis process in stressed cells by degradation of p53 tumor suppressor gene. Our strategy consists in using E6 siRNA complexed with pegylated lipoplexes. The addition of hydrophilic polymer around the nanoparticles is crucial to use them by vaginal application on account of cervicovaginal mucus. Physicochemical characteristics were evaluated and in vitro assays were performed to evaluate transfection potential, E6 mRNA extinction and p53 re-expression. Cationic liposomes DOTAP/Cholesterol/DOPE 1/0.75/0.5 (N/P 2.5) with or without 50% DSPE-PEG2000 and associated with siE6 have demonstrated good physicochemical characteristics in terms of complexation, size, surface charge and stability. Both lipoplexes have been tested on CaSki cell line (HPV 16+) with 50 nM and 100 nM of siE6. Lipoplexes formulations induce 30–40% of E6 mRNA extinction and induce the re-expression of p53. In conclusion, pegylated anti-E6 lipoplexes have demonstrated their efficiency to cross the cellular membrane and to release siRNA into the cytoplasm confirmed by final p53 protein production.
Keywords: Liposomes; siRNA; Lipoplexes; HPV; E6 oncoprotein; Cervical cancer;