International Journal of Pharmaceutics (v.440, #1)

Editorial by Maria J. Blanco-Prieto (1-2).

Drug delivery to inflamed colon by nanoparticles: Comparison of different strategies by Régis Coco; Laurence Plapied; Vincent Pourcelle; Christine Jérôme; David J. Brayden; Yves-Jacques Schneider; Véronique Préat (3-12).
For inflammatory bowel disease (IBD) treatment, local delivery of molecules loaded in nanoparticles to the inflamed colon could be a promising strategy. The aim of this study was to investigate how drug-loaded polymeric nanoparticles target the site of inflammation and to analyse the influence of different colon-specific delivery strategies. Three different polymeric nanoparticles were formulated using ovalbumin (OVA) as a model drug. pH-sensitive nanoparticles were made with Eudragit® S100. Mucoadhesive nanoparticles were created with trimethylchitosan (TMC). A mix of polymers, PLGA, PEG-PLGA and PEG-PCL, were used to obtain a sustained drug delivery. Furthermore, ligands targeting immune cells (i.e. mannose) or the inflamed colon (i.e. a specific peptide) were grafted on the PEG chain of PCL. Interaction of nanoparticles with the intestinal epithelium was explored using Caco-2 monolayers designed to mimic an inflamed epithelium and then visualized using confocal laser microscopy. TMC nanoparticles had the highest apparent permeability for OVA in the untreated model. However, in the inflamed model, there were no difference between TMC, PLGA-based and Eudragit® nanoparticles. The uptake of nanoparticles in the inflamed mouse colon was assessed in a horizontal diffusion chamber. Mannose-grafted PLGA nanoparticles showed the highest accumulation of OVA in inflamed colon. Based on these results, active targeting of macrophages and dendritic cells may be a promising approach for targeting the colon in IBD.
Keywords: Nanoparticles; Intestinal barrier; Colonic delivery; Intestinal inflammation; Trimethylchitosan; PLGA; Eudragit®;

PEGylated-PLGA microparticles containing VEGF for long term drug delivery by Teresa Simón-Yarza; Fabio R. Formiga; Esther Tamayo; Beatriz Pelacho; Felipe Prosper; María J. Blanco-Prieto (13-18).
The potential of poly(lactic-co-glycolic) acid (PLGA) microparticles as carriers for vascular endothelial growth factor (VEGF) has been demonstrated in a previous study by our group, where we found improved angiogenesis and heart remodeling in a rat myocardial infarction model (Formiga et al., 2010). However, the observed accumulation of macrophages around the injection site suggested that the efficacy of treatment could be reduced due to particle phagocytosis.The aim of the present study was to decrease particle phagocytosis and consequently improve protein delivery using stealth technology. PEGylated microparticles were prepared by the double emulsion solvent evaporation method using TROMS (Total Recirculation One Machine System). Before the uptake studies in monocyte-macrophage cells lines (J774 and Raw 264.7), the characterization of the microparticles developed was carried out in terms of particle size, encapsulation efficiency, protein stability, residual poly(vinyl alcohol) (PVA) and in vitro release. Microparticles of suitable size for intramyocardial injection (5 μm) were obtained by TROMS by varying the composition of the formulation and TROMS conditions with high encapsulation efficiency (70–90%) and minimal residual PVA content (0.5%). Importantly, the bioactivity of the protein was fully preserved. Moreover, PEGylated microparticles released in phosphate buffer 50% of the entrapped protein within 4 h, reaching a plateau within the first day of the in vitro study. Finally, the use of PLGA microparticles coated with PEG resulted in significantly decreased uptake of the carriers by macrophages, compared with non PEGylated microparticles, as shown by flow cytometry and fluorescence microscopy.On the basis of these results, we concluded that PEGylated microparticles loaded with VEGF could be used for delivering growth factors in the myocardium.
Keywords: PEG; PLGA; Macrophage uptake; VEGF; Protein delivery;

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system by Eduardo Ansorena; Erkuden Casales; Alejandro Aranda; Esther Tamayo; Elisa Garbayo; Cristian Smerdou; Maria J. Blanco-Prieto; Maria S. Aymerich (19-26).
Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.
Keywords: GDNF; SFV expression system; Protein expression; Protein purification; Glycosylated recombinant protein; BHK cells;

Vaccine delivery carriers: Insights and future perspectives by J.F. Correia-Pinto; N. Csaba; M.J. Alonso (27-38).
Vaccination is undoubtedly the most effective health intervention for disease prevention and eradication. Nevertheless, currently there is still a need for improving immunization coverage worldwide. A promising strategy to achieve this goal nowadays relies on the use of delivery carriers capable of inducing an effective immune response and providing improved stability, safety and cost effectiveness. This article focuses on analyzing the critical aspects in the design of these carriers, and reviewing the state of the art of currently marketed formulations and those in advanced clinical development. These vaccine delivery carriers include emulsions, liposomes and polymeric particulate carriers. Finally, particular attention is given to the evolution in the design of polymeric nanocarriers, which have been receiving increasing attention and hold promise to generate novel platforms for needle-free administration and single-dose vaccination.
Keywords: Antigen delivery; Adjuvant; Nanocarrier; Mucosal immunization; Nanovaccine;

Expression without boundaries: Cell-free protein synthesis in pharmaceutical research by Marco G. Casteleijn; Arto Urtti; Sanjay Sarkhel (39-47).
Proteins are an increasingly important class of new drugs. Pharmaceutical proteins are usually expressed in cell based systems in the development phase and in production, and although cell free methods have recently emerged they have not been used widely for therapeutic protein development or production. Cell free expression methodology is well suited for pharmaceutical protein expression and engineering and will probably become more commonly used in the future. Cell free expression allows protein engineering in high throughput format, flexible strategies for glycosylation and chemical conjugation, and allows easy use of unnatural amino acids as building blocks of proteins. Thus, cell free expression can be used to modify protein solubility, stability, and pharmacokinetics of therapeutic proteins. Likewise, it is potentially useful in protein development for biomaterial matrices, nanoparticles, and vaccines. This review illustrates the potential of cell free expression in pharmaceutical protein research and development while highlighting both advantages and limitations of the method.
Keywords: Protein engineering; Cell free expression; Protein expression; High throughput; Protein development;

Diabetes is a metabolic disease and is a major cause of mortality and morbidity in epidemic proportions. A type I diabetic patient is dependent on daily injections of insulin, for survival and also to maintain a normal life, which is uncomfortable, painful and also has deleterious effects. Extensive efforts are being made worldwide for developing noninvasive drug delivery systems, especially via oral route. Oral route is the most widely accepted means of administration. However it is not feasible for direct delivery of peptide and protein drugs. To overcome the gastro-intestinal barriers various types of formulations such as polymeric micro/nanoparticles, liposomes, etc. are investigated. In the recent years lot of advances have taken place in developing and understanding the oral peptide delivery systems. Simultaneously, the development and usage of other peptides having anti-diabetic potentials are also considered for diabetes therapy. In this review we are focusing on the advances reported during the past decade in the field of oral insulin delivery along with the possibility of other peptidic incretin hormones such as GLP-1, exendin-4, for diabetes therapy.
Keywords: Diabetes; Nanoparticles; Oral delivery; Insulin; Exendin-4; Glucagon like peptide-1;

Influence of acylation on the adsorption of GLP-2 to hydrophobic surfaces by Charlotte Pinholt; Sebastian J. Kapp; Jens T. Bukrinsky; Susanne Hostrup; Sven Frokjaer; Willem Norde; Lene Jorgensen (63-71).
Acylation of proteins with a fatty acid chain has proven useful for prolonging the plasma half-lives of proteins. In formulation of acylated protein drugs, knowledge about the effect of acylation with fatty acids on the adsorption behaviour of proteins at interfaces will be valuable. The aim of this work was to study the effect of acylation on the adsorption of GLP-2 from aqueous solution to a hydrophobic surface by comparing the adsorption of the 3766 Da GLP-2 with that of a GLP-2 variant acylated with a 16-carbon fatty acid chain through a β-alanine linker. Adsorption of GLP-2 and acylated GLP-2 were studied with isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. Furthermore, the effect of acylation of GLP-2 on the secondary structure was studied with Far-UV CD. Acylation was observed to have several effects on the adsorption of GLP-2. Acylation increased the amount of GLP-2 adsorbing per unit surface area and decreased the initial adsorption rate of GLP-2. Finally, acylation increased the strength of the adsorption, as judged by the lower fraction desorbing upon rinsing with buffer.
Keywords: Acylation; Protein adsorption; GLP-2; ITC; Reflectometry; TIRF;

Comparative analysis of protein expression of three stem cell populations: Models of cytokine delivery system in vivo by Stephane Roche; Gianluca D’Ippolito; L. Adriana Gomez; Thomas Bouckenooghe; Sylvain Lehmann; Claudia N. Montero-Menei; Paul C. Schiller (72-82).
Several mechanisms mediate the regenerative and reparative capacity of stem cells, including cytokine secretion; therefore these cells can act as delivery systems of therapeutic molecules. Here we begin to address the molecular and cellular basis of their regenerative potential by characterizing the proteomic profile of human embryonic stem cells (hESCs), mesenchymal stem cells (hMSCs) and marrow isolated adult multilineage inducible (MIAMI) cells, followed by analysis of the secretory profile of the latter stem cell population. Proteomic analysis establishes the closer relationship between hMSCs and MIAMI cells, while hESCs are more divergent. However, MIAMI cells appear to have more proteins in common with hESCs than hMSCs. Proteins characteristic of hMSCs include transgelin-2, phosphatidylethanolamine-binding protein 1 (PEBP1), Heat-Shock 20 kDa protein (HSP20/HSPβ6), and programmed cell death 6-interacting protein (PDC6I) among others. MIAMI cells are characterized by the high level expression of ubiquitin carboxyl-terminal hydrolase isoenzyme L1 (UCHL1), 14-3-3 zeta, HSP27 (HSPβ1), and tropomyosin 4 and 3. For hESC, elongation factor Tu (EFTu), isocitrate dehydrogenase (IDH1) and the peroxiredoxins 1, 2, and 6 (PRDX1, PRDX2, and PRDX6) were the most characteristic. Secretome analysis indicates that MIAMI cells secrete higher levels of vascular endothelial growth factor (VEGF), Fractalkine, Interleukin-6, interlukin-8, and growth related oncogene (GRO), compared to hMSCs. These soluble mediators are known to play key roles in angiogenesis, arteriogenesis, atheroprotection, immunomodulation, neuroprotection, axonal growth, progenitor cell migration, and prevention of apoptosis. All these roles are consistent with a reparative pro-survival secretory phenotype. We further discuss the potential of these cells as therapeutic vehicles.
Keywords: Cytokine; Delivery systems; Stem cells;

Therapeutic antibodies: Market considerations, disease targets and bioprocessing by John G. Elvin; Ruairidh G. Couston; Christopher F. van der Walle (83-98).
Antibodies are well established in mainstream clinical practice and present an exciting area for collaborative research and development in industry and academia alike. In this review, we will provide an overview of the current market and an outlook to 2015, focussing on whole antibody molecules while acknowledging the next generation scaffolds containing variable fragments. The market will be discussed in the context of disease targets, particularly in the areas of oncology and immune disorders which generate the greatest revenue by a wide margin. Emerging targets include central nervous system disorders which will also stimulate new delivery strategies. It is becoming increasingly apparent that a better understanding of bioprocessing is required in order to optimize the steps involved in the preparation of a protein prior to formulation. The latter is outside the scope of this review and nor is it our intention to discuss protein delivery and pharmacokinetics. The challenges that lie ahead include the discovery of new disease targets and the development of robust bioprocessing operations.
Keywords: Monoclonal antibody; Market; Oncology; Immune disorder; Biopharmaceutical; Bioprocessing;

The in vitro/in vivo characterization of antacid-insulin co-encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles is presented here. The optimized nanoparticle composition has 1% surfactant (didodecyl dimethylammonium bromide) and 2% antacid (magnesium hydroxide or zinc carbonate) in the size range ∼136–143 nm with ∼81–85% entrapment of insulin at a 4% (w/w) initial load to that of polymer. Molecular characterization using circular dichroism, fluorescence and Fourier transform infrared spectroscopy showed that the structural integrity of insulin was maintained during formulation. Furthermore, the encapsulated insulin was well protected under in vitro simulated gastric and intestinal fluids. Nanoparticle insulin results in six fold increase in oral bioavailability to that of plain insulin in healthy rats. In diabetic rats, a 120 IU/kg oral dose of insulin nanoparticles achieved an equivalent blood glucose lowering effect to a 20 IU/kg subcutaneous (sc) dose of insulin solution, the nadir in blood glucose concentration occurring 24 h and 1 h post-administration, respectively. Both sc insulin and oral nanoparticle insulin partially attenuated hyperglycemia-induced inflammation caused by tumor necrosis factor α, but not by interleukin-6 or C-reactive protein; on the other hand, subcutaneous insulin was found to be more effective on lipid profile measured in the form of high density lipoprotein, cholesterol and triglyceride. Successful oral insulin could be beneficial in type II complications.
Keywords: Biodegradable; Hyperglycemia; Inflammation; Nanoparticles; Oral delivery;

Translation of therapeutic proteins to pharmaceutical products is often encumbered by their inadequate physicochemical and biopharmaceutical properties, namely low stability and poor bioavailability. Over the last decades, several academic and industrial research programs have been focused on development of biocompatible polymers to produce appropriate formulations that provide for enhanced therapeutic performance. According to their physicochemical properties, polymers have been exploited to obtain a variety of formulations including biodegradable microparticles, 3-dimensional hydrogels, bioconjugates and soluble nanocomposites. Several soluble polymers bearing charges or hydrophobic moieties along the macromolecular backbone have been found to physically associate with proteins to form soluble nanocomplexes. Physical complexation is deemed a valuable alternative tool to the chemical bioconjugation. Soluble protein/polymer nanocomplexes formed by physical specific or unspecific interactions have been found in fact to possess peculiar physicochemical, and biopharmaceutical properties. Accordingly, soluble polymeric systems have been developed to increase the protein stability, enhance the bioavailability, promote the absorption across the biological barriers, and prolong the protein residence in the bloodstream. Furthermore, a few polymers have been found to favour the protein internalisation into cells or boost their immunogenic potential by acting as immunoadjuvant in vaccination protocols.
Keywords: Proteins; Soluble polymers; Supramolecular systems; Soluble nanocomposites; Nanocomplexes; Protein delivery;