Considerations for the delivery of STING ligands in cancer immunotherapy.

Several studies have shown the importance of the cGAS-STING pathway in antigen-presenting cells for anti-cancer immunity. Cyclic GMP-AMP (cGAMP) - STING ligand is a negatively charged dinucleotide prone to degradation by hydrolases. Once administered in its soluble form, high doses are needed which in turn may cause side effects such as T cell apoptosis. Moreover, due to its negative charge, transfection of cGAMP into negatively-charged membrane cells is hampered. In order to achieve successful transfection and protection from enzymatic degradation there is a need for a suitable carrier for cGAMP. In this review, we therefore describe currently reported carriers for cGAMP, and correlate their characteristics to the effect they cause. To achieve targeted delivery to the tumor microenvironment, the route of administration and physicochemical parameters of the particles (containing a carrier and cGAMP) such as size and charge need to be determined. Therefore, the choice of the particle formulation and its impact on the preclinical outcome will be discussed.

Equivalence of glatiramer acetate products: challenges in assessing pharmaceutical equivalence and critical clinical performance attributes.

INTRODUCTION: This review discusses the challenges to characterize and evaluate the peptide based drug glatiramer acetate (GA) and its follow-on products used for treatment of multiple sclerosis patients. AREAS COVERED: GA is a highly complex mixture of peptides consisting of four amino acids. The various (physico)-chemical approaches and bioassays used for characterizing this complex drug product are described. It is not possible to link data from preclinical performance to outcomes observed in clinical trials as no critical attributes suitable for predicting the clinical performance in MS patients have been identified yet. The limited insight into the precise mechanism(s) of action of GA may explain why these critical clinical performance attributes still have not been identified. EXPERT OPINION: The complexity of GA and lack of understanding of critical clinical performance attributes leads to a number of issues to be resolved as they hamper industry and regulatory bodies in designing and evaluating follow-on/generic applications of GA. The following questions are waiting to be addressed: Preclinical characterization vs clinical outcome: what is the relation? What are possible biomarkers? How to choose the right patient group? What is the experience with existing follow-on versions? Is there a place for GA 'betters'? How to evaluate existing and draft new guidance documents and pharmacopoeial monographs?

Association of chitosan and aluminium as a new adjuvant strategy for improved vaccination.

The use of particulate adjuvants offers an interesting possibility to enhance and modulate the immune responses elicited by vaccines. Aluminium salts have been extensively used as vaccine adjuvants, but they lack the capacity to induce a strong cellular and mucosal immune response. Taking this into consideration, in this study we designed a new antigen delivery system combining aluminium salts with chitosan. Chitosan-aluminium nanoparticles (CH-Al NPs) exhibited a mean diameter of 280nm and a positive surface charge. The newly developed CH-Al NPs are more stable at physiological environment than classical CH NPs, showing no cytotoxic effects and revealing potential as a delivery system for a wide range of model antigens. In vivo studies showed that mice immunized with hepatitis B surface antigen (HBsAg)-containing CH NPs display high anti-HBsAg IgG titers in the serum, as well as the highest antigen-specific IgG on vaginal washes. Furthermore, in contrast to mice receiving antigen alone, mice immunized with the particulate adjuvant were able to elicit IgG2c antibody titers and exhibited higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we established that CH-Al NPs, combining two immunostimulants to enhance both humoral and cellular immune responses, are a safe and promising system for antigen delivery. Our findings point towards their potential in future vaccination approaches.

Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

RGD peptide sequences are known to regulate cellular activities by interacting with α5ß1, αvß5 and αvß3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 µg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

Arming embolic beads with anti-VEGF antibodies and controlling their release using LbL technology.

Transarterial chemoembolization (TACE) is used to treat various types of hypervascular tumors such as hepatocellular carcinoma and renal cancer. However, embolization and blocking of blood vessels nourishing a tumor mass evokes an angiogenic response due to the secretion of vascular endothelial growth factor (VEGF), which results in the formation of new blood vessels and eventually limitation in therapeutic efficacy. The presented work investigates the feasibility of loading the clinically used embolic beads (DC Bead®) with Bevacizumab (BEV), an anti-VEGF antibody, and control its release kinetics via Layer-by-Layer (LbL) coating. This strategy has the aim to achieve high, localized and sustained concentrations of BEV at the tumor site and reduce drug exposure in the systemic circulation. High loading of BEV on lyophilized beads of about 76mg BEV/bead vial was achieved. LbL coating was carried out by depositing alternating layers of the biocompatible polymers alginate and poly-L-lysine. Coating was proven successful by monitoring the reversal of zeta potential after addition of each layer. Morphological changes of the bead surface before and after coating were illustrated using SEM imaging. Moreover, release profiles from different formulations were studied and results showed that optimizing the number of deposited layers effectively slows the release of BEV for three days. Activity of released BEV was studied in different 2D and 3D cell based assays. Released BEV fractions showed comparable activity to fresh BEV solution used as control after 3days. In conclusion, our results suggest the opportunity for loading anti-VEGF antibodies on commercially available embolic beads to increase the efficacy of TACE of hypervascular tumors.

Intranasal Administration of Novel Chitosan Nanoparticle/DNA Complexes Induces Antibody Response to Hepatitis B Surface Antigen in Mice.

The generation of strong pathogen-specific immune responses at mucosal surfaces where hepatitis B virus (HBV) transmission can occur is still a major challenge. Therefore, new vaccines are urgently needed in order to overcome the limitations of existing parenteral ones. Recent studies show that this may be achieved by intranasal immunization. Chitosan has gained attention as a nonviral gene delivery system; however, its use in vivo is limited due to low transfection efficiency mostly related to strong interaction between the negatively charged DNA and the positively charged chitosan. We hypothesize that the adsorption of negatively charged human serum albumin (HSA) onto the surface of the chitosan particles would facilitate the intracellular release of DNA, enhancing transfection activity. Here, we demonstrate that a robust systemic immune response was induced after vaccination using HSA-loaded chitosan nanoparticle/DNA (HSA-CH NP/DNA) complexes. Furthermore, intranasal immunization with HSA-CH NP/DNA complexes induced HBV specific IgA in nasal and vaginal secretions; no systemic or mucosal responses were detected after immunization with DNA alone. Overall, our results show that chitosan-based DNA complexes elicited both humoral and mucosal immune response, making them an interesting and valuable gene delivery system for nasal vaccination against HBV.

Chitosan as a starting material for wound healing applications.

Chitosan and its derivatives have attracted great attention due to their properties beneficial for application to wound healing. The main focus of the present review is to summarize studies involving chitosan and its derivatives, especially N,N,N-trimethyl-chitosan (TMC), N,O-carboxymethyl-chitosan (CMC) and O-carboxymethyl-N,N,N-trimethyl-chitosan (CMTMC), used to accelerate wound healing. Moreover, formulation strategies for chitosan and its derivatives, as well as their in vitro, in vivo and clinical applications in wound healing are described.

Optimized synthesis of O-carboxymethyl-N,N,N-trimethyl chitosan.

We present here the synthesis of a highly O-carboxymethylated chitosan derivative. First, an improved protocol for the two-step synthesis of N-trimethyl chitosan (TMC) from chitosan was developed, yielding a maximum degree of quaternization (DQ) of up to 46.6%. Successively, the chitosan derivative O-carboxymethyl-N-trimethyl chitosan (CMTMC) was synthesized from the TMC obtained by applying an optimized synthesis pathway. In contrast to previous reports, the optimized protocol was shown to yield very high rates (>85%) of O-carboxymethylation of CMTMC, as shown by (1)H NMR and heteronuclear single quantum correlation ((1)H-(13)C HSQC). Finally, in vitro cytocompatibility (viability >80%) of the polymer was demonstrated using human fibroblasts.

Encapsulation of VEGF165 into magnetic PLGA nanocapsules for potential local delivery and bioactivity in human brain endothelial cells.

Angiogenesis is an important repairing mechanism in response to ischemia. The administration of pro-angiogenic proteins is an attractive therapeutic strategy to enhance angiogenesis after an ischemic event. Their labile structures and short circulation times in vivo are the main obstacles that reduce the bioactivity and dosage of such proteins at the target site. We report on poly(d,l-lactic-co-glycolic acid) (PLGA) nanocapsules (diameter < 200 nm) containing bioactive vascular endothelial growth factor-165 (VEGF165) in the inner core and superparamagnetic iron oxide nanoparticles (SPIONs) embedded in the polymeric shell. The system showed good encapsulation efficiencies for both VEGF165 and SPIONs and a sustained protein release over 14 days. In vitro studies confirmed protein bioactivity in the form of significantly increased proliferation in human microvascular brain endothelial cell cultures once the protein was released. Through magnetic resonance imaging (MRI) measurements we demonstrated excellent T2 contrast image properties with r2 values as high as 213 mM-1 s-1. In addition, magnetic VEGF165-loaded PLGA nanocapsules could be displaced and accumulated under an external magnetic field for guiding and retention purposes. We therefore suggest that using VEGF165-loaded magnetic PLGA nanocapsules may become a new targeted protein-delivery strategy in the development of future pro-angiogenic treatments, as for instance those directed to neurorepair after an ischemic event.

Developing and advancing dry powder inhalation towards enhanced therapeutics.

Enhanced therapeutics are drug products derived from existing generic drugs that provide additional benefits to the patients and the healthcare system. Enhanced therapeutics are considered to be an important and relatively low risk source of innovation. Pulmonary drug delivery is the major delivery route to treat chronic respiratory diseases and has been proven as a potential delivery route for complex drugs that cannot be delivered orally. Development of dry powder inhalation systems targets the delivery of fine drug particles to the deep lung surface by a combination of drug formulation, primary packaging and a device, whereby each contributes to the overall performance. Various methodologies for the non-clinical and clinical performance testing of orally inhaled products have been proposed and applied with variable success. Regulatory pathways have been developed and applied since. Considerable efforts have been made during the past decade to understand and optimize pulmonary drug delivery including their efficient commercial manufacturing. Pulmonary drug delivery remains an area of future innovation in the effective treatment of pulmonary diseases as well as the systemic delivery of systemically active complex drugs.

Toll-like receptor-2 agonist functionalized biopolymer for mucosal vaccination.

The objective of this study was to provide a new water-soluble chitosan derivative being functionalized with a Toll-like receptor-2 (TLR-2) agonist. At first, we synthesized the water-soluble TLR-2 agonist omega-amido-[N(alpha)-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]-cysteinyl]-alpha-amino poly(ethylene glycol) (Pam(3)Cys-PEG-NH(2)), which was characterized by (1)H and (13)C NMR as well as mass spectroscopy. Secondly, Pam(3)Cys-PEG-NH(2) was then successfully grafted to 6-O-carboxymethyl-N,N,N-trimethyl chitosan polymers (CM-TMC) using EDC/NHS as condensing agents. The copolymer was analysed by means of (1)H and (13)C NMR and FTIR spectroscopy. (13)C NMR spectroscopy did not deliver evidence that an amide bond was formed between CM-TMC and Pam(3)Cys-PEG-NH(2). However, (1)H NMR and FTIR spectroscopy demonstrated clearly that successful grafting took place. Based upon these results, this new TLR-2 functionalized biopolymer merits further investigations as material for vaccine delivery systems.

Feasibility study on the retention of superporous hydrogel composite polymer in the intestinal tract of man using scintigraphy.

In recent years, many complex oral drug delivery systems have been developed using various polymers in order to achieve better drug targeting and drug absorption in the intestinal tract. Superporous hydrogel (SPH) and SPH composite (SPHC)-based drug delivery systems were also developed for the targeted delivery of peptide drugs into the intestinal tract. In the present study, the retention time of SPHC polymer is studied in man using the scintigraphy technique. To that purpose, SPHC polymers were radiolabelled with Tc-99m and administered orally in an enteric-coated gelatin capsule. The location of the radiolabelled polymer was monitored in five healthy volunteers while the subjects were sitting in front of a large field of view gamma camera. The results showed that enteric-coated gelatin capsules remained in the stomach for 75 to 150 min after oral administration to fasted volunteers and that the SPHC polymers thereafter attached to the upper part of the small intestine for at least 45 to 60 min due to their mechanical fixation properties. No discomfort was observed in any of the volunteers after oral administration of these polymers, which indicates that they are safe to be applied for oral drug delivery systems in man.

Influence of methylation process on the degree of quaternization of N-trimethyl chitosan chloride.

N-Trimethyl chitosan chloride (TMC) is a soluble chitosan derivative that shows effective enhancing properties for peptide and protein drug transport across mucosal membranes. TMC was synthesized by reductive methylation of chitosan in an alkaline environment at elevated temperature. The number of methylation process steps and the base used in the process was demonstrated to affect the degree of quaternization of the primary amino group and methylation of 3- and 6-hydroxyl groups. 1H-Nuclear magnetic resonance spectra showed that the degree of quaternization of TMC was higher when using sodium hydroxide as the base compared to using dimethyl amino pyridine. The degrees of quaternization as well as O-methylation of TMC increased with the number of reaction steps. O-Methylation resulted in decreased solubility of TMC. The high degree of quaternization of TMC with a low degree of O-methylation was prepared by employing one reaction step with two subsequent addition steps and a controlled alkaline environment of the mixture reaction.

Identification of an internalising peptide in differentiated Calu-3 cells by phage display technology; application to gene delivery to the airways.

Differentiated, human submucosal-gland carcinoma, Calu-3 cell monolayers were used as in vitro model for the airway epithelium. Internalised phage were selected from a recombinant pComb8 phage library by repetitive cycles of bio-panning on Calu-3 monolayers, protease K degradation, cell-lysis and amplification. After four selection rounds, sequence analysis of 15 enriched phage colonies revealed two clones of 73 and 27% abundancy, named IB1 and IB2, respectively. The IB2 sequence was eliminated due to a frame shift. IB1-phage internalisation at 4 degrees C was significantly lower (P < 0.05) than at 37 degrees C, suggesting involvement of a receptor-mediated endocytosis pathway. The IB1 peptide was synthesised, biotinylated and complexed to streptavidin. IB1/streptavidin-complexes co-administrated with PEI/DNA-polyplexes, enhanced polyplex transfection efficiency, dose dependently, by 6- and 4-fold in Calu-3 cells. IB1/Alexa488-streptavidin complexes were used for confocal laser-scanning microscopy (CLSM) visualisation and showed basolateral localisation in membrane associated and internalising vesicles. This study demonstrates the potential of phage display technology for identification of internalising peptide-epitopes that can enhance gene delivery efficiency in differentiated airway epithelial cells.

Intestinal absorption of human insulin in pigs using delivery systems based on superporous hydrogel polymers.

In this in vivo study, novel delivery systems based on superporous hydrogel (SPH) and SPH composite (SPHC) polymers were used to improve the intestinal absorption of insulin in healthy pigs. Six female pigs of approximately 35 kg body weight were used. A cannula was inserted into the jugular vein for blood sampling and a silicone fistula in the duodenum for administration of gelatin capsules containing the delivery systems or insulin solutions. The delivery systems consisted of two components, (1) conveyor system made of SPH and SPHC; (2) core containing insulin. The core was inserted either into the conveyor system (core inside, c.i.) or attached to the surface of conveyor system (core outside, c.o.). The following intestinal formulations were investigated: c.i., c.o. and intraduodenal (i.d.) administration of insulin solutions. Subcutaneous (s.c.) injection of insulin was also investigated for reasons of comparison. Blood samples were taken and analyzed for insulin and glucose concentrations. Relative bioavalibility values of 1.3+/-0.4 and 1.9+/-0.7% were achieved for c.o. and c.i. administrations, respectively. The bioavalibility for i.d. administration of insulin solution was 0.5+/-0.2%. These results indicate that the absorption of insulin was slightly increased using SPH/SPHC-based delivery systems. Furthermore, a large variability was observed, probably due to physiological and metabolic changes during the experiments. Blood glucose levels were slightly decreased after the c.o. and c.i administrations, whereas these levels did not decrease after i.d. administration of insulin solutions. In conclusion, SPH/SPHC-based delivery systems are able to enhance the intestinal absorption of insulin and are, therefore, considered as promising systems for peroral peptide drug delivery. However, insulin delivery from these delivery systems under in vivo have to be improved.

Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines.

Quaternized modifications of chitosan present characteristics that might be useful in DNA condensing and efficient gene delivery. Trimethylated chitosan (TMO) was synthesized from oligomeric chitosan (<20 monomer units). TMOs spontaneously formed complexes (chitoplexes) with RSV-alpha3 luciferase plasmid DNA. These complexes were characterized by photon correlation spectroscopy and were investigated for their ability to transfect COS-1 and Caco-2 cell lines in the presence and absence of fetal calf serum and compared with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulphate) lipoplexes. Additionally, their effect on the viability of the respective cell cultures was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results showed that quaternized chitosan oligomers were able to condense DNA and form complexes with a size ranging from 200 to 500 nm. Chitoplexes proved to transfect COS-1 cells, however, to a lesser extent than DOTAP-DNA lipoplexes. The quaternized oligomer derivatives appeared to be superior to oligomeric chitosan. The presence of fetal calf serum (FCS) did not affect the transfection efficiency of the chitoplexes, whereas the transfection efficiency of DOTAP DNA complexes was decreased. Cells remained 100% viable in the presence of chitosan oligomers whereas viability of DOTAP treated cells decreased to approximately 50% in both cell lines. Both DOTAP-DNA lipoplexes and chitoplexes resulted in less transfection efficiency in Caco-2 cell cultures than in COS-1 cells; however quaternized chitosan oligomers proved to be superior to DOTAP. Effects on the viability of Caco-2 cells were similar to the effects observed in COS-1 cells. We conclude that trimethylated chitosan-DNA complexes present suitable characteristics and the potential to be used as gene delivery vectors.

Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model.

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.

Lectin-functionalized liposomes for pulmonary drug delivery: interaction with human alveolar epithelial cells.

In this study the interaction of lectin-functionalized liposomes with two different alveolar epithelial cell culture models was evaluated. Plant lectins were coupled to liposomes exploiting the avidin/biotin technology. In contrast to lectin-free liposomes, lectin functionalized liposomes specifically bound to A549 cells, a tumor-derived cell line. Using this cell line, temperature-dependent binding assays as well as confocal laser scanning microscopy (CLSM) revealed that the lectin liposomes were only bound but not taken up by these cells. In contrast to these findings, confocal images of human alveolar epithelial cells in primary culture incubated together with lectin liposomes indicated binding as well as cellular uptake. Fluorescein-isothiocyanate (FITC)-labeled dextrans (Mw 40,000 Da), encapsulated in lectin-functionalized liposomes and incubated with monolayers of primary cultured human alveolar epithelial cells appeared to be localized intracellularly by CLSM. This suggests that lectin-mediated bioadhesion and uptake of liposomal carriers may provide a useful technology for improved delivery of hydrophilic macromolecules to the alveolar epithelium.

Evidence of P-glycoprotein mediated apical to basolateral transport of flunisolide in human broncho-tracheal epithelial cells (Calu-3).

1. Transepithelial transport of flunisolide was studied in reconstituted cell monolayers of Calu-3, LLC-PK1 and the MDR1-P-glycoprotein transfected LLC-MDR1 cells. 2. Flunisolide transport was polarized in the apical (ap) to basolateral (bl) direction in Calu-3 cells and was demonstrated to be ATP-dependent. In LLC-MDR1 cells, flunisolide was transported in the bl to ap direction and showed no polarization in LLC-PK1 cells. 3. Non-specific inhibition of cellular metabolism at low temperature (4 degrees C) or by 2-deoxy-D-glucose (2-d-glu) and sodium azide (NaN(3)) abolished the polarized transport. Polarized flunisolide transport was also inhibited by the specific Pgp inhibitors verapamil, SDZ PSC 833 and LY335979. 4. Under all experimental conditions and in the presence of all used inhibitors, no decrease in the TransEpithelial Electrical Resistance (TEER) values was detected. From all inhibitors used, only the general metabolism inhibitors 2-deoxy-D-glucose and NaN(3), decreased the survival of Calu-3 cells. 5. Western blotting analysis and confocal laser scanning microscopy demonstrated the presence of MDR1-Pgp at mainly the basolateral side of the plasma membrane in Calu-3 cells and at the apical side in LLC-MDR1 cells. Mass spectroscopy studies demonstrated that flunisolide is transported unmetabolized across Calu-3 cells. 6. In conclusion, these results show that the active ap to bl transport of flunisolide across Calu-3 cells is facilitated by MDR1-Pgp located in the basolateral plasma membrane.