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Purpose: Personalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose., Methods: Fifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8 + T-cells in vitro., Results: All SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200 nm), PDI (< 0.3) and zeta potential (22-30 mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8 + T-cells, indicating improved immunological activity of the SLPs., Conclusion: Cationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines.

Purpose: To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice., Methods: DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls., Results: LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles., Conclusion: Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.

Purpose: The aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT)., Methods: A novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 μm, or at 400 μm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization., Results: The applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 μm), -volumes (1-100 μL) and -rates (up to 60 μL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold)., Conclusion: Intradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.

Purpose: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics., Methods: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy., Results: Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC., Conclusions: These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

Purpose: The aim of the study was to develop a cheap and fast method to produce hollow microneedles and an applicator for injecting vaccines into the skin at a pre-defined depth and test the applicability of the system for dermal polio vaccination., Methods: Hollow microneedles were produced by hydrofluoric acid etching of fused silica capillaries. An electromagnetic applicator was developed to control the insertion speed (1-3 m/s), depth (0-1,000 μm), and angle (10°-90°). Hollow microneedles with an inner diameter of 20 μm were evaluated in ex vivo human skin and subsequently used to immunize rats with inactivated poliovirus vaccine (IPV) by an intradermal microinjection of 9 μL at a depth of 300 μm and an insertion speed of 1 m/s. Rat sera were tested for IPV-specific IgG and virus-neutralizing antibodies., Results: Microneedles produced from fused silica capillaries were successfully inserted into the skin to a chosen depth, without clogging or breakage of the needles. Intradermal microinjection of IPV induced immune responses comparable to those elicited by conventional intramuscular immunization., Conclusions: We successfully developed a hollow microneedle technology for dermal vaccination that enables fundamental research on factors, such as insertion depth and volume, and insertion angle, on the immune response.

Purpose: The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of rhEPO copy products being used in Thailand., Methods: Twelve rhEPO copy products were purchased from pharmacies in Thailand, shipped under controlled cold chain conditions to the Netherlands and characterized using (1) high performance size-exclusion chromatography, (2) asymmetrical flow field-flow fractionation, (3) sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with (4) Western blotting and additionally tested for (5) host cell protein impurities as well as (6) endotoxin contamination., Results: Some of the tested rhEPO copy products showed high aggregate levels and contained a substantial amount of protein fragments. Also, one of rhEPO copy products had a high endotoxin level, exceeding the FDA limit., Conclusions: Our observations show that some of the tested copy products on the Thai market differ significantly from the originator rhEPO product, Epogen®. This comparison study supports a link between the quality attributes of copy rhEPO products and their immunogenicity.

Purpose: To determine the immunogenicity of diphtheria toxoid (DT) formulated in two types of vesicles following transcutaneous immunization (TCI) of mice onto microneedle array-treated skin., Methods: DT-containing cationic liposomes or anionic surfactant-based vesicles were prepared by extrusion and sonication. The physicochemical properties were characterized in terms of size, ζ-potential, vesicle elasticity and antigen association. TCI was performed by applying formulations onto intact or microneedle array-pretreated mice skin, using cholera toxin as an adjuvant. Subcutaneous and intradermal immunizations were as control. Immune responses were evaluated by IgG and neutralizing antibody titers, and the immune-stimulatory properties were assessed using cultured dendritic cells., Results: Stable DT-containing cationic liposomes (∼150 nm) and anionic vesicles (∼100 nm) were obtained. Incorporation of Span 80 increased liposome elasticity. About 90% and 77% DT was associated with liposomes and vesicles, respectively. TCI of all formulations resulted in substantial antibody titers only if microneedle pretreatment was applied. Co-administration of cholera toxin further augmented the immune responses of TCI. However, vesicle formulations didn't enhance the immunogenicity on either intact or microneedle-treated skin and showed low stimulatory activity on dendritic cells., Conclusions: Microneedle pretreatment and cholera toxin, but not antigen association to vesicles, enhances the immunogenicity of topically applied DT.

Purpose: To investigate degradation kinetics of oxytocin as a function of temperature and pH, and identify the degradation products., Materials and Methods: Accelerated degradation of oxytocin formulated at pH 2.0, 4.5, 7.0 and 9.0 was performed at 40, 55, 70 and 80 degrees C. Degradation rate constants were determined from RP-HPLC data. Formulations were characterized by HP-SEC, UV absorption and fluorescence spectroscopy. Degradation products were identified by ESI-MS/MS., Results: The loss of intact oxytocin in RP-HPLC was pH- and temperature-dependent and followed (pseudo) first order kinetics. Degradation was fastest at pH 9.0, followed by pH 7.0, pH 2.0 and pH 4.5. The Arrhenius equation proved suitable to describe the kinetics, with the highest activation energy (116.3 kJ/mol) being found for pH 4.5 formulations. At pH 2.0 deamidation of Gln(4), Asn(5), and Gly(9)-NH2, as well as combinations thereof were found. At pH 4.5, 7.0 and 9.0, the formation of tri- and tetrasulfide-containing oxytocin as well as different types of disulfide and dityrosine-linked dimers were found to occur. Beta-elimination and larger aggregates were also observed. At pH 9.0, mono-deamidation of Gln(4), Asn(5), and Gly(9)-NH2 additionally occurred., Conclusions: Multiple degradation products of oxytocin have been identified unequivocally, including various deamidated species, intramolecular oligosulfides and covalent aggregates. The strongly pH dependent degradation can be described by the Arrhenius equation.

Purpose: To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivo using confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated beta-cyclodextrin and sodium taurodihydrofusidate) on this transport was studied., Methods: Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope., Results: Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated beta-cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated beta-cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed., Conclusions: Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.

Purpose: The assessment of the effects of nasal absorption enhancers on the rat nasal epithelium and membrane permeability in vivo after a single nasal dose of the enhancers., Methods: The release of marker compounds (protein, cholesterol and acid phosphatase) from the nasal epithelium was measured using a lavage technique. The nasal membrane permeability was determined after intravenous administration of a systemic tracer (FITC-albumin)., Results: The effects of the absorption enhancers could be classified into four categories. The first consisted of HP beta CD (5%), DM beta CD (2%) and RAMEB (2%) and was not different from the control (physiological saline). For the second category, DM beta CD (5%), effects were significantly higher than for the control. The third category, SGC (1%), was more active than DM beta CD (5%) but less active than the last group. The fourth, most membrane damaging, category consisted of STDHF (1%), laureth-9 (1%) and LPC (1%). Administration of these three enhancers also resulted in release of acid phosphatase, indicating that severe membrane damage occurred. The release of cholesterol from nasal epithelium was largely dependent on the cholesterol solubilisation of the absorption enhancers. The amount of cholesterol released by laureth-9 and LPC was the largest., Conclusions: The results of this in vivo study are in agreement (i.e. similarity in rank order) with morphological and ciliotoxicity studies of nasal absorption enhancers, demonstrating that this in vivo model is a valuable tool to classify nasal absorption enhancers according to their effects on the rat nasal epithelium.

The nasal absorption of insulin using dimethyl-beta-cyclodextrin (DM beta CD) as an absorption enhancer in rabbits was studied. The nasal administration of insulin/DM beta CD liquid formulations did not result in significant changes in serum insulin and blood glucose concentrations. In contrast, previous experiments in rats showed that the addition of DM beta CD to the liquid nasal formulation resulted in an almost-complete insulin absorption, with a concomitant strong hypoglycaemic response. Apparently, the effect of the cyclodextrin derivative on insulin absorption differs between animal species following nasal delivery of insulin/DM beta CD solutions. On the other hand, nasal administration of the lyophilized insulin/DM beta CD powder dosage form in rabbits resulted in increased serum insulin concentrations, and a maximum decrease in blood glucose of about 50%. The absolute bioavailability of the nasally administered insulin/DM beta CD powder was 13 +/- 4%, compared to 1 +/- 1% for both an insulin/DM beta CD liquid and an insulin/lactose powder formulation. It is concluded that insulin powder formulations with DM beta CD as an absorption enhancer are much more effective than liquid formulations.

The absorption enhancing effect of alpha-, beta-, and gamma-cyclodextrin (CD), dimethyl-beta-cyclodextrin (DM beta CD), and hydroxypropyl-beta-cyclodextrin (HP beta CD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DM beta CD to the insulin solution resulted in a high bioavailability, 108.9 +/- 36.4% (mean +/- SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% alpha-CD gave rise to an insulin bioavailability of 27.7 +/- 11.5% (mean +/- SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the alpha-CD-containing solution as compared to the DM beta CD preparation. The other CDs, HP beta CD (5%), beta-CD (1.8%), and gamma-CD (5%), did not have significant effects on nasal insulin absorption. DM beta CD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DM beta CD is a potent enhancer of nasal insulin absorption in rats.