Intradermal Vaccination with PLGA Nanoparticles via Dissolving Microneedles and Classical Injection Needles.

PURPOSE: A dissolving microneedle array (dMNA) is a vaccine delivery device with several advantages over conventional needles. By incorporating particulate adjuvants in the form of poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) into the dMNA, the immune response against the antigen might be enhanced. This study aimed to prepare PLGA-NP-loaded dMNA and to compare T-cell responses induced by either intradermally injected aqueous-PLGA-NP formulation or PLGA-NP-loaded dMNA in mice. METHODS: PLGA NPs were prepared with microfluidics, and their physicochemical characteristics with regard to encapsulation efficiencies of ovalbumin (OVA) and CpG oligonucleotide (CpG), zeta potentials, polydispersity indexes, and sizes were analysed. PLGA NPs incorporated dMNA was produced with three different dMNA formulations by using the centrifugation method, and the integrity of PLGA NPs in dMNAs was evaluated. The immunogenicity was evaluated in mice by comparing the T-cell responses induced by dMNA and aqueous formulations containing ovalbumin and CpG (OVA/CpG) with and without PLGA NP. RESULTS: Prepared PLGA NPs had a size of around 100 nm. The dMNA formulations affected the particle integrity, and the dMNA with poly(vinyl alcohol) (PVA) showed almost no aggregation of PLGA NPs. The PLGA:PVA weight ratio of 1:9 resulted in 100% of penetration efficiency and the fastest dissolution in ex-vivo human skin (< 30 min). The aqueous formulation with soluble OVA/CpG and the aqueous-PLGA-NP formulation with OVA/CpG induced the highest CD4 + T-cell responses in blood and spleen cells. CONCLUSIONS: PLGA NPs incorporated dMNA was successfully fabricated and the aqueous formulation containing PLGA NPs induce superior CD4+ and CD8+ T-cell responses.

Comprehensive evaluation of microneedle-based intradermal adalimumab delivery vs. subcutaneous administration: results of a randomized controlled clinical trial.

AIMS: To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single subcutaneous (s.c.) dose using a conventional needle. METHODS: In this single-centre double-blind, placebo-controlled, double-dummy clinical trial in 24 healthy adults we compared 40 mg adalimumab (0.4 mL) administered i.d. using a hollow microneedle with a s.c. dose using a conventional needle. Primary parameters were pain, acceptability and local tolerability; secondary parameters safety, pharmacokinetics and immunogenicity. We explored usability of optical coherence tomography, clinical photography, thermal imaging, and laser speckle contrast imaging to evaluate skin reaction after i.d. injections. In vitro protein analysis was performed to assess compatibility of adalimumab with the hollow microneedle device. RESULTS: While feasible and safe, injection pain of i.d. adalimumab was higher compared to s.c. adalimumab (35.4 vs. 7.9 on a 100-point visual analogue scale). Initial absorption rate and relative bioavailability were higher after i.d. adalimumab (time to maximum plasma concentration = 95 h [47-120]; Frel = 129% [6.46%]) compared to s.c. adalimumab (time to maximum plasma concentration = 120 h [96-221]). Anti-adalimumab antibodies were detected in 50% and 83% of the subjects after i.d. and s.c. adalimumab, respectively. We observed statistically significantly more erythema and skin perfusion after i.d. adalimumab, compared to s.c. adalimumab and placebo injections (P < .0001). Cytokine secretion after whole blood lipopolysaccharide challenge was comparable between administration routes. CONCLUSIONS: Intradermal injection of adalimumab using hollowing microneedles was perceived as more painful and less accepted than s.c. administration, but yields a higher relative bioavailability with similar safety and pharmacodynamic effects.

Coated and Hollow Microneedle-Mediated Intradermal Immunization in Mice with Diphtheria Toxoid Loaded Mesoporous Silica Nanoparticles.

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.

Cationic Liposomes: A Flexible Vaccine Delivery System for Physicochemically Diverse Antigenic Peptides.

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.

Mesoporous Silica Nanoparticle-Coated Microneedle Arrays for Intradermal Antigen Delivery.

PURPOSE: To develop a new intradermal antigen delivery system by coating microneedle arrays with lipid bilayer-coated, antigen-loaded mesoporous silica nanoparticles (LB-MSN-OVA). METHODS: Synthesis of MSNs with 10-nm pores was performed and the nanoparticles were loaded with the model antigen ovalbumin (OVA), and coated with a lipid bilayer (LB-MSN-OVA). The uptake of LB-MSN-OVA by bone marrow-derived dendritic cells (BDMCs) was studied by flow cytometry. The designed LB-MSN-OVA were coated onto pH-sensitive pyridine-modified microneedle arrays and the delivery of LB-MSN-OVA into ex vivo human skin was studied. RESULTS: The synthesized MSNs demonstrated efficient loading of OVA with a maximum loading capacity of about 34% and the lipid bilayer enhanced the colloidal stability of the MSNs. Uptake of OVA loaded in LB-MSN-OVA by BMDCs was higher than that of free OVA, suggesting effective targeting of LB-MSN-OVA to antigen-presenting cells. Microneedles were readily coated with LB-MSN-OVA at pH 5.8, yielding 1.5 µg of encapsulated OVA per microneedle array. Finally, as a result of the pyridine modification, LB-MSN-OVA were effectively released from the microneedles upon piercing the skin. CONCLUSION: Microneedle arrays coated with LB-MSN-OVA were successfully developed and shown to be suitable for intradermal delivery of the encapsulated protein antigen.

Determination of Depth-Dependent Intradermal Immunogenicity of Adjuvanted Inactivated Polio Vaccine Delivered by Microinjections via Hollow Microneedles.

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.

Layer-by-Layer Assembly of Inactivated Poliovirus and N-Trimethyl Chitosan on pH-Sensitive Microneedles for Dermal Vaccination.

The aim of this work was to coat pH-sensitive microneedle arrays with inactivated polio vaccine (IPV) particles and N-trimethyl chitosan chloride (TMC) via electrostatic interactions, and assess the immunogenicity of the vaccine after topical application of the coated microneedles in rats. The surface of 200 µm long microneedles was first chemically modified with pH-sensitive (pyridine) groups and then coated with negatively charged IPV and a positively charged polymer (TMC). To obtain a sufficient high antigen dose, 10 layers of IPV were alternately coated with TMC. The binding of IPV and TMC onto pH-sensitive microneedles was quantified and visualized by using fluorescently labeled TMC and IPV. The release of IPV and TMC from the microneedles was evaluated in ex vivo human skin by fluorescence and the immunogenicity of (unlabeled) IPV was assessed after topical application of the coated microneedles in rats. pH-sensitive microneedles were homogeneously coated with 10 layers of both IPV and TMC, resulting in 45 D antigen units IPV and 700 ng TMC per microneedle array. Fluorescence microscopy imaging revealed that both IPV and TMC were released into ex vivo human skin upon application of the coated microneedles. Finally, in vivo application of IPV-TMC-coated pH-sensitive microneedles in rats led to the induction of IPV specific antibody responses, illustrating that they are practically applicable. Topical administration of pH-sensitive microneedles coated with polyelectrolyte multinanolayers of antigens and oppositely charged polymers may be a useful approach for microneedle-based vaccination.

Chemical modifications of silicon surfaces for the generation of a tunable surface isoelectric point.

The aim of this work was to generate a tunable surface isoelectric point (sIEP), where the surface is modified with two molecules: a weak base (pyridine), carrying a pH dependent positive charge, and a derivative of a strong acid (sulfate), carrying a permanent negative charge in a physiologically relevant pH range. To this end, silicon surfaces were modified with 3-aminopropyltriethoxysilane. These amine-modified surfaces were subsequently derivatized into pyridine- or sulfate-modified surfaces. Then, the surface pKa of pyridine-modified surfaces was determined by a fluorescent nanoparticle adhesion assay (FNAA). Next, these values were used to calculate in which ratio the chemicals must be present in the reaction mixture to generate a mixed pyridine/sulfate-modified surface with a target sIEP. After preparing surfaces with a target sIEP, an FNAA with positively and negatively charged nanoparticles was used to verify the sIEP of the generated surfaces. The FNAA revealed that pyridine-modified surfaces had a pKa of 6.69 ± 0.18. When an sIEP was generated, negative nanoparticles bound to surfaces at pH values below the sIEP and positive nanoparticles bound at pH values above the sIEP. Furthermore, we found sIEP values of 5.97 ± 0.88 when we aimed for an sIEP of 6.2, and 7.12 ± 0.21 when we aimed for an sIEP of 7.1. Finally, the pH dependent binding and release of a negatively and positively charged (bio)polymer was investigated for a target sIEP of 7. A negatively charged polymer (poly(I:C)) was bound at a pH < sIEP and released at a pH > sIEP with a release efficiency of 85 ± 9% and a positively charged polymer (trimethyl chitosan) bound at a pH > sIEP and released at a pH < sIEP with a release efficiency of 72 ± 9%. In conclusion, we established a method for preparing modified silicon surfaces with a tunable sIEP, which can be used for pH-dependent binding and release of biomacromolecules.

Novel hollow microneedle technology for depth-controlled microinjection-mediated dermal vaccination: a study with polio vaccine in rats.

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.

Delayed vaccine-induced CD8+ T cell expansion by topoisomerase I inhibition mediates enhanced CD70-dependent tumor eradication.

BACKGROUND: The survival of patients with cervical cancer who are treated with cisplatin in conjunction with the topoisomerase I inhibitor topotecan is enhanced when compared with patients treated with only one of these chemotherapeutics. Moreover, cisplatin-based and T cell-based immunotherapy have been shown to synergize, resulting in stronger antitumor responses. Here, we interrogated whether topotecan could further enhance the synergy of cisplatin with T cell-based cancer immunotherapy. METHODS: Mice bearing human papilloma virus 16 (HPV16) E6/E7-expressing TC-1 tumors were vaccinated with HPV16 E7 long peptides and additionally received chemotherapy consisting of cisplatin and topotecan. We performed an in-depth study of this combinatorial chemoimmunotherapy on the effector function and expansion/contraction kinetics of vaccine-induced CD8+ T cells in the peripheral blood and tumor microenvironment (TME). In addition, we interrogated the particular role of chemotherapy-induced upregulation of costimulatory ligands by tumor-infiltrated myeloid cells on T cell proliferation and survival. RESULTS: We show that E7 long peptide vaccination combined with cisplatin and topotecan, results in CD8+ T cell-dependent durable rejection of established tumors and 94% long-term survival. Although topotecan initially repressed the expansion of vaccine-induced CD8+ T cells, these cells eventually expanded vigorously, which was followed by delayed contraction. These effects associated with the induction of the proliferation marker Ki-67 and the antiapoptosis molecule Bcl-2 by intratumoral tumor-specific CD8+ T cells, which was regulated by topotecan-mediated upregulation of the costimulatory ligand CD70 on myeloid cells in the TME. CONCLUSIONS: Taken together, our data show that although treatment with cisplatin, topotecan and vaccination initially delays T cell expansion, this combinatorial therapy results eventually in a more robust T cell-mediated tumor eradication due to enhancement of costimulatory molecules in the TME.

Efficient fabrication of thermo-stable dissolving microneedle arrays for intradermal delivery of influenza whole inactivated virus vaccine.

Dissolving microneedle arrays (dMNAs) can be used to deliver vaccines via the intradermal route. Fabrication of dMNAs using centrifugation is the most common preparation method of dMNAs, but it results in a substantial loss of antigens. In order to solve the issue of antigen waste, we engineered an automatic dispensing system for dMNA preparation. Here, we report on the fabrication of influenza whole inactivated virus (WIV) vaccine-loaded dMNAs (WIV dMNAs) by using the automatic dispensing system. Prior to the dispensing process, polydimethylsiloxane (PDMS) moulds were treated with oxygen plasma to increase surface hydrophilicity. WIV dMNAs were prepared with 1% (w/v) trehalose and pullulan (50 : 50 weight ratio). During the dispensing process, reduced pressure was applied to the PDMS mould via a vacuum chamber to make microneedle cavities airless. After producing dMNAs, WIV was quantified and 1.9 µg of WIV was loaded per dMNA, of which 1.3 µg was in the microneedle tips. Compared to the centrifugation method, this automatic dispensing system resulted in a 95% reduction of antigen waste. A hemagglutination assay confirmed that WIV dMNA maintained the stability of the antigen for at least four weeks of storage, even at room temperature or at 37 °C. The WIV dMNAs displayed 100% penetration efficiency in human skin, and 83% of the microneedle volume was dissolved in the skin within 10 minutes. In a vaccination study, mice immunised with WIV dMNAs showed similar IgG levels to those that received WIV intramuscularly. In conclusion, using the automatic dispensing system for dMNA production strongly reduced antigen waste and yielded dMNAs with excellent physical, mechanical, and immunological properties.

Fluorescent nanoparticle adhesion assay: a novel method for surface pKa determination of self-assembled monolayers on silicon surfaces.

Since the computer industry enables us to generate smaller and smaller structures, silicon surface chemistry is becoming increasingly important for (bio-)analytical and biological applications. For controlling the binding of charged biomacromolecules such as DNA and proteins on modified silicon surfaces, the surface pK(a) is an important factor. Here we present a fluorescent nanoparticle adhesion assay as a novel method to determine the surface pK(a) of silicon surfaces modified with weak acids or bases. This method is based upon electrostatic interactions between the modified silicon surface and fluorescent nanoparticles with an opposite charge. Silicon slides were modified with 3-aminopropyltriethoxysilane (APTES) and were further derivatized with succinic anhydride. Layer thickness of these surfaces was determined by ellipsometry. After incubating the surfaces with an amine-reactive fluorescent dye, fluorescence microscopy revealed that the silicon surfaces were successfully modified with amine- and carboxyl-groups. Two surface pK(a) values were found for APTES surfaces by the fluorescent nanoparticle adhesion assay. The first surface pK(a) (6.55 ± 0.73) was comparable with the surface pK(a) obtained by contact angle titration (7.3 ± 0.8), and the second surface pK(a) (9.94 ± 0.19) was only found by using the fluorescent nanoparticle adhesion assay. The surface pK(a) of the carboxyl-modified surface by the fluorescent nanoparticle adhesion assay (4.37 ± 0.59) did not significantly differ from that found by contact angle titration (5.7 ± 1.4). In conclusion, we have developed a novel method to determine the surface pK(a) of modified silicon surfaces: the fluorescent nanoparticle adhesion assay. This method may provide a useful tool for designing pH-dependent electrostatic protein and particle binding/release and to design surfaces with a pH-dependent surface charge for (bio-)analytical lab-on-a-chip devices or drug delivery purposes.

Quantification of Lipid and Peptide Content in Antigenic Peptide-loaded Liposome Formulations by Reversed-phase UPLC using UV Absorbance and Evaporative Light Scattering Detection.

Antigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical translation. In this work we describe a reversed phase ultra-performance liquid chromatography (RP-UPLC) method that separates lipids (DOTAP, DOPC and their degradation products) and two physicochemically different peptides within 12 min. Samples were prepared by dilution in a 1:1 (v/v) mixture of methanol and water. Peptide quantification was done via UV detection and lipids were quantified by an evaporative light scattering detector (ELSD), both coupled to the RP-UPLC system, with high precision (RSD < 3.5%). We showed that the presence of lipids and peptides did not mutually influence their quantification. Limit of detection (LOD) and limit of quantification (LOQ), as determined in the ICH guidelines, were 6 and 20 ng for DOTAP, 12 ng and 40 ng for DOPC, 3.0 ng and 8.0 ng for peptide A and 2.4 ng and 7.2 ng for the more hydrophobic peptide B. Finally, lipid degradation of DOTAP and DOPC was monitored in peptide loaded DOTAP:DOPC liposomes upon storage at 4 °C and 40 °C.

Evaluation of the high-pressure extrusion technique as a method for sizing plasmid DNA-containing cationic liposomes.

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56-150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles.

Cationic Nanoparticle-Based Cancer Vaccines.

Cationic nanoparticles have been shown to be surprisingly effective as cancer vaccine vehicles in preclinical and clinical studies. Cationic nanoparticles deliver tumor-associated antigens to dendritic cells and induce immune activation, resulting in strong antigen-specific cellular immune responses, as shown for a wide variety of vaccine candidates. In this review, we discuss the relation between the cationic nature of nanoparticles and the efficacy of cancer immunotherapy. Multiple types of lipid- and polymer-based cationic nanoparticulate cancer vaccines with various antigen types (e.g., mRNA, DNA, peptides and proteins) and adjuvants are described. Furthermore, we focus on the types of cationic nanoparticles used for T-cell induction, especially in the context of therapeutic cancer vaccination. We discuss different cationic nanoparticulate vaccines, molecular mechanisms of adjuvanticity and biodistribution profiles upon administration via different routes. Finally, we discuss the perspectives of cationic nanoparticulate vaccines for improving immunotherapy of cancer.

Engineering of an automated nano-droplet dispensing system for fabrication of antigen-loaded dissolving microneedle arrays.

Dissolving microneedle arrays (dMNAs) are promising devices for intradermal vaccine delivery. The aim of this study was to develop a reproducible fabrication method for dMNAs based on an automated nano-droplet dispensing system that minimizes antigen waste. First, a polymer formulation was selected to dispense sufficiently small droplets (<18 nL) that can enter the microneedle cavities (base diameter 330 µm). Besides, three linear stages were assembled to align the dispenser with the cavities, and a vacuum chamber was designed to fill the cavities with dispensed droplets without entrapped air. Lastly, the dispenser and stages were incorporated to build a fully automated system. To examine the function of dMNAs as a vaccine carrier, ovalbumin was loaded in dMNAs by dispensing a mixture of ovalbumin and polymer formulation, followed by determining the ovalbumin loading and release into the skin. The results demonstrate that functional dMNAs which can deliver antigen into the skin were successfully fabricated via the automatic fabrication system, and hardly any antigen waste was encountered. Compared to the method that centrifuges the mould, it resulted in a 98.5% volume reduction of antigen/polymer solution and a day shorter production time. This system has potential for scale-up of manufacturing to an industrial scale.

Antigen Uptake After Intradermal Microinjection Depends on Antigen Nature and Formulation, but Not on Injection Depth.

The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.

Nanoporous microneedle arrays seamlessly connected to a drug reservoir for tunable transdermal delivery of memantine.

Conventional transdermal drug patches have been on the market since 1997 but their applicability for drug delivery is limited: currently only nearly two dozen of molecules have been approved by the regulatory authorities for transdermal administration and have reached the market. The possibilities for drug delivery via the skin can be improved and expanded by using microneedle patch technologies. However, most microneedle patches focus on the delivery of low amounts of drugs that are generally very potent due to the small dimensions of the microneedle systems. In this study nanoporous microneedle arrays (npMNAs) were combined with a liquid drug reservoir. The parameters that influence the diffusion of memantine from the drug reservoir through the npMNAs in an acceptor solution were investigated. Based on these results a model was developed to predict the diffusion of low-molecular-weight drugs as a function of npMNA properties (i.e., backplate thickness and surface area) and reservoir properties (i.e., volume and drug concentration). This generated an in silico model to predict the release of low-molecular-weight drug from a drug reservoir through a microneedle array into receptor solution, showed a good correlation with the delivery of memantine in a preclinical minipig study. The drug release rates by the npMNAs can be tuned and allow for both zero and first order release kinetics. Summarizing, this work shows that the npMNA technology is a versatile drug delivery system. The npMNAs can be combined with a (seamlessly connected) external drug reservoir and this integrated drug delivery system can be used to deliver at least 9 mg of memantine over 72 h in a preclinical minipig study.

Liposome-Based Drug Delivery Systems in Cancer Immunotherapy.

Cancer immunotherapy has shown remarkable progress in recent years. Nanocarriers, such as liposomes, have favorable advantages with the potential to further improve cancer immunotherapy and even stronger immune responses by improving cell type-specific delivery and enhancing drug efficacy. Liposomes can offer solutions to common problems faced by several cancer immunotherapies, including the following: (1) Vaccination: Liposomes can improve the delivery of antigens and other stimulatory molecules to antigen-presenting cells or T cells; (2) Tumor normalization: Liposomes can deliver drugs selectively to the tumor microenvironment to overcome the immune-suppressive state; (3) Rewiring of tumor signaling: Liposomes can be used for the delivery of specific drugs to specific cell types to correct or modulate pathways to facilitate better anti-tumor immune responses; (4) Combinational therapy: Liposomes are ideal vehicles for the simultaneous delivery of drugs to be combined with other therapies, including chemotherapy, radiotherapy, and phototherapy. In this review, different liposomal systems specifically developed for immunomodulation in cancer are summarized and discussed.

Thermosensitive hydrogels as sustained drug delivery system for CTLA-4 checkpoint blocking antibodies.

Thermosensitive poloxamer 407 (P407) hydrogels were evaluated as slow release system for optimizing CTLA-4 therapy. Slow release reduces systemic antibody levels and potentially mitigates the side effects of CTLA-4 therapy. The 25% P407 hydrogel is injectable at room temperature and depots are established quickly after subcutaneous injection. Scanning electron microscopy revealed the porous structure of the hydrogel, average pore surface was 1335 µm2. Release studies were optimized using the human IgG antibody. IgG was easily incorporated in the hydrogel by simple mixing and no antibodies were lost during preparation. In vitro, hydrogels showed low burst release within the first 24 h. Total IgG load was gradually released within 120 h. In vitro cytotoxicity assays showed that P407 is not cytotoxic and induces no immune activation by itself. In vivo, P407 hydrogels significantly reduced serum IgG levels, were biocompatible and were broken down 1 week after injection. Finally, local hydrogel delivery of anti-CTLA-4 antibodies near established tumors effectively slowed down tumor growth, whilst significantly reduced serum anti-CTLA-4 levels. Altogether, P407 hydrogels represent promising delivery systems for the optimization of CTLA-4 blocking therapy.