Engineered microneedles for transcutaneous vaccine delivery

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (p. 151-165).Immunization is a powerful approach for the prevention and control of infectious disease, however despite the successes of modem vaccine development, there remain several notable obstacles for the advancement of vaccine-mediated improvements in global healthcare. Many of the current limitations in vaccine availability and administration are the result of obligate needle-based delivery, which in addition to contributing to reduced speed, ease, and compliance in administration, has been shown to contribute to reduced overall safety due to needle re-use and needle-based injuries. Needle-based vaccine delivery to immunologically passive tissues such as muscle may limit efficacy, thus motivating the targeting of more inherently potent immune-competent sites. These inherent limitations of needle-based vaccination on global health have led to a strong impetus to develop needle-free vaccination strategies which have the potential to improve vaccine efficacy and availability, enhance the ease, speed, and safety of vaccine administration, and reduce vaccination associated costs world-wide. Here we present the design and preclinical testing of several parallel materials strategies for the noninvasive delivery of subunit vaccines to the skin. We have utilized laser ablative micro-molding of poly(dimethylsiloxane) to generate bio resorbable poly(lactide-co-glycolide) micro-structured skin patches bearing -100 micron-scale needles arrayed across their surface. Upon topical application, these 'microneedle arrays' are able to safely, and painlessly insert into the immune-competent epidermal skin layers to generate microscopic conduits through which otherwise impermeant vaccines and therapeutics are able to passage into the body. We have leveraged this approach in combination with layer-by-layer (LbL) directed assembly to generate vaccine-loaded conformal coatings on the surface of these microneedle arrays, which are then delivered into the skin through topical patch application. The construction of coatings containing antigen-expressing plasmid DNA (pDNA), together with immune-stimulatory RNA, and degradable cationic polymers provided tunable control over vaccine dosage, rapid and effective vaccine delivery in murine and primate skin models, and potent immunogenicity against a model HIV antigen in mice. In this case, DNA vaccine delivery was able to elicit strong functional CD8' T cell and humoral responses matching or exceeding the potency of in vivo electroporation, currently the most promising approach for clinical DNA delivery in humans. Further efforts have explored the use of LbL for encapsulation and delivery of soluble and particulate protein subunit vaccines, giving enhanced generation of diverse and potent humoral responses in mice. In other work, we have developed an approach enabling rapid delivery of micron-scale degradable polymer matrices or hydrogel depots using dissolvable composite microneedle structures for the delivery of vaccines with programmable kinetics. These efforts have demonstrated the potential of persistent vaccine release on tuning immune potency following non-invasive microneedle delivery, including induction of potent effector and memory CD8* T cell responses and more powerful and diverse antigen-specific humoral responses. Finally, we have developed an approach for simple loading and delivery of clinically advanced recombinant adenoviral vaccine vectors from sugar-glass coatings on bioresorbable microneedles. Formulation in microneedle coatings improved vaccine stability at room temperature and preclinical testing of these vaccine patches in mice and nonhuman primates demonstrated equivalent immunogenicity compared to parenteral injection, eliciting strong systemic and disseminated mucosal CD8' and CD4* T cell responses to a model HIV antigen. These cellular responses were correlated with a similarly potent systemic and mucosal humoral response, together suggesting the utility of this approach for non-invasive adenoviral immunization in a model close to humans. Together these results strongly demonstrate the potential of materials engineering strategies for the effective formulation, delivery, and release of recombinant vaccines by microneedle patches targeting the skin. In addition to the significant practical advantages enabled by microneedle delivery including improved safety, convenience, and storage, we have shown that advanced formulation strategies paired with controlled release are able to initiate humoral and cellular adaptive immunity more potently than possible through parenteral injection. Comprehensive tests in both mice and primates have suggested that these principles may be broadly applied to enhance various recombinant vaccination strategies potentially targeting numerous disease targets. Finally, initial tests performed in nonhuman primates have indicated the promise of engineered microneedle approaches for successful translation to humans. Overall, these findings provide a strong basis for the continued development of similar vaccination strategies for the comprehensive transformation of conventional vaccination enabling significant vaccine-mediated improvements in global health.by Peter C. DeMuth.Ph.D

Composite Dissolving Microneedles for Coordinated Control of Antigen and Adjuvant Delivery Kinetics in Transcutaneous Vaccination

Transcutaneous administration has the potential to improve therapeutics delivery, providing an approach that is safer and more convenient than traditional alternatives, while offering the opportunity for improved therapeutic efficacy through sustained/controlled drug release. To this end, a microneedle materials platform is demonstrated for rapid implantation of controlled-release polymer depots into the cutaneous tissue. Arrays of microneedles composed of drug-loaded poly(lactide-co-glycolide) (PLGA) microparticles or solid PLGA tips are prepared with a supporting and rapidly water-soluble poly(acrylic acid) (PAA) matrix. Upon application of microneedle patches to the skin of mice, the microneedles perforate the stratum corneum and epidermis. Penetration of the outer skin layers is followed by rapid dissolution of the PAA binder on contact with the interstitial fluid of the epidermis, implanting the microparticles or solid polymer microneedles in the tissue, which are retained following patch removal. These polymer depots remain in the skin for weeks following application and sustain the release of encapsulated cargos for systemic delivery. To show the utility of this approach the ability of these composite microneedle arrays to deliver a subunit vaccine formulation is demonstrated. In comparison to traditional needle-based vaccination, microneedle delivery gives improved cellular immunity and equivalent generation of serum antibodies, suggesting the potential of this approach for vaccine delivery. However, the flexibility of this system should allow for improved therapeutic delivery in a variety of diverse contexts.Massachusetts Institute of Technology. Ragon Institute of MGH, MIT and HarvardNational Institutes of Health (U.S.) (Award AI095109)United States. Army Research Office (Contract W911NF-07-D-0004

Polymer multilayer tattooing for enhanced DNA vaccination

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.Howard Hughes Medical Institute (Investigator)Ragon Institute of MGH, MIT, and HarvardNational Institutes of Health (U.S.) (NIH AI095109)United States. Dept. of Defense. Institute for Soldier Nanotechnologies (contract W911NF-07-D-0004)United States. Dept. of Defense. Institute for Soldier Nanotechnologies (contract W911NF-07-0004

Stable Patterns of Gene Expression Regulating Carbohydrate Metabolism Determined by Geographic Ancestry

Background: Individuals of African descent in the United States suffer disproportionately from diseases with a metabolic etiology (obesity, metabolic syndrome, and diabetes), and from the pathological consequences of these disorders (hypertension and cardiovascular disease). Methodology/Principal Findings: Using a combination of genetic/genomic and bioinformatics approaches, we identified a large number of genes that were both differentially expressed between American subjects self-identified to be of either African or European ancestry and that also contained single nucleotide polymorphisms that distinguish distantly related ancestral populations. Several of these genes control the metabolism of simple carbohydrates and are direct targets for the SREBP1, a metabolic transcription factor also differentially expressed between our study populations. Conclusions/Significance: These data support the concept of stable patterns of gene transcription unique to a geographic ancestral lineage. Differences in expression of several carbohydrate metabolism genes suggest both genetic and transcriptional mechanisms contribute to these patterns and may play a role in exacerbating the disproportionate levels o

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Формирование эмоциональной культуры как компонента инновационной культуры студентов

Homozygosity has long been associated with rare, often devastating, Mendelian disorders1 and Darwin was one of the first to recognise that inbreeding reduces evolutionary fitness2. However, the effect of the more distant parental relatedness common in modern human populations is less well understood. Genomic data now allow us to investigate the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity, ROH), which are inferred to be homozygous along their complete length. Given the low levels of genome-wide homozygosity prevalent in most human populations, information is required on very large numbers of people to provide sufficient power3,4. Here we use ROH to study 16 health-related quantitative traits in 354,224 individuals from 102 cohorts and find statistically significant associations between summed runs of homozygosity (SROH) and four complex traits: height, forced expiratory lung volume in 1 second (FEV1), general cognitive ability (g) and educational attainment (nominal p<1 × 10−300, 2.1 × 10−6, 2.5 × 10−10, 1.8 × 10−10). In each case increased homozygosity was associated with decreased trait value, equivalent to the offspring of first cousins being 1.2 cm shorter and having 10 months less education. Similar effect sizes were found across four continental groups and populations with different degrees of genome-wide homozygosity, providing convincing evidence for the first time that homozygosity, rather than confounding, directly contributes to phenotypic variance. Contrary to earlier reports in substantially smaller samples5,6, no evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein (LDL) cholesterol, or ten other cardio-metabolic traits. Since directional dominance is predicted for traits under directional evolutionary selection7, this study provides evidence that increased stature and cognitive function have been positively selected in human evolution, whereas many important risk factors for late-onset complex diseases may not have been

New loci for body fat percentage reveal link between adiposity and cardiometabolic disease risk

To increase our understanding of the genetic basis of adiposity and its links to cardiometabolic disease risk, we conducted a genome-wide association meta-analysis of body fat percentage (BF%) in up to 100,716 individuals. Twelve loci reached genome-wide significance (P<5 × 10−8), of which eight were previously associated with increased overall adiposity (BMI, BF%) and four (in or near COBLL1/GRB14, IGF2BP1, PLA2G6, CRTC1) were novel associations with BF%. Seven loci showed a larger effect on BF% than on BMI, suggestive of a primary association with adiposity, while five loci showed larger effects on BMI than on BF%, suggesting association with both fat and lean mass. In particular, the loci more strongly associated with BF% showed distinct cross-phenotype association signatures with a range of cardiometabolic traits revealing new insights in the link between adiposity and disease risk

New loci for body fat percentage reveal link between adiposity and cardiometabolic disease risk

To increase our understanding of the genetic basis of adiposity and its links to cardiometabolic disease risk, we conducted a genome-wide association meta-analysis of body fat percentage (BF%) in up to 100,716 individuals. Twelve loci reached genome-wide significance (P <5 x 10(-8)), of which eight were previously associated with increased overall adiposity (BMI, BF%) and four (in or near COBLL1/GRB14, IGF2BP1, PLA2G6, CRTC1) were novel associations with BF%. Seven loci showed a larger effect on BF% than on BMI, suggestive of a primary association with adiposity, while five loci showed larger effects on BMI than on BF%, suggesting association with both fat and lean mass. In particular, the loci more strongly associated with BF% showed distinct cross-phenotype association signatures with a range of cardiometabolic traits revealing new insights in the link between adiposity and disease risk.Peer reviewe

Releasable Layer-by-Layer Assembly of Stabilized Lipid Nanocapsules on Microneedles for Enhanced Transcutaneous Vaccine Delivery

Here we introduce a new approach for transcutaneous drug delivery, using microneedles coated with stabilized lipid nanocapsules, for delivery of a model vaccine formulation. Poly(lactide-co-glycolide) microneedle arrays were coated with multilayer films via layer-by-layer assembly of a biodegradable cationic poly(β-amino ester) (PBAE) and negatively charged interbilayer-cross-linked multilamellar lipid vesicles (ICMVs). To test the potential of these nanocapsule-coated microneedles for vaccine delivery, we loaded ICMVs with a protein antigen and the molecular adjuvant monophosphoryl lipid A. Following application of microneedle arrays to the skin of mice for 5 min, (PBAE/ICMV) films were rapidly transferred from microneedle surfaces into the cutaneous tissue and remained in the skin following removal of the microneedle arrays. Multilayer films implanted in the skin dispersed ICMV cargos in the treated tissue over the course of 24 h in vivo, allowing for uptake of the lipid nanocapsules by antigen presenting cells in the local tissue and triggering their activation in situ. Microneedle-mediated transcutaneous vaccination with ICMV-carrying multilayers promoted robust antigen-specific humoral immune responses with a balanced generation of multiple IgG isotypes, whereas bolus delivery of soluble or vesicle-loaded antigen via intradermal injection or transcutaneous vaccination with microneedles encapsulating soluble protein elicited weak, IgG1-biased humoral immune responses. These results highlight the potential of lipid nanocapsules delivered by microneedles as a promising platform for noninvasive vaccine delivery applications.Howard Hughes Medical Institute (Investigator)United States. Dept. of Defense (W911NF-07-D-0004)Ragon Institute of MGH, MIT, and HarvardNational Institutes of Health (U.S.) (NIH AI095109)United States. Army Research Office. Institute for Soldier Nanotechnologie

Graphene Multilayers as Gates for Multi-Week Sequential Release of Proteins from Surfaces

The ability to control the timing and order of release of different therapeutic drugs will play a pivotal role in improving patient care and simplifying treatment regimes in the clinic. The controlled sequential release of a broad range of small and macromolecules from thin film coatings offers a simple way to provide complex localized dosing in vivo. Here we show that it is possible to take advantage of the structure of certain nanomaterials to control release regimes from a scale of hours to months. Graphene oxide (GO) is a two-dimensional charged nanomaterial that can be used to create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. Protein-loaded polyelectrolyte multilayer films were fabricated using layer-by-layer assembly incorporating a hydrolytically degradable cationic poly(β-amino ester) (Poly1) with a model protein antigen, ovalbumin (ova), in a bilayer architecture along with positively and negatively functionalized GO capping layers for the degradable protein films. Ova release without the GO layers takes place in less than 1 h but can be tuned to release from 30 to 90 days by varying the number of bilayers of functionalized GO in the multilayer architecture. We demonstrate that proteins can be released in sequence with multi-day gaps between the release of each species by incorporating GO layers between protein loaded layers. In vitro toxicity assays of the individual materials on proliferating hematopoietic stem cells (HSCs) indicated limited cytotoxic effects with HSCs able to survive for the full 10 days of normal culture in the presence of Poly1 and the GO sheets. This approach provides a new route for storage of therapeutics in a solid-state thin film for subsequent delivery in a time-controlled and sequential fashion.Singapore-MIT Alliance for Research and Technolog