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

Buchbesprechungen aus Botanik und Naturschutz in Hessen Bd. 8

Unter anderem folgende Publikationen werden rezensiert: Adler et al.: Exkursionsflora von Österreich. Bestimmungsbuch für alle in Österreich wildwachsenden sowie die wichtigsten kultivierten Gefäßpflanzen (Farnpflanzen und Samenpflanzen) mit Angaben über ihre Ökologie und Verbreitung. Jahn & Schönfelder: Exkursionsflora für Kreta. Wirth: Die Flechten Baden-Württembergs. Adolphi: Neophytische Kultur- und Anbaupflanzen als Kulturflüchtlinge des Rheinlandes. Arbeitsgemeinschaft für Fledermausschutz in Hessen (Herausgeber): Die Fledermäuse Hessens. Sautter: Untersuchungen zur Diasporen- und Samenökologie in bedrohten Pflanzengesellschaften sandiger Böden. Denz: Natürliche Habichtskraut-Traubeneichenwälder bodensaurer Felsstandorte und ihre Vegetationskomplexe im Rheinischen Schiefergebirge und weiteren silikatischen Mittelgebirgen Wißkirchen: Verbreitung und Ökologie von Flußufer-Pioniergesellschaften (Chenopodion rubri) im mittleren und westlichen Europa. Goebel: Die Vegetation der Wiesen, Magerrasen und Rieder im Rhein-Main-Gebiet. Gilbert: Städtische Ökosysteme. Garve: Atlas der gefährdeten Farn- und Blütenpflanzen in Niedersachsen und Bremen

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

Genome-wide meta-analysis associates HLA-DQA1/DRB1 and LPA and lifestyle factors with human longevity

Genomic analysis of longevity offers the potential to illuminate the biology of human aging. Here, using genome-wide association meta-analysis of 606,059 parents’ survival, we discover two regions associated with longevity (HLA- DQA1/DRB1 and LPA). We also validate previous suggestions that APOE, CHRNA3/5, CDKN2A/B, SH2B3 and FOXO3A influence longevity. Next we show that giving up smoking, educational attainment, openness to new experience and high-density lipoprotein (HDL) cholesterol levels are most positively genetically correlated with lifespan while susceptibility to coronary artery disease (CAD), cigarettes smoked per day, lung cancer, insulin resistance and body fat are most negatively correlated. We suggest that the effect of education on lifespan is principally mediated through smoking while the effect of obesity appears to act via CAD. Using instrumental variables, we suggest that an increase of one body mass index unit reduces lifespan by 7 months while 1 year of education adds 11 months to expected lifespan

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

Location, location, location: the role of objective neighborhood characteristics for perceptions of control

Introduction: Control beliefs can protect against age-related declines in functioning. It is unclear whether neighborhood characteristics shape how much control people perceive over their life. This article studies associations of neighborhood characteristics with control beliefs of residents of a diverse metropolitan area (Berlin, Germany). Methods: We combine self-report data about perceptions of control obtained from participants in the Berlin Aging Study II (N = 507, 60-87 years, 51% women) with multisource geo-referenced indicators of neighborhood characteristics using linear regression models. Results: Findings indicate that objective neighborhood characteristics (i.e., unemployment rate) are indeed tied to perceptions of control, in particular, how much control participants feel others have over their lives. Including neighborhood characteristics in part doubled the amount of explained variance compared with a reference model covarying for demographic characteristics only (from R2 = 0.017 to R2 = 0.030 for internal control beliefs; R2 = 0.056 to R2 = 0.102 for external control beliefs in chance; R2 = 0.006 to R2 = 0.030 for external control beliefs in powerful others). Discussion/Conclusion: Findings highlight the importance of access to neighborhood resources for control beliefs across old age and can inform interventions to build up neighborhood characteristics which might be especially helpful in residential areas with high unemployment

Differences in biocompatibility of microneedles from cyclic olefin polymers with human endothelial and epithelial skin cells

Microneedles are promising devices for transdermal delivery and diagnostic applications, due to their minimally invasive and painless nature of application. However, so far, applications are limited to small scale research projects. Material selection and production for larger projects remain a challenge. In vitro testing using human cell culture could bridge the gap between cost effective screening of suitable materials and concerns for safety and ethics. In this study, materials were tested for effects on viability and morphology of human endothelial cells and keratinocytes. In addition, materials were assessed for their potential to influence cellular differentiation and barrier formation. Elution-based testing of inflammatory markers revealed no negative effects in all applied tests, whereas the assessment of differentiation markers on cells in direct contact with the material showed differences and allowed the selection of candidate materials for future medical device applications. This study illustrates that elution-based biocompatibility testing can paint an incomplete picture. Advanced staining techniques and cell types specific for the application of the medical device improve material selection to reduce and replace animal testing at an early stage in the development process. © 2018 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 505–512, 2019

Vaccine delivery with microneedle skin patches in nonhuman primates

Transcutaneous drug delivery from planar skin patches is effective for small-molecule drugs and skin-permeable vaccine adjuvants. However, to achieve efficient delivery of vaccines and other macromolecular therapeutics into the skin, penetration of the stratum corneum is needed. Topically applied skin patches with micron-scale projections ('microneedles') pierce the upper layers of the skin and enable vaccines that are coated on or encapsulated within the microneedles to be dispersed into the skin. Although millimeter-scale syringes have shown promise for vaccine delivery in humans and technologies, such as the Dermaroller (Dermaroller, Wolfenbüttel, Germany), exist for creating microscale punctures in the skin for delivery of solutions of therapeutics, solid microprojection microneedles coated with dry vaccine formulations offer a number of valuable features for vaccination, including reduced risk of blood-borne pathogen transmission or needle-stick injury, the potential for vaccine administration by minimally trained personnel or even self administration and the use of solid-state vaccine formulations that may reduce or eliminate cold-chain requirements in vaccine distribution. Recent studies in mice have demonstrated the ability of microneedles to effectively deliver vaccines to the skin, eliciting protective immunity to influenza, hepatitis C and West Nile virus.Ragon Institute of MGH, MIT and HarvardMassachusetts Institute of TechnologyHarvard UniversityNational Institutes of Health (U.S.) (AI095109)National Institutes of Health (U.S.) (AI096040)National Institutes of Health (U.S.) (AI095985)National Institutes of Health (U.S.) (AI078526)National Institutes of Health (U.S.) (AI060354)United States. Dept. of Defense (Contract W911NF-07-D-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

Musculoskeletal modelling of the Nile crocodile (Crocodylus niloticus) hindlimb: Effects of limb posture on leverage during terrestrial locomotion.

We developed a three-dimensional, computational biomechanical model of a juvenile Nile crocodile (Crocodylus niloticus) pelvis and hindlimb, composed of 47 pelvic limb muscles, to investigate muscle function. We tested whether crocodiles, which are known to use a variety of limb postures during movement, use limb orientations (joint angles) that optimise the moment arms (leverages) or moment-generating capacities of their muscles during different limb postures ranging from a high walk to a sprawling motion. We also describe the three-dimensional (3D) kinematics of the crocodylian hindlimb during terrestrial locomotion across an instrumented walkway and a treadmill captured via X-ray Reconstruction of Moving Morphology (biplanar fluoroscopy; 'XROMM'). We reconstructed the 3D positions and orientations of each of the hindlimb bones and used dissection data for muscle lines of action to reconstruct a focal, subject-specific 3D musculoskeletal model. Motion data for different styles of walking (a high, crouched, bended and two types of sprawling motion) were fed into the 3D model to identify whether any joints adopted near-optimal poses for leverage across each of the behaviours. We found that (1) the hip adductors and knee extensors had their largest leverages during sprawling postures and (2) more erect postures typically involved greater peak moment arms about the hip (flexion-extension), knee (flexion) and metatarsophalangeal (flexion) joints. The results did not fully support the hypothesis that optimal poses are present during different locomotory behaviours because the peak capacities were not always reached around mid-stance phase. Furthermore, we obtained few clear trends for isometric moment-generating capacities. Therefore, perhaps peak muscular leverage in Nile crocodiles is instead reached either in early/late stance or possibly during swing phase or other locomotory behaviours that were not studied here, such as non-terrestrial movement. Alternatively, our findings could reflect a trade-off between having to execute different postures, meaning that hindlimb muscle leverage is not optimised for any singular posture or behaviour. Our model, however, provides a comprehensive set of 3D estimates of muscle actions in extant crocodiles which can form a basis for investigating muscle function in extinct archosaurs